hepatocellular carcinoma ( hcc ) is the fifth most common cancer and the third most frequent cause of death of cancer in the world ( 1 - 3 ) . most hcc cases are diagnosed in advanced stage of disease , limiting the options of treatment . while liver transplantation offers improved survival , other treatments such as chemotherapy , may also help to control disease progression . furthermore , the main risk factors of hccs are infection with hepatitis b or c , alcoholism , consumption of aflatoxin - contaminated food , liver cirrhosis and type ii diabetes . therefore , the importance of these risk factors varies widely ; for example , in countries like china where hepatitis b is endemic ( 5 ) , hepatitis b is the predominant cause of hcc . whereas in countries , such as the us , the major cause of hcc is cirrhosis and alcoholism ( 6 ) . early detection of hcc and elimination of risk factors could reduce hcc - related mortality . for early hcc diagnosis or predicting disease progression , we need to understand molecular mechanism and pathogenesis of hcc . hcc development like any other cancer occurs because of silence of tumor suppressor genes or activation of oncogenes . these risk factors individually or together alter gene expression or mutation to cause hepatocytes to replicate at a higher rate and/or resistance to apoptosis , and in turn transform normal hepatocytes to hcc , although the precise mechanism remains to be defined ( 1 , 7 , 8) . our research focused on high - mobility group box 1 ( hmgb1 ) in hcc . altered hmgb1 expression could lead to tumor cell survival , expansion and metastasis ( 9 ) , and overexpression of hmgb1 has been associated with all of central hallmarks of cancer ( 9 - 11 ) . hmgb1 is secreted or released by necrotic cells , activated macrophages , mature dendritic cells and natural killer cells and functions to promote cell migration , differentiation , systemic inflammation and immunity ( 12 - 14 ) . nucleus - expressed hmgb1 protein integrates and stabilizes the nucleosome ( 15 , 16 ) , also facilitates dna organization and regulation of gene transcriptional activation ( 10 , 11 ) . the nuclear localization of hmgb1 protein is regulated by posttranslational modifications , hyperacetylation on lysine residues , which induces hmgb1 to translocate from the nucleus to the cytosol ( 9 ) . previous studies have reported that hmgb1 protein is upregulated in different types of cancer , such as breast cancer ( 17 , 18 ) , melanoma ( 19 , 20 ) , gastric cancer ( 21 ) , non - hodgkin lymphoma ( 22 ) , colorectal cancer ( 23 , 24 ) and liver cancer ( 25 - 27 ) and hmgb1 expression is associated with cancer invasion and metastasis . therefore , hmgb1 is believed to be an oncogene in different cancers ( 12 - 14 , 16 , 28 ) . previous studies have shown that overexpression of hmgb1 induced nf-b activity , thereby leading to overexpression of inhibitor of apoptosis protein-2 ( c - iap2 ) , a target gene of nf-b ( 23 , 29 ) . c - iap2 is a key antiapoptotic factor and overexpressed in a variety of human cancers , which is associated with drug resistance and cancer recurrence . c - iap2 is a potent antiapoptotic factor , which inhibits apoptosis through its interaction with a variety of cytokines ( 30 ) . c - iap2 directly associates with caspase-3 , -7 and -9 to attenuate apoptotic signal transduction , thereby inhibiting the incidence of apoptosis and promoting tumorigenesis ( 13 , 14 , 31 , 32 ) . a previous study hypothesized that hmgb1 acts as an oncoprotein to inhibit tumor cell apoptosis ( 33 ) . in this study , we analyzed expression of hmgb1 and c - iap2 levels in hcc tissue specimens , and then assessed the prognostic significance of hmgb1 and c - iap2 proteins in patients with hcc . to detect the level of hmgb1 and c - iap2 mrna and protein in paired tumor and para - tumor tissue samples ( defined as  3 cm distance from the tumor edge ) , we collected fresh tissues from 68 patients with hcc who