Positive Expression of SMYD2 is Associated with Poor Prognosis in Patients with Primary Hepatocellular Carcinoma
2018. 2018

Shan-Ru Zuo
Department of Center Clinical Pharmacology and Pharmacy
Central South University
The Third Xiangya HospitalChangshaChina

Xiao-Cong Zuo
Yang He
Department of Center Clinical Pharmacology and Pharmacy
Central South University
The Third Xiangya HospitalChangshaChina

Wei-Jin Fang
Department of Center Clinical Pharmacology and Pharmacy
Central South University
The Third Xiangya HospitalChangshaChina

Chun-Jiang Wang
Department of Center Clinical Pharmacology and Pharmacy
Central South University
The Third Xiangya HospitalChangshaChina

Heng Zou
Department of Center Clinical Pharmacology and Pharmacy
Central South University
The Third Xiangya HospitalChangshaChina

Department of General Surgery
The Second Xiangya Hospital
Central South University
ChangshaChina

Pan Chen
Department of Hepatobiliary Surgery
The Affiliated Cancer Hospital of Xiangya School of Medicine
Central South University
ChangshaChina

Ling-Fei Huang
Department of Obstetrics and Gynecology
Taizhou Hospital of Zhe Jiang Province
TaizhouChina

Li-Hua Huang
Center for Experimental Medicine Research
Department of Center Clinical Pharmacology and Pharmacy
The Third Xiangya Hospital of Central
The Third Xiangya Hospital
Central South University
ChangshaChina

South University
No.172 Tongzipo Road, YueLu District Changsha410013Hunan P.R.China

Hong Xiang
Center for Experimental Medicine Research
Department of Center Clinical Pharmacology and Pharmacy
The Third Xiangya Hospital of Central
The Third Xiangya Hospital
Central South University
ChangshaChina

South University
No.172 Tongzipo Road, YueLu District Changsha410013Hunan P.R.China

Shi-Kun Liu
Department of Center Clinical Pharmacology and Pharmacy
Central South University
The Third Xiangya HospitalChangshaChina

PhD.Shi-Kun Liu
Positive Expression of SMYD2 is Associated with Poor Prognosis in Patients with Primary Hepatocellular Carcinoma

Journal of Cancer
922018. 201810.7150/jca.22218Received: 2017.08.03; Accepted: 2017.10.24;321 J Jo ou ur rn na al l o of f C Ca an nc ce er r Research Paper  Corresponding author: (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.SMYD2hepatocellular carcinomaimmunohistochemistryprognosis
Purpose: SET and MYND domain-containing protein2 (SMYD2), a histone lysine methyltransferases, is a candidate human oncogene in multiple tumors. However, the expression dynamics of SMYD2 in hepatocellular carcinoma (HCC) and its clinical/prognostic significance are unclear. Methods: The SMYD2 expression profile was examined by quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry (IHC) in HCC tissues and matched adjacent non-tumorous tissues. SMYD2 was silenced in HCC cell lines to determine its role in tumor proliferation and cell cycle progression, and the possible mechanism. Spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data. Results: The SMYD2 expression in HCC tissues were significantly up-regulated at both mRNA and protein levels as compared with the matched adjacent non-tumorous tissues. By IHC, positive expression of SMYD2 was examined in 122/163 (74.85%) of HCC and in 10/59 (16.95%) of tumor-adjacent tissues. Positive expression of SMYD2 was correlated with tumor size, vascular invasion, differentiation and TNM stage (P < 0.05). In univariate survival analysis, a significant association between positive expression of SMYD2 and shortened patients' survival was found (P < 0.05). Importantly, SMYD2 expression together with vascular invasion (P < 0.05) provided significant independent prognostic parameters in multivariate analysis. Functionally, SMYD2 silenced markedly inhibited cell proliferation and cell cycle progression in SMMC-7721 cell. Conclusions: Our findings provide evidences that positive expression of SMYD2 in HCC may be important in the acquisition of an aggressive phenotype, and it is an independent biomarker for poor prognosis of patients with HCC.

Introduction

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor that accounts for 80% of all liver cancer cases worldwide [1]. HCC is particularly problematic in China, where the incidence of HCC is much higher than that in other Asian countries due to endemic Hepatitis B virus infection [2,3]. At present, liver transplantation and surgical liver resection are the curative treatments for early-stage HCC. Cellular and molecular mechanisms that contribute to the progression of liver cancer from an acute or a chronic form to metastatic disease are not fully understood [1]. Therefore, the identification Ivyspring International Publisher of new possible targets for preventing the initiation and progression of HCC is urgent and must be improved.

SET and MYND domain-containing protein 2 (SMYD2), a histone lysine methyltransferases, was first identified as one of the SMYD family members, containing a SET domain and a MYND domain [4]. SMYD2 methylates H3K36 and functions as a transcriptional regulator in cooperation with the Sin3A and HDAC1 histone deacetylase complex [4,5]. It was initially identified expression in muscle tissues and might be associated with muscle cells differentiation [6]. Recently, emerging evidence suggests that SMYD2 inhibits tumor suppressor proteins p53 [7], Rb [5,8], PTEN [9] and enhances the poly (ADP-ribose) activity of the oncogenic protein PARP1 in cancer cells [10]. Moreover, SMYD2 also plays promoting roles in the development and progression of various tumors. The mRNA and protein expression levels of SMYD2 have been reported to be overexpressed in bladder carcinoma [5], oesophageal squamous cell carcinoma (ESCC) [11], paediatric acute lymphoblastic leukaemia [12], gastric cancer [13] and HPV-unrelated head and neck squamous cell carcinoma(HNSCC) [14]. Moreover, SMYD2 expression has been found to be associated with the poor prognosis of ESCC, paediatric acute lymphoblastic leukaemia, gastric cancer and HPV-unrelated head and neck squamous cell carcinoma(HNSCC) [11][12][13][14]. However, to date, there has been no report on the clinical and prognostic significance of SMYD2 in patients with HCC. These findings prompted us to investigate the effects of SMYD2 overexpression and activation in HCC.

In the present study, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry (IHC) were utilized to examine the expression dynamics of SMYD2 in a cohort of HCC and matched adjacent non-tumorous tissues. We found that the expression level of SMYD2 in human HCC was significantly elevated, and the increased expression level of SMYD2 was associated with unfavorable clinical features and the poor prognosis of the HCC patients. Our in vitro studies demonstrated that SMYD2 promoted the tumor growth of HCC by facilitating cell proliferation and cell cycle progression. Our results indicated that SMYD2 is a promising biomarker of HCC, and can potentially serve as a therapeutic target of HCC.

Materials and Methods

Clinical samples and data

Clinical specimens derived from 163 patients, including 143 males and 20 females who were diagnosed with primary HCC, were examined in the present study. All patients underwent surgical resection at the Department of Hepatobiliary Surgery of Xiangya Hospital (Changsha, China) during January 2003 to December 2012. All patients did not receive any chemotherapy or embolization during the perioperative period. The clinicopathological characteristics are summarized in Table 1. Prior patient consent and approval from the Institutional Research Ethics Committee were obtained for the use of these clinical materials for research purposes. The tumor specimens were obtained from the paraffin blocks of 163 primary HCCs. We also obtained 59 samples, in paraffin blocks, tumor-adjacent hepatocellular tissue from the same HCC patients. Tumor grade and stage were defined according to the criteria of the World Health Organization and the sixth edition of the TNM classification of the International Union Against Cancer (UICC, 2002). Patient follow-up was terminated on Jannuary, 2012. Median follow-up was 18 months (range, 1-93). HCC-related death [15] was defined if there was a computed tomography scan increase of >25% in the sum of recurrent tumor index lesions' cross-sectional areas or new onset of, or increase in, either recurrence, vascular invasion, or metastasis within the last 6 months of life, irrespective of immediate cause of death. Overall survival (OS) was defined as the time interval between the date of resection and the date of death or the last observation.

Immunohistochemistry

Paraffin-embedded samples were cut into 4-μm thick slices. The slides were dried overnight at 37°C, dewaxed in xylene, rehydrated with graded alcohol, and immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Antigen retrieval was carried out in a microwave oven with 10 mM citrate buffer (pH 6.0) for 20 min. The slides were incubated with 10% normal goat serum at room temperature for 30 min to reduce nonspecific reactions. Subsequently, the slides were incubated overnight at 4°C with rabbit polyclonal antibody against SMYD2 (1:50; Proteintech Group, Chicago, USA), overnight at 4°C. After rinsing four times with 0.01 mol/L phosphate-buffered saline (PBS; pH 7.4) for 10 min, primary antibody was detected using a secondary antibody (Maxim, FuZhou, China) for 1 h at room temperature and stained with 3,3-diaminobenzidine (DAB) after washing in PBS again. Finally, the sections were counterstained with Maxim's hematoxylin, dehydrated, and mounted.

