breast cancer and other malignancies result from stepwise genetic alterations of normal host cells and , possibly , from epigenetic changes in the behavior of not only malignant cells but also host cells that interact with the tumor , such as immune , vascular , and stromal cells [ 1 , 2 ] . the term epigenetic refers to information which is transmitted from the parental genome to the next generation of cells which is not encoded by the primary dna sequence . epigenetic mechanisms are essential for the regulation of gene expression and genome integrity in normal cells . nearly 50% of the genes that cause familial forms of cancer undergo methylation - associated silencing in various sporadic forms of cancer , once they are mutated in the germ line . the response gene to complement 32 ( rgc32 ) protein , recently named chromosome 13 open reading frame 15 , is a cell cycle regulator induced by activation of complements [ 5 , 6 ] . human rgc32 is found on the long arm of chromosome 13 and is mapped in the interval of 13q1213q14 . the rgc32 gene product plays a dual role in both cell proliferation and tumor suppressor in certain types of cancers . it is downregulated in high - grade prostate intraepithelial neoplasia , invasive prostate cancer , multiple myeloma , and drug - resistant glioblastoma , but upregulated in others , including cutaneous t cell lymphoma and ovarian and breast cancer [ 8 , 9 ] . overexpression of rgc32 leads to reorganization of cytoskeleton and promotion of cell migration , which could be an important mechanism of rgc32 in progression of cancer metastasis . however , promoter hypermethylation plays a chief role in cancer through transcriptional silencing of crucial growth regulators such as tumor suppressor genes . recently , kim et al . have reported that rgc32 is subjected to epigenetic silencing in non - small - cell lung cancers ( nsclcs ) . they found that the rgc32 dna methylation is associated with low or undetectable levels of rgc32 mrna expression in malignant and corresponding nonmalignant lung tissues . their findings suggest that transcriptional inactivation of rgc32 expression may be caused by promoter methylation of that gene . therefore , given the proof that rgc32 functions as a tumor suppressor gene in certain types of cancers , in this study , we analyzed the promoter methylation status of rgc32 and evaluated its correlation with its gene expression in breast cancer . this study included 63 breast paraffin - embedded tumor samples and 63 adjacent nontumor tissues from the same patients . the inclusion criteria were female patient with primary breast cancer and the availability of the paraffin - embedded tissue along with patients ' clinicopathologic data . patients previously treated with neoadjuvant or adjuvant therapy as well as those missing clinicopathologic data , for example , her2 , er , pr , and nodal status , were excluded . an informed consent was obtained from all subjects , and ethical committee of zahedan university of medical sciences approved our study . dna was extracted from tissues using the standard protocol by proteinase k treatment and salting - out extraction protocol as described previously [ 12 , 13 ] . the quality and integrity of the dna were checked by electrophoresis on 0.8% agarose gel , quantitated spectrophotometrically , and stored at 20c till further use . the dna samples were treated with sodium bisulfite , which converts unmethylated c to u. however , when the methylation occurs at the c residues , they will withstand the treatment . the protocol applied for bisulfite modification of dna one g ) , naoh solution was added to a final concentration of 0.3  m. denaturation of the dna strands occurred effectively after incubation of the mix at 50c for 15  min . this mixture was then blended with 50  l of 2% low melting point ( lmp ) agarose and incubated at 50c for 15  min . a 15  l drop of this mixture was pipetted into 300  l cold mineral oil ( sigma ) . the agarose / dna drop quickly hardened in the oil and agarose beads shaped once they were incubated at 4c for 30  min . aliquots of 700  l of a 5  m bisulfite reagent ( 5  m sodium bisulfite , merck ; 125  mm hydroquinone , merck ; ph = 5.0 ) were added to each reaction tube containing a single bead . the tube was gently inverted to move the