underwent hepatectomy between may 2011 and may 2013 from the second and third clinical medical college of harbin medical university ( harbin , china ) . diagnosis of hcc in all patients was confirmed by histopathologic examination and no tumor cells were found in para - cancerous tissue samples . complete clinicopathological data from each patient was collected and surgical specimens were processed immediately after resection . the specimens were frozen in liquid nitrogen and transferred to a -80 c freezer or fixed in 4% formalin followed by paraffin embedding . this study was approved by the medical ethics committee of the second affiliated hospital of harbin medical university . expression of hmgb1 and c - iap2 proteins in paired tumor and para - tumor tissue samples of 68 patients was examined by immunohistochemistry . the samples were fixed by 4% buffered formalin , embedded in paraffin and cut into 4 m - thick sections . the sections were then baked at 70 c overnight , and de - waxed in xylene and rehydrated in a graded concentration of ethanol and washed with phosphate buffered saline ( pbs ) . the sections were then subjected to antigen retrieval in a high pressure cooker with a citric acid buffer and slowly cooled to room temperature . after , sections were incubated in 3% h2o2-methanol for 10 minutes , then incubated overnight at 4 c with a rabbit polyclonal antibody against human hmgb1 at a dilution of 1:40 ( boster biological technology co. , ltd . , wuhan , china ) or a mouse polyclonal antibody against human c - iap2 at a dilution of 1:10 ( german r&d company ) , respectively . subsequently , tissues were treated with horseradish peroxidase conjugated secondary antibody ( zhongshan jinqiao biotechnology co. , ltd , beijing , china ) , and the color was developed using diaminobenzidine tetrahydrochloride ( dab ) and counterstained with hematoxylin . a known hmgb1 positive liver cancer specimen was used as a positive control , while in negative control samples the antibody was replaced by pbs . immunostained sections were reviewed and scored under a light microscope ( shanghai optical instrument factory , shanghai , china ) by two independent investigators ( bi mr and chen ly ) , blinded to the treatment group . to score expression of hmgb1 and c - iap2 protein , 10 microscopic fields (  400 ) we ranked the staining scores into four categories : 1 ,  25% , as no staining / background of negative controls ( - ) ; 2 , 26 - 50% , as weak staining ( + ) ; 3 , 51 - 75% , as moderate staining ( + + ) ; and 4 , 76 - 100% , as intense staining ( + + + ) . we defined ( - ) , ( + ) as low expression and ( + + ) and ( + + + ) as overexpression . a hundred milligram of fresh tissue was grounded into powder in liquid nitrogen and 400 l lysis buffer ( 50 mmol / l tris - hcl at ph 7.4 , 150 mmol / l nacl , 1% tritonx-100 , 1 mmol / l pmsf , 1 mmol / l edta , and 0.25% sodium deoxycholate ) was added to the sample . the mixtures were then centrifuged at 12000 rpm and 4 c for 10 minutes and one - fifth volume of 5  laemmli s sample buffer ( 62.5 mm tris base , 10% ( w / v ) glycerol , 2.3% sodium dodecyl sulphate , and 5% 2-mercaptoethanol ) was added to the supernatant . after denaturing at 95 c in a water bath , protein samples were quantified and separated by 12% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and transferred to a pvdf membrane ( millipore , schwalbach , germany ) . after blocking with 5% non - fat milk for two hours at room temperature , membranes were incubated overnight at 4 c with an anti - hmgb1 antibody at a dilution of 1:400 ( boster biological technology ) , anti - c - iap2 antibody at a dilution of 1:200 ( german r & d ) or anti--actin antibody at a dilution of 1:800 ( santa cruz biotechnology , heidelberg , germany ) . followed by incubation with an antimouse or rabbit horseradish peroxidase conjugated igg ( zhongshan jinqiao biotechnology , beijing , china ) . the membranes were incubated