Two independent pathologists blinded to the clinicopathological information performed the analysis of IHC for SMYD2. Immunopositive staining was evaluated in randomly selected five areas of the tissue section. For SMYD2 protein expression, sections were scored as positive if epithelial cells showed immunostaining in the nucleus/cytoplasm when observed. The tissue sections were scored based on the percentage of SMYD2 expressing cells in the total population (proportion score 0-10% = 0, 1-25% = 1, 26-50% = 2, 51-75% = 3 and 76-100% = 4) and scoring SMYD2 staining intensity as negative = 0, mild = 1, moderate = 2, intense = 3 using high-power (×400) microscopy. Finally, expression of SMYD2 was graded based on the sum of the intensity score and the proportion score: positive expression (intensity multiply proportion scores > 6) or negative expression (intensity multiply proportion scores ≤6).

HCC cell lines and cell cultures

The human HCC cell lines MHCC-97H, MHCC-97L and HepG2 were obtained from the Jennio Biological Technology (Guangzhou, China) and SMMC-7721 was obtained from the KeyGen Biological Technology (Nanjing, China). MHCC-97H, MHCC-97L and SMMC-7721 Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. HepG2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. All cells were grown in a humidified incubator at 37°C with 5% CO2.

Paired tumor and adjacent tissues

Ten pairs of HCC tissues and matched adjacent non-tumorous tissues were selected from the Department of Hepatobiliary Surgery of Xiangya Hospital, and then frozen and stored in -80℃ refrigerator until used to compare the expression levels of SMYD2 mRNA.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from the 10 pairs of HCC tissue and matched adjacent non-tumorous tissues using TRIZOL reagent (OMEGA, USA). RNA was reverse-transcribed using ReverTra Ace Qpcr RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer's instructions. The SMYD2 sense primer was 5'-ATCTCCTGTACCCAACGGAAG-3', and the antisense primer was 5'-CACCTTGGCCTTATCCTTG TCC-3'. For the β-actin gene, the sense primer was 5'-TGGCACCCAGCACAATGAA-3', and the antisense primer was 5'-CTAAGTCATAGTCCGCCT AGAAGCA-3'. qRT-PCR was done using SYBR Green PCR master mix (TOYOBO, Osaka, Japan) in a total volume of 20 μl on the ABI 7500 thermocycler (Thermo Fisher Scientific, Inc., USA) as follows: 95°C for 1 min, 40 cycles of 95°C for 15s, and 60°C for 20s. A dissociation procedure was performed to generate a melting curve for confirmation of amplification specificity. β-actin was used as the reference gene. The relative levels of gene expression were represented as △Ct=Ctgene-Ctreference, and the fold change of gene expression was calculated by the 2 -△△Ct Method. Experiments were repeated in triplicate.

Western blotting

Equal amount of whole-cell lysates were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad, USA). This was followed by incubation with primary rabbit polyclonal antibody against human SMYD2 (1:1500) (Abcam, Cambridge, MA), cyclin D1 (1:10000) (Abcam, Cambridge, MA). The immunoreactive proteins were detected with enhanced chemiluminescent detection reagents (Millipore Corporation, USA) according to the manufacturer's instructions. The membranes were stripped and reblotted with a rabbit monoclonal anti-GAPDH antibody (Bioworld Technology, Minneapolis, USA) as a loading control.

Small interfering RNA transfection

siRNA oligonucleotide duplexes and siNegative control (siNC) were synthesized by GenePharma, Suzhou. Co., Itd. (Suzhou, China). The siRNA sequences are described in Table 2. SiRNA duplexes (final concentration, 50nM) were transfected into HCC cell lines with an HVJ Envelope (HVJ-E) Vector Kit GenomONE-Neo (Ishikawa Sangyo KaishaLtd., Osaka, Japan) by modified methods described in the manufacturer's instructions and by Tsuchiya et al. [16]. Briefly, HVJ-E (10ul) was mixed with 5ul of siRNA solution and 3ul of Reagent B. After centrifugation, the pellet was resuspended in 27ul of buffer, followed by the addition of 8ul of Reagent C. The siRNA-HVJ-E mixture was combined with HCC cells and centrifuged at 12,000 rpm for 20 min at 4℃. The cells were resuspended in 1 ml of culture media and incubated for 72h.

EdU assay

EdU assays was used to assess cell proliferation. Briefly, cells were cultured in 96-well plates and transfected with siSMYD2 or siNC. Cell proliferation was evaluated on 24h, 48h and 72h. Then, cells were incubated with 50 μl of EdU (Ribobio Co., LTD, Guangzhou, China) for additional 2 h at 37 °C. Cells were fixed with 4% formaldehyde for 30 min, incubated with glycine (2 mg/ml) for 5 min and treated with 0.5% Triton X-100 for 10 min to permeabilize cells. After being washed with phosphate-buffered saline, cells were incubated with Apollo reaction cocktail for 30 min and treated twice with 0.5% Triton X-100. DNA was stained with Hoechst 33342 stain for 30 min and visualized/calculated with Operetta (Perkin Elmer, USA). Six groups of confluent cells were randomly selected from each sample image.

Flow cytometry

For cell cycle analysis, cells were collected at the indicated time points. Cells (1 × 10 6 ) were washed with PBS and fixed with cold 70% ethanol at 4°C overnight. Then, cells were treated with RNase and stained with propidium iodide (PI, Wellbio, China). The DNA content of the cells was quantified using a flow cytometer (BD FACSCalibur, USA). In total, 10,000 nuclei were examined in the flow cytometer, and DNA histograms were analyzed by ModFit software (Verity Software House, USA).

Statistical analysis

Data were analyzed using SPSS18.0 software (SPSS Inc.). Significant associations between SMYD2 expression and clinicopathological parameters were assessed using a χ2 test. Survival curves were plotted by Kaplan-Meier analysis and compared by the log-rank test. Cox regression analysis was carried out to assess the significance of variables for survival. Data were expressed as mean ± SD, and the t-test was used to determine the significance of differences between two groups. All tests carried out were two-sided. P < 0.05 was considered statistically significant.

Results

qRT-PCR and Western blotting analysis of SMYD2 expression in HCC tissues and cell lines

SMYD2 exhibited a markedly higher expression in HCC tumor tissues than in matched adjacent non-tumorous tissues, as determined by qRT-PCR ( Figure 1A). SMMC-7721, HepG2, MHCC-97H, and MHCC-97L cells were detected by Western blot analysis to determine the SMYD2 expression in different liver cancer cell lines. The results exhibit as following (in descending order of their SMYD2 levels): SMMC-7721, MHCC-97L, HepG2 and MHCC-97H. Almost positive results were obtained in different liver cancer cell lines ( Figure 1B).

IHC analysis of SMYD2 expression in paraffin-embedded of HCC tissues

The expression of SMYD2 protein was determined by IHC in paraffin-embedded of HCC tissues containing 163 cases of HCC tissues and 59 specimens of matched adjacent non-tumorous tissues. Using the criteria described earlier, positive expression of SMYD2 was detected in 74.85% (122/163) of HCC tissues, while only 16.95% (10/59) of matched adjacent non-tumorous tissues had positive staining (Figure 2A-2D).

Association of SMYD2 expression with HCC clinicopathological features

The association between SMYD2 expression in HCC detected by IHC and several known clinicopathological features were studied further. SMYD2 levels were inversely correlated with tumor size, vascular invasion, differentiation and TNM stage (P < 0.05, Table 1). There was no significant association between SMYD2 expression and other clinicopathological features, such as patient gender, age, AFP level, and Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), cirrhosis, tumor encapsulation, tumor number and distant metastasis (P > 0.05, Table 1).

Relationship between clinicopathological variables, SMYD2 expression, and HCC patient survival: univariate survival analysis

To confirm the representativeness of the HCC in our study, we analyzed the established prognostic predictors of the overall survival (OS) in our cohort. Kaplan-Meier analysis demonstrated a significant impact of well-known clinicopathological prognostic parameters on patient survival, such as vascular invasion, differentiation and TNM stage (P < 0.05, Table 3). Assessment of the overall survival curves showed that patients with positive staining of SMYD2 had significantly reduced OS (P <0.01, Table 3, Figure  3). These data suggest that SMYD2 may be involved in the development and progression of HCC, and can serve as a promising predictor of the prognosis of HCC.