bead into the aqueous phase and was incubated at 55c in a water bath for 418 treatments were stopped by equilibrations against 1  ml of 1x te ( 2  15  min ) followed by desulphonation in 500  ml of 0.2  m naoh ( 2  10  min ) . finally , beads were washed with 1  ml 1x te ( tris - edta ) buffer followed by equilibrations against 1  ml of ddh2o ( 1  15  min ) . the beads were used directly for the pcr or kept at 20c for several weeks without any loss of quality . methylation status of the promoter region of rgc32 gene was determined by a nested methylation - specific polymerase chain reaction ( nested - msp ) , which boosts the sensitivity to detect the hypermethylated promoter by more than 50-fold . the primers used for the first stage of the msp distinguish the bisulfite - modified template but do not discriminate methylated and unmethylated templates . primers used for rgc32 nested - msp were nested - forward ( f ) : gggtaaatatttggggttgtaat , nested - reverse ( r ) : ttcaaccctaccaatcccttc ; methylated - f : tcgcggttttagggcgggcgc , methylated - r : ccgctcccaacacgatccgcg ; unmethylated - f : ttgtggttttagggtgggtgt and unmethylated - r ccactcccaacacaatccaca . the cycling conditions for the stage 1 of the nested - msp were 95c for 10  min followed by 30 cycles of denaturation at 95c for 15  s , annealing for 30  s at 60c , extension at 72c for 45  s , and final extension at 72c for 10  min . the pcr product of the first stage ( 282  bp ) was diluted 1  :  50 and subjected to the second stage of the nested - msp using two pairs of primers , one specific for the methylated alleles and another specific for the unmethylated as described previously by kim et al . . the pcr conditions for the second stage of the nested - msp were complete denaturation of dna at 95c for 10  min and 35 cycles involving denaturation at 95c for 15  s , annealing for 30  s at 63c , 45  s extension at 72c , and final extension at 72c for 10  min . the pcr products were verified on 2% agarose gels containing 0.5  g / ml ethidium bromide , and a photograph showing different methylation patterns was taken ( figure 1 ) . the amplicons size for both methylated and unmethylated allele was 194  bp . to check the accuracy of our experiments , we repeated dna methylation measurement randomly in 10% of samples . total rna was isolated from formalin fixed paraffin - embedded tissue samples using a rneasy ffpe kit according to the manufacturer 's instructions . cdna synthesis performed applying revertaid first strand cdna synthesis kit ( fermentas ) based on the manufacturer 's procedure . quantitative reverse transcriptase - pcr ( qrt - pcr ) for rgc32 was performed using the lightcycler abi 7500 system and maxima sybr green / rox ( fermentas ) . specific primers for mrna amplification of rgc32 were used as previously described by schlick et al . . reaction volumes of 20  l consisted of 10  m forward primer , 10  m reverse primer , 12.5  l maxima sybr green / rox , and 3  l of cdna as pcr template . gene expression was quantified by the comparative ct method , normalizing ct values to the housekeeping gene gapdh and calculating relative expression values . the following program conditions were applied for qrt - pcr running : 95c for 10 seconds followed by 40 cycles of 95c for 10 seconds and 60c for 1  min . expression levels were normalized against gapdh , which was amplified in the same run and following the same procedure described above . the primers sequences for q - pcr analysis of gapdh ( 173  bp ) and rgc32 ( 105  bp ) were gapdh - f : ttgccatcaatgaccccttca and gapdh - r : cgccccacttgattttgga ; rgc32-f : agccttcattgctgatcttga and rgc32-r : gcaggtcctcggaactttct . the statistical analyses of the data were done using the spss 18.0 software ( spss inc . the association between methylation patterns was assessed by computing the odds ratio ( or ) and 95% confidence intervals ( 95% ci ) from logistic regression analyses . kruskal - wallis one - way analysis of variance or one - way anova test was used to assess possible association between methylation patterns and covariates in this study . the promoter methylation status of the rgc32 gene was examined on the dna samples of 63 sporadic breast cancer tumors ( average age : 46.2  10.1 years ) and 63 adjacent noncancerous tissues of the same patients . as presented