with an ecl western blotting detection system ( zhongshan jinqiao biotechnology ) for autoradiography against x - ray films ( zhongshan jinqiao biotechnology ) . total rna was isolated from tumor and para - tumor tissue samples using a trizol reagent ( invitrogen , carlsbad , ca ) according to the manufacture s instruction . rna samples of 0.5 g were reversely transcribed into cdna using a reverse transcriptase and the cdna samples were amplified by qpcr using taqman fluorescent probes ( ambion , austin , tx , usa ) for hmgb1 ( 5'-atatggcaaaagcggacaag-3 ' and 5'-gcaacatcaccaatggacag-3 ' ) , c - iap2 ( 5'-tccgtcaagttcaagccagtt-3 ' and 5'-tctcctgggctgtctgatgtg-3 ' ) , or -actin ( 5'-gcgggaaatcgtgcgtgacatt-3 ' and 5'-gatggagttgaaggtagtttcgtg-3 ' ) . pcr conditions were an initial 95 c for 10 minutes , 40 cycles of 95 c for 15 seconds and 60 c for one minute . in the last pcr cycle , during the temperature rise from 55 c to 95 c , the melt curves were collected at 0.5 c intervals . each experiment was performed in duplicate , repeated three times and the mean fold - change in hmgb1 and c - iap2 mrna levels was calculated using the 2 method . all data were presented as mean  standard deviation ( sd ) of the results obtained from three independent experiments . comparison between the two groups was performed using student s t - test for continuous data and chi - square test for categorical data . all data were analyzed using spss version 13.0 statistical software ( spss , chicago , il , usa ) . to detect the level of hmgb1 and c - iap2 mrna and protein in paired tumor and para - tumor tissue samples ( defined as  3 cm distance from the tumor edge ) , we collected fresh tissues from 68 patients with hcc who underwent hepatectomy between may 2011 and may 2013 from the second and third clinical medical college of harbin medical university ( harbin , china ) . diagnosis of hcc in all patients was confirmed by histopathologic examination and no tumor cells were found in para - cancerous tissue samples . complete clinicopathological data from each patient was collected and surgical specimens were processed immediately after resection . the specimens were frozen in liquid nitrogen and transferred to a -80 c freezer or fixed in 4% formalin followed by paraffin embedding . this study was approved by the medical ethics committee of the second affiliated hospital of harbin medical university . expression of hmgb1 and c - iap2 proteins in paired tumor and para - tumor tissue samples of 68 patients was examined by immunohistochemistry . the samples were fixed by 4% buffered formalin , embedded in paraffin and cut into 4 m - thick sections . the sections were then baked at 70 c overnight , and de - waxed in xylene and rehydrated in a graded concentration of ethanol and washed with phosphate buffered saline ( pbs ) . the sections were then subjected to antigen retrieval in a high pressure cooker with a citric acid buffer and slowly cooled to room temperature . after , sections were incubated in 3% h2o2-methanol for 10 minutes , then incubated overnight at 4 c with a rabbit polyclonal antibody against human hmgb1 at a dilution of 1:40 ( boster biological technology co. , ltd . , wuhan , china ) or a mouse polyclonal antibody against human c - iap2 at a dilution of 1:10 ( german r&d company ) , respectively . subsequently , tissues were treated with horseradish peroxidase conjugated secondary antibody ( zhongshan jinqiao biotechnology co. , ltd , beijing , china ) , and the color was developed using diaminobenzidine tetrahydrochloride ( dab ) and counterstained with hematoxylin . a known hmgb1 positive liver cancer specimen was used as a positive control , while in negative control samples the antibody was replaced by pbs . immunostained sections were reviewed and scored under a light microscope ( shanghai optical instrument factory , shanghai , china ) by two independent investigators ( bi mr and chen ly ) , blinded to the treatment group . to score expression of hmgb1 and c - iap2 protein , 10 microscopic fields (  400 ) we ranked the staining scores into four categories : 1 ,  25% , as no staining / background of negative controls ( - ) ; 2 , 26 - 50% , as weak staining ( + ) ; 3 , 51 - 75% , as moderate staining ( + + ) ; and 4 , 76 - 100% , as intense staining ( + + + ) . we defined ( - ) , ( + ) as low expression and ( + + ) and ( + + + ) as overexpression . a hundred milligram of fresh tissue was grounded into powder in liquid nitrogen and 400 l lysis buffer ( 50 mmol / l tris - hcl at ph 7.4 , 150 mmol / l nacl , 1% tritonx-100 , 1 mmol / l pmsf , 1 mmol / l edta , and 0.25% sodium deoxycholate ) was added to the sample . the mixtures were then centrifuged at 12000 rpm and 4 c for 10 minutes and one - fifth volume of 5  laemmli s sample buffer ( 62.5 mm tris base , 10% ( w / v ) glycerol , 2.3% sodium dodecyl sulphate , and 5% 2-mercaptoethanol ) was added to the supernatant . after denaturing at 95 c in a water bath , protein samples were quantified and separated by 12% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and transferred to a pvdf membrane ( millipore , schwalbach , germany ) . after blocking with 5% non - fat milk for two hours at room temperature , membranes were incubated overnight at 4 c with an anti - hmgb1 antibody at a dilution of 1:400 ( boster biological technology ) , anti - c - iap2 antibody at a dilution of 1:200 ( german r & d ) or anti--actin antibody at a dilution of 1:800 ( santa cruz biotechnology , heidelberg , germany ) . followed by incubation with an antimouse or rabbit horseradish peroxidase conjugated igg ( zhongshan jinqiao biotechnology , beijing , china ) . the membranes were incubated with an ecl western blotting detection system ( zhongshan jinqiao biotechnology ) for autoradiography against x - ray films ( zhongshan jinqiao biotechnology ) . total rna was isolated from tumor and para - tumor tissue samples using a trizol reagent ( invitrogen , carlsbad , ca ) according to the manufacture s instruction . rna samples of 0.5 g were reversely transcribed into cdna using a reverse transcriptase and the cdna samples were amplified by qpcr using taqman fluorescent probes ( ambion , austin , tx , usa ) for hmgb1 ( 5'-atatggcaaaagcggacaag-3 ' and 5'-gcaacatcaccaatggacag-3 ' ) , c - iap2 ( 5'-tccgtcaagttcaagccagtt-3 ' and 5'-tctcctgggctgtctgatgtg-3 ' ) , or -actin ( 5'-gcgggaaatcgtgcgtgacatt-3 ' and 5'-gatggagttgaaggtagtttcgtg-3 ' ) . pcr conditions were an initial 95 c for 10 minutes , 40 cycles of 95 c for 15 seconds and 60 c for one minute . in the last pcr cycle , during the temperature rise from 55 c to 95 c , the melt curves were collected at 0.5 c intervals . each experiment was performed in duplicate , repeated three times and the mean fold - change in hmgb1 and c - iap2 mrna levels was calculated using the 2 method . all data were presented as mean  standard deviation ( sd ) of the results obtained from three independent experiments . comparison between the two groups was performed using student s t - test for continuous data and chi - square test for categorical data . all data were analyzed using spss version 13.0 statistical software ( spss , chicago , il , usa ) . immunohistochemistry results showed that hmgb1 protein was highly expressed in tumor tissue samples compared to para - tumor tissue samples ( 85.3% vs. 32.3% ,  = 46.10 , p < 0.01 ; table 1 ) . as shown in figure 1 a , strong staining of hmgb1 was observed in the cytoplasm and weaker staining was observed in the nuclei of tumor cells , resulting in high expression of hmgb1 protein in tumor tissue samples in 44 of 68 hcc cases ( 64.7% ; table 1 ) . in figure 1 b , only 22 of 68 hcc cases displayed expression of hmgb1 in paired para - tumor tissue samples ( 32.3% ; table 1 ) , among which 11 cases showed weak staining in the cytoplasm and nuclei of paired para - tumor cells . furthermore , immunohistochemistry results showed that expression