Independent prognostic factors of HCC: multivariate Cox regression analysis

The expression of SMYD2 as well as other clinical pathological parameters that were significant in univariate analysis (vascular invasion, differentiation, TNM stage and SMYD2 expression), was further examined in multivariate analysis. Positive expression of SMYD2 was found to be an independent prognostic factor for poor overall survival (relative risk: 4.00, 95% confidence interval: 2.03-7.91, P <0.01, Table 4). Of the other parameters, vascular invasion was also demonstrated as independent prognostic factors for overall survival (P < 0.05, Table 4).

SMYD2 promotes the growth of HCC cells in vitro

To explore the functional of SMYD2 in HCC, we performed cell proliferation assays using siRNA specific for SMYD2 and investigated whether silence of SMYD2 would suppress proliferation of liver cancer cell lines. We chose the SMMC-7721 cell line for these assays, because it had the highest amount of SMYD2 protein ( Figure 1B). Expression of SMYD2 protein in this cell line was efficiently knocked down 72 h after the transient introduction of SMYD2-specific siRNA (siRNA-SMYD2) ( Figure 4A) than with the control siRNA (siRNA-NC). Subsequently, the results of the EdU assays showed that the cell proliferation of the SMMC-7721 cells was significantly decreased after SMYD2 knockdown (P <0.05, Figure 4B). In addition, cell cycle assays showed that knockdown of SMYD2 significantly increased the percentage of cells in the G0/G1 phase (P <0.05), and decreased the percentage of cells in the S phase (P <0.05) ( Figure 4C). These data suggest that SMYD2 promotes the growth of HCC cells by regulating cell proliferation and cell cycle progression in vitro.

SMYD2 regulates the expression of cyclin D1 in HCC cells

Cyclin D1 has been found to be overexpressed in HCC [17,18], and plays an important role in regulating cell cycle progression [19]. Therefore, we ascertained whether SMYD2 regulates the expression of cyclin D1 in HCC cells. The results of the Western blot analyses showed that the expression of cyclin D1 was significantly reduced after downregulation of the expression of SMYD2 (P <0.05, Figure 5). These data indicate that SMYD2 may regulate the expression of cyclin D1 in HCC cells.

Discussion

In spite of significant advances in its diagnosis and treatment, HCC remains one of the most lethal tumors in China [20]. Therefore, it is of great importance to explore HCC-specific biomarkers and effective therapeutic targets, which may promote the early diagnosis and improve the efficacy of molecular-targeted drugs for HCC. Several SMYD family members have been reported to be involved in oncogenesis and tumor progression. For example, Liu et al. reported that overexpression of SMYD3 promoted tumor cell proliferation and was a predictor of poor prognosis in gastric cancer [21], and others have shown that SMYD4 acts as a tumor suppressor in breast cancer [22]. Despite this existing body of knowledge, the role of SMYD2 in the progression of HCC has not been explored.

In the present study, we measured SMYD2 expression in primary HCC samples by qPCR. Similar to previous studies on oesophageal squamous cell carcinoma (ESCC), bladder carcinoma, paediatric acute lymphoblastic leukaemia, gastric cancer and HPV-unrelated head and neck squamous cell carcinoma(HNSCC) [5,[11][12][13][14], we found that SMYD2 expression in HCC tumor tissue was significantly increased compared with matched adjacent non-tumorous tissue. Importantly, we found that the positive expression of SMYD2 was associated with poor prognosis and reduced survival periods for HCC patients. Multivariate analysis showed that the positive expression of SMYD2 protein expression could be used as an independent prognostic predictor for HCC patients. Our results indicate that the expression level of SMYD2 protein might provide useful information in the evaluation prognosis and follow up schedule guiding for HCC patients. In addition, Silence of endogenous SMYD2 expression using a specific siRNA suppressed the proliferation and cell cycle progression of SMMC-7721 cells. CyclinD1 was also down-regulated under that condition. Taken together, these findings may suggest that SMYD2 has an oncogenic role in the HCC cell line. Cyclin D1 functions as a well-recognized regulator of cell cycle progression and proliferation, and has been found to be deregulated in many types of human cancers, including HCC [23][24][25]. In the present study, we confirmed that SMYD2 regulated the expression of cyclin D1 in HCC cells using specific siRNA. SMYD2 as a lysine methyltransferase (KMT) have been validated suppress p53 at Ly370. PTEN protein was shown to be methylated at lysine 313 in breast cell lines [9], and methylated RB at lysine 860 in bladder cancer cell lines [5]. Therefore, SMYD2 might act through different mechanisms in different cancer types.

In summary, the present study confirmed for the first time that SMYD2 is overexpressed in HCC tissues. Patients with positive expression of SMYD2 had adverse clinicopathological features and poorer prognosis. Functionally, SMYD2 can promote the growth of HCC cells by promoting cell proliferation and cell cycle progression. Furthermore, SMYD2 can regulate the expression of cyclin D1 in HCC cells. Therefore, the present study demonstrated that SMYD2 can serve as a promising biomarker for the early diagnosis and prognostic prediction of HCC patients, and can potentially act as an effective therapeutic target of HCC.

Figure 1 .
1Expression of SMYD2 in HCC tumor tissues, matched adjacent non-tumorous tissues and cell lines. (A) qRT-PCR analysis was performed to analyze SMYD2 expression in 10 pairs of HCC tissues and matched adjacent non-tumorous tissues, the data are shown the mean of 2 -△△Ct , and the expression of SMYD2 in HCC is significantly higher than that in matched adjacent non-tumorous tissues (P <0.05); (B) Western blot assay was performed to analyze SMYD2 expression in liver cancer cell lines (MHCC-97H, MHCC-97L, HepG2, and SMMC-7721). C: HCC tumor tissues, N: matched adjacent non-tumorous tissues

Figure 2 .
2Representative immunohistochemical staining of SMYD2 in HCC tissues and matched adjacent non-tumorous tissue. (A) Negative staining of SMYD2 in matched adjacent non-tumorous tissue with the magnifying power of 400×. (B) Low. (C) medium and (D) high expression of SMYD2 in HCC tissues.

Figure 3 .
3Kaplan-Meier survival analysis of the correlation between SMYD2 expression in patients with HCC and OS. Patients with positive SMYD2 expression (solid line) exhibited decreased OS.

Figure 4 .
4Suppression of SMYD2 expression inhibits the cell proliferation and cell cycle progression in SMMC-7721 cells. (A) SMYD2-specific siRNA significantly inhibited the levels of SMYD2 protein in the SMMC-7721 cells. (B) Cell proliferation as assessed by EdU assay was inhibited after SMYD2 knockdown. (C) Cell cycle assays demonstrated that SMYD2 silience increased the percentage of SMMC-7721 cells in the G0/G1 phase while decreased the percentage of SMMC-7721 cells in the S phase. n=3 independent repeats with similar results. *P <0.05 by t-test.

Figure 5 .
5SMYD2 regulates the expression of cyclin D1 in HCC cells. SMYD2 knockdown in SMMC-7721 cells significantly reduced the protein level of cyclin D1. *P <0.05 by t-test.

Table 1 .
1Correlation of SMYD2 expression in tissue with patients' clinicopathological variables in 163 cases of HCCVariables
All cases
(N=163)

SMYD2 expression (%)
Positive
expression
(N=122)

Negative
expression
(N=41)

p-value a

Age(years)
0.88
≤50 b
97
73(75.25)
24(24.74)
>50
66
49(74.24)
17(25.76)
Gender
0.07*
Male
143
104(72.73)
39(27.23)
Female
20
18(90.00)
2(10.00)
AFP(ug/L)
0.81
≤20
61
45(73.77)
16(26.23)
>20
102
77(75.49)
25(24.51)
HBV
0.38
Negative
22
7(31.82)
15(68.18)
Positive
141
105(74.47)
36(25.53)
HCV
0.99*
Negative
157
117(74.52)
40(25.48)
Positive
6
5(83.33)
1(16.67)
Cirrhosis
0.68
No
75
55(73.33)
20(26.67)
Yes
88
67(76.14)
21(23.86)
Tumor size(cm)
0.04
≤5
60
57(95.0)
3(5.00)
>5
103
65(63.11)
38(36.89)
Tumor encapsulation
0.45
None
75
54(72.00)
21(28.00)
Complete
88
68(77.23)
20(22.73)
Tumor number
0.51*
Single
149
110(73.83)
39(26.17)
Multiple
14
12(85.71)
2(14.29)
Vascular invasion
0.01*
No
123
86(69.92)
37(30.08)
Yes
40
36(90.00)
4(10.00)
Distant metastasis
>0.99*
Negative
158
118(74.68)
40(25.32)
Positive
5
4(80.00)
1(20.00)
Differentiation
<0.01
Poor-moderate
127
105(82.68)
22(17.32)
Well
36
19(52.78)
17(47.22)
TNM stage
0.04
Ⅰ
99
68(68.69)
31(31.31)
Ⅱ-Ⅲ-Ⅳ
64
54(84.38)
10(15.63)

a Chi-square test. b mean age. *Fisher exact test.