in table 2 , the mm phenotype was more frequent in breast cancer tumors than noncancerous tumors ( 4.8% versus 0% ) , but no significant difference was found between two groups ( or = 2.30 , 95% ci = 0.955.54 ) . we also examined the rgc32 methylation status in 63 blood samples of breast cancer patients and we found that all samples were unmethylated ( data were not shown ) . considering the impact of different covariates in the current study , we found no associations between rgc32 promoter methylation and patients ' age ( p = 0.332 ) , period age ( p = 0.541 ) , menopausal age ( p = 0.197 ) , tumor grade ( p = 0.611 ) , stage ( p = 0.092 ) , nodal metastasis ( p = 0.245 ) , estrogen receptor ( p = 0.377 ) , progesterone receptor ( p = 0.489 ) , and her2 ( p = 0.932 ) . the qpcr analysis showed that the rgc32 mrna expression level was higher in breast cancerous tissues than in noncancerous breast tissues ( p < 0.001 ) . the ratios of gapdh / rgc32 mrna were 1.073  0.045 and 0.959  0.036 in breast cancerous and adjacent noncancerous tissues , respectively ( figure 2 ) . rgc32 expression level was neither associated with its dna methylation pattern nor correlated to clinicopathologic characteristics of the patients ( p > 0.05 ) . in the current study , we found no significant difference between promoter methylation of rgc32 gene in breast tumor tissues and adjacent nontumor tissues . however , the rgc32 mrna was overexpressed in breast cancer tissues compared to adjacent noncancerous tissues , but it was not associated with dna methylation pattern of rgc32 gene . moreover , neither methylation pattern nor expression level of rgc32 was associated with clinical characteristics of patients . concerning rgc32 expression , our findings support the results of different studies reporting that rgc32 is upregulated in malignancies of ovary , lung , breast , and colon [ 8 , 16 ] . localized rgc32 protein in various carcinomas including lung , colon , and breast and found that rgc32 is overexpressed in areas adjacent to the tumor carcinomas . similarly , our findings showed that rgc32 is overexpressed in breast tumors compared to adjacent nontumor tissues which highlights the plausible role of rgc32 in the tumorigenesis . in alignment with our finding , vlaicu et al . have found that the rgc32 protein was absent from normal colon epithelial cells that were adjacent to the tumor that argues against its proposed tumor - suppressing role . rgc32 plays a key role in modulating the activity of cell cycle - specific kinases , thus regulating cell cycle progression . localized in the cytoplasm , rgc32 physically associates with cyclin - dependent kinase p34 cdc2 and plays a role in tumorigenesis and immunity [ 68 ] . overexpression of rgc32 may promote cell replication by downregulating cell cycle inhibitors and contribute to the pathogenesis of malignancies , suggesting that rgc32 participates in tumor transformation and progression . in addition , rgc32 plays an important role in regulating the acetylation of histones , a process that may possibly cause transcriptional activation and thereby support the possible function of rgc32 in tumor progression . have suggested that rgc32 regulates the acetylation of histones h2b lysine 5 ( h2bk5 ) , h2bk15 , h3k9 , h3k18 , and h4k8 . their findings proposed that rgc32 may be involved in the development of colon cancer by regulating chromatin assembly . although growing proof points to a role for rgc32 in the promotion of cell proliferation , some studies have implicated rgc32 as a tumor suppressor . kim et al .   have recently reported that rgc32 is subjected to promoter hypermethylation and that its dna methylation is associated with reduced expression of rgc32 in nsclcs . in contrast , we did not find any difference in methylation pattern of rgc32 promoter in cancerous and noncancerous breast tumors . regarding dual functions of rgc32 in a variety of carcinomas , it is speculated that these opposing reports could possibly arise from contradictory functions of rgc32 in different cell types . it is upregulated in cutaneous t cell lymphoma , colon , ovarian , and breast cancer and contributes to the cell proliferation and tumorigenesis , whereas it is downregulated in invasive prostate cancer , multiple myeloma , and drug - resistant