of c - iap2 protein was significantly higher in hcc tissue samples compared to para - tumor tissue samples ( 82.4% vs. 27.9% ,  = 46.335 , p < 0.01 ; table 1 ) . there was strong staining of c - iap2 protein in the cytoplasm of 38 ( 55.9% ) hcc cases ( figure 1 c , table 1 ) , whereas only 19 of 68 ( 27.9% ) of paired para - tumor tissue samples expressed c - iap2 protein . overall , there was only one case that demonstrated a strong expression of c - iap2 protein in para - tumor tissue samples ( figure 1 d , table 1 ) . furthermore , we verified our immunohistochemical results in hcc and para - tumor tissue samples using western blot , and the data was consistent with the immunohistochemical results , i.e. 59 of 68 hcc cases showed significant high expression of hmgb1 protein in hcc tissue samples ( t ) compared to paired para - tumor ( p ) tissue samples ( figure 2 a ) . besides , 54 of 68 hcc cases showed significant high c - iap2 expression in tumor tissue samples ( t ) compared to paired para - tumor ( p ) tissue samples ( figure 2 b ) . the difference between relative expression levels of hmgb1 and c - iap2 in tumor and para - tumor samples was statistically significant ( p < 0.01 ) . ( a ) high expression of hmgb1 in hcc tissue samples , hmgb1 immunostaining was predominantly localized in the cytoplasm of hcc cells (  400 ) . ( b ) weak expression of hmgb1 protein in the para - tumor tissue samples ( 400 ) . ( c ) c - iap2 immunostaining mainly occurs in the cytoplasm of tumor cells (  400 ) . ( d ) weak expression of c - iap2 in para - tumor tissue samples (  400 ) . ( a ) representative western blot data on hmgb1 expression in cases and a chart on the relative expression levels of hmgb1 for tumor and para - tumor samples . ( b ) representative western blot data on c - iap2 expression in cases and a chart on the relative expression levels of hmgb1 for tumor and para - tumor samples . lane t corresponds to the expression of hmgb1 and c - iap2 in hcc samples and lane p corresponds to the expression of hmgb1 and c - iap2 in paired para - tumor tissue samples . we found that expression of hmgb1 was significantly associated with tumor multiplicity (  = 4.225 , p < 0.05 ) , tumor size (  = 10.748 , p < 0.05 ) , alpha fetoprotein ( afp ) level (  = 4.83 , p < 0.05 ) and advanced tnm stage (  = 7.0584 , p < 0.05 ) , while high expression of c - iap2 was significantly associated with tumor size (  = 9.862 , p < 0.05 ) , afp level (  = 9.998 , p < 0.05 ) , and advanced tnm stage (  = 8.228 , p < 0.05 ; table 2 ) . however , there was no significant association between expression of hmgb1 or c - iap2 protein and other variables ( such as age , gender and viral infection ) ( table 2 ) . abbreviations : hbv , hepatitis b virus ; hcv , hepatitis c virus ; afp , alpha - fetoprotein . data are presented as ( n. % ) . to further assess the overexpression of hmgb1 and c - iap2 mrna and proteins in hcc tissue samples , we performed qrt - pcr in fresh hcc and paired para - tumor tissue samples . expression of hmgb1 mrna was 2.209 fold higher in hcc tissue than paired para - tumor tissue ( t = 5.976 , p < 0.01 , figure 3 a ) , while expression of c - iap2 mrna was 2.769 fold higher in tumor tissue than paired para - tumor tissue samples ( t = 9.507 , p < 0.01 , figure 3 b ) . the expression of hmgb1 mrna was 2.209 fold higher in hcc tissue than paired para - tumor tissue ( t = 5.976 , p < 0.01 ) , ( b ) qrt - pcr analysis of c - iap2 mrna . the expression of c - iap2 mrna was 2.769 fold higher in tumor tissue than paired para - tumor tissue samples ( t = 9.507 , p < 0.01 ) . we associated the expression of hmgb1 and c - iap2 proteins in hcc tissue samples using spearman 's test , since hmgb1 may act as an oncoprotein to inhibit cell apoptosis ( 34 ) , while c - iap2 is a key antiapoptotic factor . using immunohistochemical analysis , 46 of 68 hcc cases showed high expression of hmgb1 in tumor tissue samples , 45 of 68 hcc cases showed