Table 2 .
2siRNA Sequences.siRNA Name
Sequence
siNegative control
Sense
UUCCCGAACGUGUCACGU
Antisense
ACGUGACACGUUCGGAGAA
siSMYD2 no.1
Sense
GAUUUGAUUCAGAGUGACA
Antisense
UGUCACUCUGAAUCAAAUC
siSMYD2 no.2
Sense
GGUUAAGAGAUUCUUAUUU
Antisense
AAAUAAGAAUCUCUUAACC

Table 3 .
3Univariate analysis of factors associated with overall survival of 163 patients with HCCOS
univariate analysis
HR
95%CI
p-value
Age(>50vs.≤50)
1.15
0.77-1.37
0.47
Gender (male vs. female)
1.61
0.92-2.81
0.10
AFP (≤20 vs.>20)
0.77
0.52-1.14
0.19
HBV (positive vs. negative)
1.03
0.59-1.78
0.93
HCV (positive vs. negative)
0.69
0.22-2.19
0.53
Cirrhosis (yes vs. no)
1.34
0.90-1.99
0.15
Tumor size(cm)(>5 vs. ≤5)
1.19
0.80-1.77
0.39
Tumor encapsulation (none vs. complete) 0.74
0.49-1.10
0.13
Tumor number (multiple vs. single)
0.95
0.51-1.78
0.88
Vascular invasion (yes vs. no)
2.55
1.51-4.31
<0.01
Distant metastasis (positive vs. negative) 0.56
0.21-1.47
0.24
Differentiation (poor-moderate vs. well)
1.74
1.13-2.70
0.01
TNM stage(Ⅱ-Ⅲ-Ⅳvs.Ⅰ)
1.62
1.08-2.44
0.02
SMYD2 expression (positive vs. negative) 2.89
1.87-4.46
<0.01

Table 4 .
4Multivariateanalysis of prognostic factors on overall
survival (Cox regression model)

OS
multivariate analysis
HR
95%CI
p-value
Vascular invasion (yes vs. no)
1.64
1.06-2.53 0.03
Differentiation (poor-moderate vs. well)
1.06
0.63-1.78 0.83
TNM stage(Ⅱ-Ⅲ-Ⅳvs.Ⅰ)
1.06
0.61-1.82 0.84
SMYD2 expression (positive vs. negative)
4.00
2.03-7.91 <0.01

http://www.jcancer.org
AcknowledgementsShi-Kun Liu designed the study and revised the manuscript critically. Shan-Ru Zuo evaluated the clinical records, carried out the experimental work and drafted the manuscript. Xiao-Cong Zuo contributed for critical revision of statistical analysis and of the manuscript. Wei-Jin Fang, Chun-Jiang Wang, and Yang He contributed for data interpretation and drafted the manuscript. Heng Zou, Pan Chen and Ling-Fei Huang help to collect the samples and participate in the design of the study. Li-Hua Huang and Hong Xiang contributed to carry out the immunohistochemistry assays. This work was supported by Program for New Century Excellent Talents in University (grant number NCET-13-0605, 2013)(to Z.X.C), the New Xiangya Talent Project of the Third Xiangya Hospital of Central South University(grant number 20150218, 2015)(to Z.X.C), and National Natural Science Foundation of China (grant number 81102512) (to Z.X.C).Competing InterestsThe authors have declared that no competing interest exists.
Cellular and molecular mechanisms of hepatocellular carcinoma: an update. R N Aravalli, E N Cressman, C J Steer, Arch Toxicol. 87Aravalli RN, Cressman EN, and Steer CJ. Cellular and molecular mechanisms of hepatocellular carcinoma: an update. Arch Toxicol. 2013; 87: 227-247.

Global cancer statistics. A Jemal, F Bray, M M Center, CA Cancer J Clin. 61Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin. 2011; 61: 69-90.

Hepatectomy for hepatocellular carcinoma: patient selection and postoperative outcome. R T Poon, Fan St, Liver Transpl. 10Poon RT, and Fan ST. Hepatectomy for hepatocellular carcinoma: patient selection and postoperative outcome. Liver Transpl. 2004; 10: S39-45.

Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex. M A Brown, Sims Rj 3rd, P D Gottlieb, Mol Cancer. 526Brown MA, Sims RJ 3rd, Gottlieb PD, et al. Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex. Mol Cancer. 2006; 5: 26.

RB1 methylation by SMYD2 enhances cell cycle progression through an increase of RB1 phosphorylation. H S Cho, S Hayami, G Toyokawa, Neoplasia. 14Cho HS, Hayami S, Toyokawa G, et al. RB1 methylation by SMYD2 enhances cell cycle progression through an increase of RB1 phosphorylation. Neoplasia. 2012; 14: 476-486.

Smyd1 and Smyd2 are expressed in muscle tissue in Xenopus laevis. S Kawamura, E Yoshigai, S Kuhara, Cytotechnology. 57Kawamura S, Yoshigai E, Kuhara S, et al. Smyd1 and Smyd2 are expressed in muscle tissue in Xenopus laevis. Cytotechnology. 2008; 57: 161-168.

Repression of p53 activity by Smyd2-mediated methylation. J Huang, L Perez-Burgos, B J Placek, Nature. 444Huang J, Perez-Burgos L, Placek BJ, et al. Repression of p53 activity by Smyd2-mediated methylation. Nature. 2006; 444: 629-632.

Methylation of the retinoblastoma tumor suppressor by SMYD2. L A Saddic, L E West, A Aslanian, J Biol Chem. 285Saddic LA, West LE, Aslanian A, et al. Methylation of the retinoblastoma tumor suppressor by SMYD2. J Biol Chem. 2010; 285: 37733-37740.

Dysregulation of AKT Pathway by SMYD2-Mediated Lysine Methylation on PTEN. M Nakakido, Z Deng, T Suzuki, Neoplasia. 17Nakakido M, Deng Z, Suzuki T, et al. Dysregulation of AKT Pathway by SMYD2-Mediated Lysine Methylation on PTEN. Neoplasia. 2015; 17: 367-373.

The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells. L Piao, D Kang, T Suzuki, Neoplasia. 16Piao L, Kang D, Suzuki T, et al. The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells. Neoplasia. 2014; 16: 257-264.

Overexpression of SMYD2 relates to tumor cell proliferation and malignant outcome of esophageal squamous cell carcinoma. S Komatsu, I Imoto, H Tsuda, Carcinogenesis. 30Komatsu S, Imoto I, Tsuda H, et al. Overexpression of SMYD2 relates to tumor cell proliferation and malignant outcome of esophageal squamous cell carcinoma. Carcinogenesis. 2009; 30: 1139-1146.

SMYD2 is highly expressed in pediatric acute lymphoblastic leukemia and constitutes a bad prognostic factor. L H Sakamoto, R V Andrade, M S Felipe, Leukemia Research. 38Sakamoto LH, Andrade RV, Felipe MS, et al. SMYD2 is highly expressed in pediatric acute lymphoblastic leukemia and constitutes a bad prognostic factor. Leukemia Research. 2014; 38: 496-502.

Overexpression of SMYD2 contributes to malignant outcome in gastric cancer. S Komatsu, D Ichikawa, S Hirajima, British Journal of Cancer. 112Komatsu S, Ichikawa D, Hirajima S, et al. Overexpression of SMYD2 contributes to malignant outcome in gastric cancer. British Journal of Cancer. 2014; 112: 357-364.