glioblastoma functioning as a tumor suppressor . both global hypomethylation and regional hypermethylation have been studied in a wide spectrum of cancers , but cpg site - specific dna methylation has recently been the center of attraction for cancer research . recent analysis of dna from different tissues demonstrated tissue - specific differentially methylated regions ( dmrs ) . dmrs are genomic regions with diverse methylation statuses among multiple samples ( tissues , cells , individuals , or others ) . the identification of dmrs among multiple tissues provides a comprehensive survey of epigenetic differences among human tissues [ 20 , 21 ] . for example , these methylated regions that are distinctive to a particular tissue allow individuals to distinguish between tissue types , such as semen and vaginal fluid . additionally , dmrs between cancer and normal samples ( c - dmrs ) demonstrate the aberrant methylation in cancers and may affect the oncogenic process . aberrant methylation of tdmrs has been reported in different types of cancer and may modify tumor suppressor genes and/or oncogenes . quantification of site - specific determination of cpg methylation is done by several approaches such as allele - specific bisulfite sequencing , bisulfite - pyrosequencing , and microarray - based genome - wide analysis . all these new techniques will improve our understanding of the pathophysiology of cancer [ 18 , 23 , 24 ] . although all the previous studies have enabled a broader view of the genome - wide dna methylation patterns , there still remain questions to be answered , for example , how the tdmrs are being established and what are the functions of gene - body tdmrs . determining the human tdmr profile will not only provide important insights into the normal processes of tissue - specific differentiation ; it may also identify markers of pathogenic processes , such as cancer . to the best of our knowledge this is the first study that examined the association of rgc32 promoter methylation and gene expression in breast carcinoma . the rgc32 mrna expression level was much higher in breast carcinoma tissues than it was in nontumorous tissues , but it was not associated with methylation pattern of rgc32 promoter . additionally , we found that the rgc32 promoter methylation was not different between breast tumorous tissues and nontumorous tissues , although the methylated phenotype was observed more frequently in tumorous tissues than in nontumorous breast tissues ( 4.8% versus 0 ) . all these findings suggest that rgc32 dna methylation is possibly abrupt in the cancerous tissues when compared to noncancerous tissues . the small sample size of this study may have limited the statistical power of identifying the difference between groups . therefore , larger sample size with in - depth analyses like bisulphite sequencing or cpg site - specific measurement can help in understanding the role of rgc32 dna methylation in breast cancer .
background . response gene to complement 32 ( rgc32 ) , induced by activation of complements , has been characterized as a cell cycle regulator ; however , its role in carcinogenesis is still controversial . in the present study we compared rgc32 promoter methylation patterns and mrna expression in breast cancerous tissues and adjacent normal tissues . 
 materials and methods . 
 sixty - three breast cancer tissues and 63 adjacent nonneoplastic tissues were included in our study . design . nested methylation - specific polymerase chain reaction ( nested - msp ) and quantitative pcr ( qpcr ) 
 were used to determine rgc32 promoter methylation status and its mrna expression levels , respectively . results . 
 rgc32 methylation pattern was not different between breast cancerous tissue and adjacent nonneoplastic tissue ( or = 2.30 , 95% ci = 0.955.54 ) . however , qpcr analysis displayed higher levels of rgc32 mrna in breast cancerous tissues than in noncancerous tissues ( 1.073 versus 0.959 ; p = 0.001 ) , irrespective of the promoter methylation status . the expression levels and promoter methylation of rgc32 
 were not correlated with any of patients ' clinical characteristics ( p > 0.05 ) . 
 conclusion . 
 our findings confirmed upregulation of rgc32 in breast cancerous tumors , but it was not associated with promoter methylation patterns .