a high expression of c - iap2 in tumor tissue samples , while 36 of 68 hcc cases showed both high expression of hmgb1 and c - iap2 in tumor tissue samples ( table 3 , rs = 0.878 , p < 0.01 ) , indicating that high expression of hmgb1 protein was significantly associated with enhanced levels of c - iap2 protein in hcc tissue samples . spearman s rank correlation test , rs = 0.878 , p < 0 01 . immunohistochemistry results showed that hmgb1 protein was highly expressed in tumor tissue samples compared to para - tumor tissue samples ( 85.3% vs. 32.3% ,  = 46.10 , p < 0.01 ; table 1 ) . as shown in figure 1 a , strong staining of hmgb1 was observed in the cytoplasm and weaker staining was observed in the nuclei of tumor cells , resulting in high expression of hmgb1 protein in tumor tissue samples in 44 of 68 hcc cases ( 64.7% ; table 1 ) . in figure 1 b , only 22 of 68 hcc cases displayed expression of hmgb1 in paired para - tumor tissue samples ( 32.3% ; table 1 ) , among which 11 cases showed weak staining in the cytoplasm and nuclei of paired para - tumor cells . furthermore , immunohistochemistry results showed that expression of c - iap2 protein was significantly higher in hcc tissue samples compared to para - tumor tissue samples ( 82.4% vs. 27.9% ,  = 46.335 , p < 0.01 ; table 1 ) . there was strong staining of c - iap2 protein in the cytoplasm of 38 ( 55.9% ) hcc cases ( figure 1 c , table 1 ) , whereas only 19 of 68 ( 27.9% ) of paired para - tumor tissue samples expressed c - iap2 protein . overall , there was only one case that demonstrated a strong expression of c - iap2 protein in para - tumor tissue samples ( figure 1 d , table 1 ) . furthermore , we verified our immunohistochemical results in hcc and para - tumor tissue samples using western blot , and the data was consistent with the immunohistochemical results , i.e. 59 of 68 hcc cases showed significant high expression of hmgb1 protein in hcc tissue samples ( t ) compared to paired para - tumor ( p ) tissue samples ( figure 2 a ) . besides , 54 of 68 hcc cases showed significant high c - iap2 expression in tumor tissue samples ( t ) compared to paired para - tumor ( p ) tissue samples ( figure 2 b ) . the difference between relative expression levels of hmgb1 and c - iap2 in tumor and para - tumor samples was statistically significant ( p < 0.01 ) . ( a ) high expression of hmgb1 in hcc tissue samples , hmgb1 immunostaining was predominantly localized in the cytoplasm of hcc cells (  400 ) . ( b ) weak expression of hmgb1 protein in the para - tumor tissue samples ( 400 ) . ( c ) c - iap2 immunostaining mainly occurs in the cytoplasm of tumor cells (  400 ) . ( d ) weak expression of c - iap2 in para - tumor tissue samples (  400 ) . ( a ) representative western blot data on hmgb1 expression in cases and a chart on the relative expression levels of hmgb1 for tumor and para - tumor samples . ( b ) representative western blot data on c - iap2 expression in cases and a chart on the relative expression levels of hmgb1 for tumor and para - tumor samples . lane t corresponds to the expression of hmgb1 and c - iap2 in hcc samples and lane p corresponds to the expression of hmgb1 and c - iap2 in paired para - tumor tissue samples . we found that expression of hmgb1 was significantly associated with tumor multiplicity (  = 4.225 , p < 0.05 ) , tumor size (  = 10.748 , p < 0.05 ) , alpha fetoprotein ( afp ) level (  = 4.83 , p < 0.05 ) and advanced tnm stage (  = 7.0584 , p < 0.05 ) , while high expression of c - iap2 was significantly associated with tumor size (  = 9.862 , p < 0.05 ) , afp level (  = 9.998 , p < 0.05 ) , and advanced tnm stage (  = 8.228 , p < 0.05 ; table 2 ) . however , there was no significant association between expression of hmgb1 or c - iap2 protein and other variables ( such as age , gender and viral infection ) ( table 2 ) . abbreviations : hbv , hepatitis b virus ; hcv , hepatitis c virus ; afp , alpha - fetoprotein . to further assess