SMYD2 overexpression is associated with tumor cell proliferation and a worse outcome in human papillomavirus-unrelated nonmultiple head and neck carcinomas. R Ohtomo-Oda, S Komatsu, T Mori, Human Pathology. 49Ohtomo-Oda R, Komatsu S, Mori T, et al. SMYD2 overexpression is associated with tumor cell proliferation and a worse outcome in human papillomavirus-unrelated nonmultiple head and neck carcinomas. Human Pathology. 2016; 49: 145-155.

BTB/POZ domain-containing protein 7: epithelial-mesenchymal transition promoter and prognostic biomarker of hepatocellular carcinoma. Y M Tao, J L Huang, S Zeng, Hepatology. 57Tao YM, Huang JL, Zeng S, et al. BTB/POZ domain-containing protein 7: epithelial-mesenchymal transition promoter and prognostic biomarker of hepatocellular carcinoma. Hepatology. 2013; 57: 2326-2337.

4-Hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G. Y Tsuchiya, Y Okuno, K Hishinuma, Free Radic Biol Med. 43Tsuchiya Y, Okuno Y, Hishinuma K, et al. 4-Hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G. Free Radic Biol Med. 2007; 43: 1604-1615.

Hepatocellular carcinoma results from chronic cyclin D1 overexpression in transgenic mice. N G Deane, M A Parker, R Aramandla, Cancer Res. 61Deane NG, Parker MA, Aramandla R, et al. Hepatocellular carcinoma results from chronic cyclin D1 overexpression in transgenic mice. Cancer Res. 2001; 61: 5389-5395.

Cyclin D1 overexpression in hepatocellular carcinoma. M Joo, Y K Kang, M R Kim, Liver. 21Joo M, Kang YK, Kim MR, et al. Cyclin D1 overexpression in hepatocellular carcinoma. Liver. 2001; 21: 89-95.

Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. D W Stacey, Curr Opin Cell Biol. 15Stacey DW. Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. Curr Opin Cell Biol. 2003; 15: 158-163.

Hepatocellular carcinoma: current trends in worldwide epidemiology, risk factors, diagnosis and therapeutics. M I Shariff, I J Cox, A I Gomaa, Expert Rev Gastroenterol Hepatol. 3Shariff MI, Cox IJ, Gomaa AI, et al. Hepatocellular carcinoma: current trends in worldwide epidemiology, risk factors, diagnosis and therapeutics. Expert Rev Gastroenterol Hepatol. 2009; 3: 353-367.

Overexpression of SMYD3 and matrix metalloproteinase-9 are associated with poor prognosis of patients with gastric cancer. Y Liu, H Liu, X Luo, Tumor Biology. 36Liu Y, Liu H, Luo X, et al. Overexpression of SMYD3 and matrix metalloproteinase-9 are associated with poor prognosis of patients with gastric cancer. Tumor Biology. 2015; 36: 4377-4386.

Identification of Smyd4 as a Potential Tumor Suppressor Gene Involved in Breast Cancer Development. L Hu, Y T Zhu, C Qi, Cancer Research. 69Hu L, Zhu YT, Qi C, et al. Identification of Smyd4 as a Potential Tumor Suppressor Gene Involved in Breast Cancer Development. Cancer Research. 2009; 69: 4067-4072.

BCL-3 promotes the tumor growth of hepatocellular carcinoma by regulating cell proliferation and the cell cycle through cyclin D1. K Tu, Z Liu, B Yao, Oncol Rep. 35Tu K, Liu Z, Yao B, et al. BCL-3 promotes the tumor growth of hepatocellular carcinoma by regulating cell proliferation and the cell cycle through cyclin D1. Oncol Rep. 2016; 35: 2382-2390.

Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line. C Freiburghaus, B Janicke, H Lindmark-Månsson, J Dairy Sci. 92Freiburghaus C, Janicke B, Lindmark-Månsson H, et al. Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line. J Dairy Sci. 2009; 92: 2477-2484.

Cyclin D1 triggers autonomous growth of breast cancer cells by governing cell cycle exit. R M Zwijsen, R Klompmaker, E B Wientjens, Mol Cell Biol. 16Zwijsen RM, Klompmaker R, Wientjens EB, et al. Cyclin D1 triggers autonomous growth of breast cancer cells by governing cell cycle exit. Mol Cell Biol. 1996; 16: 2554-2560.


Psychological profiling of participants at the Miss World contest via combined methodology: psychometric assessments and projective drawing

Kristina Brajovic Car email@example.com
Faculty for Media and Communication
Department for Psychology
BelgradeSerbia

Aleksandra Đurić email@example.com
Oana Pănescu

BelgradeSerbia

Faculty of Psychology and Educational Sciences
Spiru Haret University
BucharestRomania

Psychological profiling of participants at the Miss World contest via combined methodology: psychometric assessments and projective drawing
F5543A80DD5BAC246279FDB4EA8C3E6C10.26758/8.1.10self-imageinjunctionsdriversbeauty pageantadolescent
Objectives.We are discussing psychological consequences of consequences of embracing dominant cultural values.Among them, beauty and its constant visual (and superficial) presentation become imperative criteria for self-evaluation during the growing up process for young girls.Visual criteria are more strongly connected to the perception of self-image compared to the self-evaluation criteria that was relevant for adolescents in the recent past.Material and methods.On the sample of 20 young women, in the process of competition for Miss World their individual aspirations and profiles were analyzed and compared with the cultural trend.Profiling was done via Machover test and diagnostic interview in line with the drawing output as well as the psychometric evaluation based on the Driver test (35Q) dominant defense mechanisms of personality.Comparative analysis of cultural trends and individual profiles was conducted through interpretative analysis of results collected during psychological profiling of contestants through the theoretical model of Transactional analysis.Transactional analysis of the famous fairy tale Cinderella was reinterpreted in the classical Berne approach in order to make it more relevant for the current problems and issues of young girls.Results.The central theme is the self-image that is not matching calendar age.Graphical and phenomenological self-reports show that many participants perceived themselves as a child.Selfvalidation and social context interpretation come from the regressive frame of reference.Conclusions.The results strongly support the hypothesis that beauty contestants need to be carefully selected, motivated and supported in this personality challenging qualification and evaluation process based on one dimension.

Introduction

Contemporary use of the term star does not have a long history.The emergence of a modern concept and the phenomenon of stars were influenced by the occurrence of mass media, as well as the development of consumer culture in the Western capitalist societies of the twentieth century.The term "star" refers to a person who has a set of characteristics that distinguish him/her from the general population.In describing the stars, adjectives such as fame, glamor, spectacular, respectable, famous, popular and similar are most often used.

A complex categorization of celebrities was given by sociologist Chris Rojek (2001).He refers to three types of celebrities categorized according to the status of accomplished fame: status is assigned by a blood link (royal family, powerful families of politicians or business people); status is acquired by work or talent (athletes, artists, successful business people, all those with a rare talent or skill); status is attributed thanks to the media and the public's attention, whereby a person may and may not have a specific talent and quality (Graeme, 2004).This category also includes participants of beauty pageants, as well as the Miss World contestants.

Celebrities who have gained fame through the attention of the media and the public, including the participants of the beauty pageants (even for a short time) have mythological figures of contemporary culture.According to German sociologist Leo Lowenthal, this transition from the "idol of production" to the "idols of consumption" marked a change in the moral and value systemthose are the contents that the audience can easily adopt by reading magazines and watching entertainment shows (Schmid, 2014).This kind of shows enables escapism to the audience, encourages and offers an illusion of participation in this glamorous world that brings satisfaction.On the other hand, the difficulties through which people pass, for example, at the Miss World contest, provide consolation and facilitate the identification process for the audience.At one level, participants in beauty pageants are unrealistic ideals, and at the second level -faults and weaknesses can be found in them.In this way, the media create the impression that there is actually no difference between "us and them," celebrities are the same ordinary people, with the same problems as everyone else.Becoming a celebrity is, therefore, a choice available to each of us.This is one of the key factors of the prevalence and intensity of the tendency towards media glory."The idea that someone can build an identity by depicting someone else has created particular problems in contemporary society.The message that everyone can achieve a life of glamour and fame, if they choose, and make an effort, led countless people to neglect the essential elements of their lives searching for unattainable fantasy" (Salecl, 2014, p.51).

Consumer culture in many respects is referring to technology.Based on that fact consumer culture is also promoting the ability to quickly reject "the old" (goods, values, friends etc.) and even faster to accept "the new ones".According to Teodor Adorn and Max Horkheimer, culture is now subject to exchange, it is fully commodified, it has no longer usable value, and only the exchange value remains (Adorno and Horkhajmer, 1989).

Lifestyles are based on the social organization of consumption.In the contemporary culture of celebrities, dominant values and lifestyles are imposed through the media.We are living in a worldwide community made possible by media and internet, who influences the way we view men, women and family (Rada and Pănescu, 2016).Take, for example, competition for the Miss World, which offers a unique way of life and lifestyle, namely it provides accurate Instructions how people should look, behave, what kind of career they should have.This form of unification undermines the individual's identity and uniqueness.

The Miss World competition manipulates the lifestyle that relates to the exterior.Everything is spectacular, glamorous, but a certain pattern of appearance and behavior is imposed, and distributed through the media mostly to young girls, who then adopt and strive towards such a lifestyle.Today, in the culture of spectacle, instead of increasing the diversity of media content, uniformity is pursued according to the dominant form of sensationalism that gathers the widest audience.The world of spectacle has become a substitute for the grim reality that stuns people by keeping them in a state of passivity.The spectacle, which relies on the material-hedonistic value orientation, where conformity, orientation to the outward appearance, leisure life, entertainment and unrealistic desires are the basis of reality, has accomplished the tactics of its domination.It is precisely this form of cultural values that contribute to the development of narcissism (or reinforcing narcissistic predisposition) and pseudo identity in celebrities and people who look up to them and adopt their identity as their own.As for the beauty contests, the ones who look up to contestants are mostly young girls.Sigmund Freud (2011) distinguished primary and secondary narcissism.Primary narcissism is a normal development stage during childhood.Secondary narcissism occurs in adulthood.This narcissism is more of a pathological nature, because it refers to the conditions in which people are self-absorbed, incapable of empathy and interpersonal relationships, and approach people as means of meeting their needs.Narcissism is a love for oneself, for one's own personality.A moderate dose of self-love is necessary for a person to have a positive image of himself/herself.However, excessive self-esteem is considered a psychopathological phenomenon.The characteristic of pathological narcissism is that a person creates a grand self that characterizes omnipotence, expressed perfectionism, a strong motive for achievement, maintaining self-esteem by manipulation, exploitation, and underestimation of others.This self is merely a compensation for a genuine self of a narcissist personality that features a negative image of itself and the abundance of narcissistic injuries from childhood, a sense of emptiness, low self-esteem, a sense of pain, and fragmentation of a false image of oneself, which is built as a defensive mechanism.

Narcissism in the light of game analysis (Cinderella example).Here, as a symbolic illustration, we will show intrapsychic developmental challenges in the process of psychological development of the girl in the light of dynamic psychology and transactional analysis of the popular fairy tale -Cinderella.The analysis was carried out by the authors of the research as an additional qualitative research contribution and support to the interpreted conclusions of this paper.

The beginning of the drama starts with a psychological auto manipulated game "Poor me".Cinderella unquestioningly accepts her difficult fate by believing in her inferiority in relation to other family members.The way others treat her will determine her inner experience of her own value.In order to remain persistent in the role she has accepted, she must isolate all those needs that she acquired by birth -to be important to herself, to know what she feels, to openly seek and take what she needs.Fortunately, growing up and maturing nevertheless spontaneously inflict damage on her wrong decisions and negative adjustment of her personality to the unfavorable living condition.She begins to realize that a different life story, no matter how far from her, is still possible.There are "happy princes" but also "happy princesses" who do not hide in the ashes behind those whom they leave the role of "flamboyant villains" in order to preserve their innocent face and the public presentation of "martyrs".Those same villains do not lack initiative or willpower to live their lives according to their own choices.However, they are really the first to be criticized by the critics who watch the drama from the side.In spite of that, all villains are wholeheartedly accepted in the life of a Cinderella.They help her define and shape her weaknesses, fears, and insecurities, turning them into real virtues compared to the "bad" features of the people she is surrounded by.First of all, in her own eyes, she receives recognition of the identity of a "goody-good" and ceases to believe that she herself is not Ok, although others do not treat her well.I am Ok -others are not Ok, is the motto which she uses to protect herself against deeper injuries.Thanks to that, she has steadily built the identity of a "good girl" in her own eyes, but also in the eyes of the selected audience by the script.Regardless of the fact that those are birds, horses, dogs, and mice, as it is said in fairy tales, it is important that they are from the same story and share her viewpoint.After that, Cinderella is ready for new challenges of social reality.Finally, tired of the game "Poor me", she decides to take risks and openly seeks what she wants out of life: fun, opportunity, joy, play, pleasure, and related psychological needs in accordance with the development stage.Of course, she does not get what she seeks right away.Nothing new."Cannot", "Not now", "Why you," "I won't," "I can't," "I don't know" are just some of the frequent answers when a person needs the cooperation of others to realize his own intentions and ideas.Nevertheless, being free does not mean that the plan would be accomplished in any case.Even the refusal does not mean that "it shouldn't have been asked for".Anyone who is aware of what he really wants and believes he is entitled to it will find a way to get it.Even if it was with the help of the pumpkin, mice, and villas, as it is described in a fairy tale.Of course, leaving a bad life pattern does not mean jumping straight to autonomy.This is simply not the case, not even in fairy tales.So Cinderella withdrew from the game of "victim" for one night to experience how it is to be a "peasant woman".This is a metaphor, and the name of the psychological game designed by Eric Berne, not a derogatory word for a woman who is engaged in agriculture work."Peasant Woman" has no confidence in herself, uncritically believes people of high status, keeps asking for advice of people who she believes are better and smarter than her, but in the end never listens or applies this advice, she doesn't even remember it, but instead she finds a way to quickly replace them with other eminent advisers or to discredit them as ignorant, even if she doesn't understand or listen to them (Berne, 1996).In the same manner, Cinderella celebrates the importance and significance of the success and value of the people who organize the event and come to such parties.She gives herself the right only to passively observe how others enjoy.And superficially, by her appearance, fits into society, but retains the feeling that she essentially does not belong to the world she admires the most.Today, Instagram would be a lot of help for her to build an image faster and easier to fit in wherever she wants, not even having to be exposed to the challenge of personal confrontation, or conversation.

Subsequently, there is another trap of the new script and dramatic turnaround.The Cinderella has succeeded much more than her original role of the Bystander imposed upon her (Clarkson, 1996).She managed to be noticed and chosen.It was for her capacities and experiences of herself a bit too much at the time, and she avoided a spontaneous and free encounter with the prince, saving herself from the risk of intimacy by the script of Almost.He almost met her and her truth instead of the Instagram picture, but she skillfully managed to escape.There was just one shoe left but it was not enough to know who she really was and to what extent she really was not what she represented.

A persistent prince does not stop here and does not agree to the game.Love pushes him to seek life more than a game.He conducts extensive research, after which, all false arguments and false representations dissipate.Simply, advertising is not sufficient, nor necessary, when we know exactly what we want, and what we do not want.And it is confirmed in the end, regardless of the fact that the sweat, blood, and tears are spilled on the advertisement, just like in the original fairy tale.This is the painful truth of advertisers in today's marketing perspective of all-pervasive digital communications and complete control of users over media and the growing control of consumers over purchasing choices.The one who has the ability to see and to know what he wants is inspiring enough to add to the confidence of those who do not have enough of it as well.There is no ash that could hide quality, nor deception that could mask a bad offer.Cinderella, therefore, reveals her noble person and interest, accepts the risk of intimacy, leaves the game, gives up Cinderella and Instagram beauty as well, and for now, there seems to be no objection.

Narcissism and the public self.After the transaction analysis of Cinderella, a popular narrative that is still developmentally relevant, especially in the psychological development of women, we return to a wider picture.Famous persons have dual aspects of identity -personal and public, or private and social.The difference between the individual and social identity can be explained as a distinction between the private and public sphere.The problem is that changes in the content, character and expression of one sphere leave the consequences in the way of expressing another sphere.A celebrity, in order to become one, often has to look and behave in a certain way, which is culturally defined, and media support and broadcast it all for the sake of profit.This uniformity is part of the social identity, that is, the public sphere of celebrities.When this sphere becomes dominant, and when individuality is suppressed, personality problems occur, or pseudoidentity is formed, which in the material-hedonistic value orientation and culture of spectacle is often shown through narcissism and self-absorption.The prescribed rules of public life are not authentic; an individual can achieve self-actualization only through personal experience, personal growth and development.Suffocating freedom in this way, cause or open repressed impulses of hatred and aggression that affect the creation of uniformed and unhappy people, who have suppressed their individualities and the need for personal growth and development, autonomy, firm identity, and integrity.Young people, especially girls, who look up to models, who are also very young themselves, rarely realize that their sense of self-confidence is conditioned by the opinions of others about them, and the behavior of others as well.Their self-respect is conditioned by the success in "managing the image others have about them" and that is the reason why fame is so important to them.It is not only a practical means of acquiring material profit, which may have been a primary motive, but also a means of confirming the importance and value of a person, first of all in their own eyes.