the overexpression of hmgb1 and c - iap2 mrna and proteins in hcc tissue samples , we performed qrt - pcr in fresh hcc and paired para - tumor tissue samples . expression of hmgb1 mrna was 2.209 fold higher in hcc tissue than paired para - tumor tissue ( t = 5.976 , p < 0.01 , figure 3 a ) , while expression of c - iap2 mrna was 2.769 fold higher in tumor tissue than paired para - tumor tissue samples ( t = 9.507 , p < 0.01 , figure 3 b ) . the expression of hmgb1 mrna was 2.209 fold higher in hcc tissue than paired para - tumor tissue ( t = 5.976 , p < 0.01 ) , ( b ) qrt - pcr analysis of c - iap2 mrna . the expression of c - iap2 mrna was 2.769 fold higher in tumor tissue than paired para - tumor tissue samples ( t = 9.507 , p < 0.01 ) . we associated the expression of hmgb1 and c - iap2 proteins in hcc tissue samples using spearman 's test , since hmgb1 may act as an oncoprotein to inhibit cell apoptosis ( 34 ) , while c - iap2 is a key antiapoptotic factor . using immunohistochemical analysis , 46 of 68 hcc cases showed high expression of hmgb1 in tumor tissue samples , 45 of 68 hcc cases showed a high expression of c - iap2 in tumor tissue samples , while 36 of 68 hcc cases showed both high expression of hmgb1 and c - iap2 in tumor tissue samples ( table 3 , rs = 0.878 , p < 0.01 ) , indicating that high expression of hmgb1 protein was significantly associated with enhanced levels of c - iap2 protein in hcc tissue samples . spearman s rank correlation test , rs = 0.878 , p < 0 01 . in the present study , we detected expression of hmgb1 and c - iap2 mrna and proteins in hcc tissue and para - tumor tissue samples . we found an upregulation of both hmgb1 and c - iap2 mrna and proteins in hcc tissue samples compared to para - tumor tissue samples . nevertheless , some of para - tumor tissue samples expressed a low level of these proteins , which indicates that para - tumor tissue may be really normal tissues , which could have a field of carcinogenesis effects and second para - tumour tissues usually had multiple lesions , such as hepatitis , cirrhosis , premalignant lesion of hcc . therefore , all of these could contribute to low expression of these two proteins in para - tumour tissues . upregulation of hmgb1 protein was associated with tumor multiplicity , tumor size , afp level and advanced tnm stages , while expression of c - iap2 was associated with tumor size , afp level and advanced tnm stage . moreover , expression of hmgb1 protein was associated with upregulation of c - iap2 expression in hcc tissues . these data suggest that altered expression of both hmgb1 and c - iap2 proteins was associated with hcc development and tumor progression . further studies would investigate whether targets of these two proteins can be used as a novel therapeutic strategy in hcc treatment . our current immunohistochemical data showed that hmgb1 was mostly localized at the cytosol of tumor cells , which raised a question why this nuclear protein appears in the cytoplasm , but not in the nuclei of tumor cells . indeed , a previous review publication discussed that the presence of hmgb1 protein in the nucleus depends on posttranslational modifications . when the protein is not acetylated , it stays in the nucleus , but hyperacetylation on lysine residues causes it to translocate into the cytosol ( 9 ) . furthermore , hmgb1 protein is also secreted by immune cells , including monocytes , macrophages and dendritic cells ( 9 ) . in addition , our current data was consistent with previous studies showing upregulated hmgb1 levels in hcc tissues compared to para - tumor tissues ( 35 - 37 ) . previous studies demonstrated the ability of hmgb1 protein to support tumor growth and metastasis through its function as an extracellular ligand to induce cell proliferation ( 13 , 38 - 40 ) or through its pro - inflammatory properties as a cytokine ( 14 ) . a previous study demonstrated that overexpression of hmgb1 protein inhibited bak - induced cell death by inhibition of caspase-9 activation ( 23 ) . most caspase family members are promoters or effectors of apoptosis , playing important roles in the process of apoptosis ( 23 , 29 ) . caspase activity can be regulated by iap proteins , especially , c - iap2 , which is known to directly bind to caspase-3 and caspse-7 proteins and inhibit their activity and activation of pro - caspase-9 proteins . in the current study , we analyzed the expression of c - iap2 mrna and protein and found that expression of hmgb1 protein was associated with upregulation of c - iap2 protein in hcc tissues . overexpression of c - iap2 protein has been found in a variety of human cancers and associated with drug resistance and recurrence of different human cancers ( 31 , 32 , 41 ) . in the current study , we found that upregulation of c - iap2 protein in hcc tissues was associated with tumor size , afp level and advanced tnm stages . these data were consistent with a previous study in colon cancer ( 23 ) . in the recent years , there has been increasing interest in the role of hmgb1 in hcc . for example , cheng et al . ( 37 ) reported an association between serum hmgb1 level and clinicopathological features of hcc patients , i.e. higher serum hmgb1 level was associated with bigger tumor size , poor tumor differentiation and advanced tnm stages . jiang et al . ( 26 ) showed that overexpression of hmgb1 in tumor tissue samples , rather than para - tumor tissue samples , was associated with advanced tnm stage , vascular invasion and capsule invasion of hcc . recently , liu et al . ( 27 , 42 ) showed that expression of hmgb1 was significantly associated with incomplete tumor encapsulation and advanced tnm stages . in the current study using both fresh tissues samples and paraffin blocks from 68 hcc patients , we demonstrated that expression of hmgb1 was significantly associated with tumor multiplicity and size , afp level and advanced tnm stages , while expression of c - iap2 was associated with tumor size , afp level and advanced tnm stage . the current study found an association between the expression of hmgb1 and c - iap2 proteins in hcc , suggesting that c - iap2 could mediate the effects of hmgb1 on inhibition of hcc apoptosis and promotion of hcc progression . we also showed that upregulation of hmgb1 and c - iap2 was at the transcriptional level . further studies are warranted to target these proteins as novel therapeutic strategies in the treatment of hcc patients and investigate how hmgb1 regulate c - iap2 expression in hcc cells .
background : hepatocellular carcinoma ( hcc ) is one of the most important health problems in china.objectives:this study analyzed expression of high - mobility group protein b1 ( hmgb1 ) and inhibitor of apoptosis protein-2 ( c - iap2 ) proteins in hcc compared to paired para - tumor tissue samples to assess the association with hcc pathogenesis and progression.materials and methods : sixty - eight hcc and para - tumor tissue samples were collected for western blot , qrt - pcr and immunohistochemical analyses of hmgb1 and c-iap2.results:hmgb1 and c - iap2 proteins were highly expressed in hcc tissue samples [ 85.3% ( 58/68 ) and 82.4% ( 56/68 ) , respectively ] compared to para - tumor tissue samples [ 32.3% and 27.9% , respectively ] . 
 furthermore , expression of hmgb1 was significantly associated with enhanced c - iap2 expression in hcc tissue samples ( r = 0.878 , p < 0.01 ) . 
 expression of hmgb1 was associated with tumor multiplicity and size , alpha - fetoprotein ( afp ) level and advanced tnm stage , while expression of c - iap2 was associated with tumor size , afp level and advanced tnm stage.conclusions:expression of hmgb1 and c - iap2 proteins was associated with hcc development and progression , and the expression of hmgb1 and c - iap2 proteins in hcc were significantly associated with each other . 
 additionally , these proteins may show promise as biomarkers to predict hcc progression .