Methodology

Objectives

The general goal of this research was to explore in depth motivation and expectations among adolescent girls (from the age 16 to 21) that have chosen to enter a beauty pageant.Competing in sports is normally accompanied by psychological evaluation and preparation for any outcome, winning or losing.This is not the case with beauty competitions, generally speaking, at least not as a part of a standard protocol.We could say that during beauty contest, in particular where high stakes are involved (i.e.national ambassador of beauty), girls are exposed to situations which represent a stressor for them, without undergoing proper preparations or support procedures.In order to better understand the specific needs of a typical contestant, and for the purpose of proposing and developing an adequate response and support network, psychological testing was organized for the finalist, during the last phase of the competition.By that time, they all overcame the initial anxiety and adapted themselves adequately to the role.

Material and methods

Testing was performed through the application of the projective technique Human figure drawing -Machover test, followed by the semi-structured interview on the meaning presented in the drawing.Various projection drawings have been designed for assessing children's personality and psychological disorders including the tests of drawing a person (DAP).The validity and reliability of the DAP test are satisfactory (Rawley, 2005).This research protocol aimed at assessing the nature and quality of self-perception and self-awareness from the perspective of dominant life goals and values.The projective kind of technique is chosen due to the fact that it bypasses conscious resistance and does not provoke giving of socially preferred response.In addition, candidates were profiled on the basis of the two theoretically similar personality tests, based on the theory of the Transactional analysis, one of which was intended to measure dominant defense mechanisms of the personality (test Driver -35Q), while the other test, Drego Injunction scale (Drego, 1994), was intended to measure a developmental deficit in terms of accurate self and others perception, social skills, and affective fluency and stability.Transactional analysis theorists and researchers consider five such clusters are universally present regardless of gender and national identification (Stewart & Joines, 1987).In his empirical efforts to develop a scientifically valid driver instrument, Sandström (2015) created a test with the following psychometric characteristics: the values of Cronbach's alpha for the drivers as a measure of reliability for each individual sub-scale are: Hurry Up -0.83; Be Strong -0.80; Please Me -0.82; Try Hard -0.77; Be Perfect -0.73.Study of these concepts in Serbia and Croatia gave results that indicate the need for further development of the psychometric characteristics of the Drivers test (Pejić et al., 2015).Drego's scale is a descriptive clinical inventory that is often used by transactional analysis clinicians as a naturalistic method for client assessment, with strong clinical and conceptual validity in quick client description, treatment orientation and understanding based on theory.Empirical validity of Drego's scale is yet to be psychometrically tested and improved by research and clinical application in different cultures, especially outside of the Western world.

As stated above, both tests have been administered previously on the Serbian sample where this research is conducted.Joint administration of these two instruments increased the validity of each of the tests used individually and added to the possibility of a broader understanding of participants' capacity to overcome stress, their dominant interpersonal style and grounding for the sense of self.A total of 20 subjects completed all questionnaires.

Research protocol.Testing was carried out in the presence of psychologists and other subjects, that is, in a group context.The time they needed to complete the tests was 80 minutes, since they were interacting with the examiner so that all the uncertainties related to certain questions and instructions were clarified.This ensured the quality (credibility) of the obtained results and their further usability in the analysis of individual differences, as well as the similarities in the personality profile among the participants.

Results

The results are given in the form of the narrative summary, containing the crossinterpretation of the results obtained with all three psychometric tools.The reason for the application of combined methodology and narrative analysis of results was to better describe and explain the individual differences between the tested subjects, while, at the same time, narrative analysis provided an insight into some common themes and trends in all the answers given by the participants that came from the same sample group.

In what follows, we will present two illustrative narratives, from a sample of 20 participants, contestants of the Miss World Competition, as a support to the conclusions drawn on the basis of the thematic and interpretive analysis of all the profiles obtained.

Profile of the participant A.Ć. in a narrative form: High expectations of herself have been taken over by others.The basic motive is to get approval and acceptance from her environment.The participant is not set up independently in her plans.Her success and engagement are conditioned by the circumstances, permissions and encouragement that she seeks of others.Contact with reality is not her strong side; she rather leaves the assessment to others.In trying to respond successfully to others' expectations of herself, she may seem false, alienated, rude, distanced or uninterested.As for her authentic expression of personality, the drawing reveals a sense of herself as an "awkward child, stopped in the game".

Profile of the participant T.P. in a narrative form: Extremely high ambition, as well as expectations from herself.She is strict towards herself, with strong self-criticism.With this profile of personality, responsibility and reliability go together as related characteristics.She does not forget her own failures, nor does she like to be seen and treated as if she were a child (a girl).Self-perception of herself is ahead of her years (she considers herself a "

powerful woman", as she states). There is a noticeable conflict within her personality at the level of self-image. On one hand, there is a vision of a powerful woman, and on the other a graphic representation and an accompanying interview show the perception of herself as a playful, carefree and lazy child.

It could be assumed that this is exactly the side of her personality she is trying to hide in self-representation.The closeness with others is not valued as a quality, nor is it initiated.She relies more on herself.

Profile of the participant A.Z. in narrative form: Anxiety and mistrust dominate above other personality traits in this particular psychological profile. In the first place because of other people opinion and acceptance, the interviewee wants to be successful (even perfect, as she claims). It is noticeable that she appreciates the efforts more than the results, which diminishes her ambition on the long run. Self-perception is such that she sees herself as an immature person. From the drawing, we can see that human figure corresponds to the girl's appearance, without any signs that the figure would indicate the self-perception as a girl

who is growing into adulthood.The primary criterion in perception and evaluation of oneself is aesthetic in nature.In relations with others, she is presenting her flexibility, poor self-confidence, and unclear personal goals.

Profile of the participant M.N. in a narrative form: Results point to Introversion in social situations and avoidance of self-analysis and critical review of herself.Primarily, what is important to interviewee is that her performance helps her in seeking of love and acceptance from the people that are close to her.On the other hand, it seems that she does not give herself the right or permission to be open and spontaneous in social situations.Selfconfidence is unstable, "shaky" because it strongly depends on "how others see me."This is not seen in the initial impression based on her appearance by which she is trying to control and hide emotions and strives toward perfectionism.Female figures from her immediate social environment are perceived as the most important role models and support.Striving toward perfection is the dominant defense mechanism reflected in her answers and self-presentation.

Profile of the participant A.S. in a narrative form: The interviewee has not developed autonomous motivation or her own assessment criteria. She is directed toward others in that sense, which is in accordance with her adolescent phase (being the youngest among the contestants). Analysis of the drawings shows the active interpersonal conflict between two dimensions of the identity (pupil versus professionally engaged individual). In her drawing, she presented herself as an older girl, with a book in one hand and a purse in the other (the symbols of two different tendencies). Motivation is partly irrational ("to be memorable"), especially after comparative analysis of the different results (DAP, Driver and Injunction tests), which point to insecurity and low self-esteem.

The central theme dominating the majority of answers is the self-image that is not in line with the actual age.Graphical and phenomenological self-report indicates that almost all participants (16 out of 20) perceived themselves as children.That means that self-validation, as well as social context interpretation, is oriented within the regressive frame of reference.In most of the cases, "others" are perceived as threats, while also being granted more power and control compared to the control function attributed to oneself.The results of the interviews show high level for the injunctions -Don't grow up, Don't be you and Don't think.Also, dominant drivers observed are Be Please others and Try hard.The results clearly support the initial hypothesis that beauty contestants, still in their adolescent and post-adolescent years, need to be carefully selected, motivated and supported in these psychologically provocative situations, such as evaluation and qualification in one-dimensional perspective.

Discussions

Fame gives the participants of beauty contests (which include Miss World competition) the illusion of being in the center of the world, but, in fact, they are being alienated from the world and themselves as well.The material and hedonistic system of values, the culture of spectacle, the orientation to the exterior, the tendency to fulfill the abundance of desires and desires without delay, media exposure and loss of privacy in order to gain and maintain fame are dominant.All this leads to narcissism or intensifies the already existing narcissistic predisposition in which case identity is connected with the fact to what degree they are accepted by the public and exposed in the media.

Here we can speak of the functions of the internalized psychological injunction "Don't Be Important", because people with expressed narcissism as a characteristic (or mechanism of defense in this case) view their sense of identity and values through the prism and opinion of others.For this reason, they are often surrounded by people who would confirm ideas about their "Grand Self".In addition to transactional analysis with interpretation based on concept of Drivers as irrational motivators developed during formative years in reaction to a persistent exposure to social stressors and psychological challenges, we can found theoretical base for associations between sociopsychological deficit, fear of rejection and development of "Grand Self" also in the theory of interpersonal psychoanalysis (Horney, 1937;Sullivan, 1953.).Horney's classification of intrapersonal defense mechanisms, as reactions to an early experience of hostility and threat of rejection, names "self-investment into status and material profit" as one of four different ways to adapt in situations where lack of trust and hope are experienced by a person during developmental years (Chang & James, 1987).

Money and fame are social, external motives, but there are other, deeper, psychological reasons that lie at the basis of their desire for media exposure.A histrionic personality disorder is closely linked with narcissism.A histrionic personality wants to be always at the center of attention, these individuals often act aggressively and egocentrically and need to feel that "everything revolves around them."They want everyone to love them, to admire and to adore them.They really want recognition.They handle situations in which they do not get what they want with difficulty.These people are too social, often seductive, everything around them is exaggerated, especially emotions.They change their mood often; from the greatest euphoria they fall into the deepest depression.The authors of personal adaptations concept in transactional analysis (Joines and Stewart, 2015) argue that at the basis of this structure of personality lays the fear of rejection that is deeply hidden from self-awareness by intrapersonal dynamics.As children, these people did not get enough love and attention.Here you can recognize a psychological ban, do not be what you are, but be what others expect from you, because this is the only way that this person thinks he can get love and attention.

Based on the theory of life script analysis, we can say that, in time, these people create beliefs about themselves and others that only if they are liked by everyone, they would be loved.Growing up, their fears have reinforced, as well as the need to impress others.It has the only way to avoid what they are most terrified of ˗ to be alone and unloved.

The assumption is that one of the main motives that famous people have is the strong psychological need for attention.The need for provoking attention that some famous people have is almost pathological, and their symbiosis with the media is a way to get that attention.Due to the introjected "Don't Be Important" ban, attention is being sought and obtained in an inauthentic way, and thus it is not valued by the person who seeks it pathologically.

Drivers are also important concepts of Transaction Analysis in work with celebrities.Drivers are visible in behavior and represent a certain, predictable behavioral pattern adopted in childhood that has adaptive function for the personality, but inhibits authentic reactions.By conducting Driver Test 35Q, we examined the dominant drivers among the respondents.Driver "Be perfect" can be perceived through focus on appearance and obsession with oneself.The most common attributes of the "Be perfect" driver have to do with performance and appearance; being spotless, clean, with a perfect presentation for others.Narcissistic and histrionic people often manifest this driver, which they project on the outside environment.Striving for perfection or showing the person as perfect is closely linked with the phenomenon of selfie.As for selfie, actor James Franco says: "Selfie is an avatar, a mini version of us, a way to let others know who we are.At a time when the sea of information is available only with a mouse click, the power of attracting the audience is a real power.Selfie attracts attention, and attention is the key word.It is a kind of pseudo-privacy.Selfie is in the era of social networks a way to sayhey this is me" (Džejms, 2014, p.45).The results show that's exactly the same psychological effect the respondents from the sample are trying to reach, only in the traditional way (such as the Miss World competition).

Conclusion

By analyzing the professional texts (Đurić, 2015) one can conclude that the pseudo identity celebrities build provides a false and ephemeral feeling of security, value, and self-confidence.The psychological need for growth and development and the realization of all the potentials that a person possesses can be suppressed, and the need for love, attention and personal worth can be achieved in an unauthentic manner through symbiosis with the media, success can be equated with fame, but in spite of this, basic needs cannot disappear because they are immanent to human nature.

Numerous studies, as this one, suggest that this kind of pseudo-identity among famous people can cause the appearance of narcissism or reinforce the already existing narcissistic and/or histrionic personality structure, since narcissism has the character of a defensive mechanism.Transactional concepts, such as bans, drivers, psychological games, and life script analysis, aim to uncover the mechanisms of personality dysfunctions in order to provide incentives for the development of authentic forces in person.According to Eric Berne (Berne, 1996), consciousness, spontaneity, and intimacy are basic forces of the autonomous personality.

Advisory and if necessary psychotherapeutic, the strategy for the psychological problems that this research has dealt with, regarding the developmental and psychological vulnerability of young adult women in the light of the popular culture and its negative impacts on personality, implies that in addition to working on the raising the awareness of psychological bans, it is also necessary to perform the analysis of life script, as well as the decontamination of the ego state Adults.The functionality of the ego state Adults is significantly reduced under the influence of life script that is detected as a pattern within the sample, the participants of the study.Then, a therapy that follows involves working on a new decision as the antithesis of the script beliefs along with the strengthening of Nursing Parent, who should support a new decision and strengthen the ego state Child by functioning in accordance with the new decisions about a change (Erskin and Morsund, 2015).

As a reminder, all aspects, such as socio-economic, cultural and educational, are inseparable from the individual and vice versa.Personality cannot be developed in isolation, and therefore communication is a basic condition for the development of personalization.Social existence and activity are a necessary precondition for the realization of one's personal life.Therefore, the private and public sphere of personality should not be in dichotomy.Personality achieves its integration when it succeeds in developing its specific abilities and autonomy.
 (2) Belgrade business school -Higher education institution for applied studies, Department for Social studies,
Games People Play: The Basic Handbook of Transactional analysis. T Adorno, M Horkhajmer, Dijalektika prosvetiteljstva (Dialectics of Enlightenment). E Veselin Masleša Berne, Sarajevo; New YorkBallantine Books1989. 1996

Anxiety and projection as related to games and scripts. V Chang, M James, Transactional Analysis Journal. 1741987

Bystander games. P Clarkson, Transactional Analysis Journal. 2331996

G R Erskin, P Morsund, Dž, Integrativna psihoterapija na delu (Integrative psychotherapy at work), Translation from English by Milan Djurisic. Novi SadPsihopolis Institut2015

Changing systems through correlations of injunction inventories with analyses of myths and songs. P Drego, The Maastricht papers: Selections from the 20th EATA Conference. P Lapworth, Amersfoort, NetherlandsYvonne van Praag1994. 10-1 July 1994

. S Freud, 2011Prosveta, BeogradKompletan uvod u psihoanalizu (A complete introduction to psychoanalysis

Understanding celebrity. T Graeme, 2004SageLondon

The neurotic personality of our time. K Horney, 1937Norton &CoNew York

Psihologia, psihoterapia familiei şi cuplului. Teorii, cercetări, intervenţii psychotherapy of family and couples. Theories, research, interventions). C Rada, O M Pănescu, 2016Editura UniversitarăBucureşti

M Pejić, K Brajović Car, P Ellersich, The Psychometric Properties of the Driver test Q 35 in Serbian and Croatian Sample (Poster). University of Zagreb, Faculty of Humanities and Social Sciences, Department of Psychology, 22 nd International Scientific Meeting in Psychology "Ramirez and Zoran Bujas'days. Zagreb, Croatia2015. April 2015

Aggression and depression assessed through art: Using draw-a-story to identify children and adolescents at risk. Rawley S , 2005Brunner-RoutledgeNew York and Hove

Tiranija izbora (The tyranny of choice). C Rojek, R Salecl, 2001. 2014ArhipelagBeogradCelebrity, London: Reaktion Books 14

The interpersonal theory of psychiatry. H S Sullivan, 1953W.W. Norton & CoNew York, NY, US

D Čejni, Životni stilovi (Lifestyles). BeogradClio Publisher2003

V Džons, I Stjuart, Tipovi ličnosti (Types of personality). Novi SadPsihopolis Institut2015

A Đurić, Medijasfera u zagrljaju Velikog brata (Media-embraced Big Brother). BeogradČigoja štampa2015

Selfi kaže ovo sam ja (Selfie says this is me. F Džejms, February 201722

. D Schmid, Murderabilia: Consuming Fame. M/C Journal. 752004. 14 April 2017

S Sandström, Transactional Analysis Driver Test. 2015. 10 February 2015