The Research Of Aberrant SOX17, ECRG4CpG Island Methylation Regulation MRNA Expression In Breast Cancer

Background and ObjectiveBreast cancer (BC), a common malignancy of the female, is the top of incidence of malignant tumors of the female, the major diseases that endanger women’s health,in some developed cities in China. Prognosis is related to when diagnosis of breast cancer. At present, the diagnosis of breast cancer is still relying on breast ultrasound, mammography and other imaging tests. CA-153, as a tumor marker in screening for breast cancer, has been widely used, but its low sensitivity often leads to misdiagnosis, therefore, a new, high sensitivity, specificity diagnostic method is badly in need.DNA methylation is a regulatity mechanism of gene expression and a contributor to carcinogenetic process. Aberrant CpG island methylation of tumor-related gene is an early and frequent event in carcinogenctic proeess and plays an important role in the development of cancer. Researches on CpG island methylation of specific genes may be a new perspective to clarify the molecular mechanism of BC.Some studies have shown that abnormal methylation of SOX17gene the frequent event in the process of lung cancer, and is an important mechanism of SOX17genes downregulated. Inhibition of SOX17by microRNA141and methylation activates the WNT signaling pathway in esophageal cancer; Expression of ECRG4, a novel esophageal cancer-related gene, is downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma. It also has been reported that SOX17gene CpG island have higher frequency of hypermethylation in BC tumor tissues compared to adjacent non-cancerous tissues by methylation qualitative method.In this study, we quantify the CpG island methylation levels of SOX17and ECRG4in36tumor tissues and adjacent non-cancerous tissues by MassArray platform; we also detect SOX17and ECRG4mRNA levels in the36paired samples by real-time PCR to investigate the relationship between CpG island methylation and mRNA expression. The methylation levels of SOX17and ECRG4in different groups of age, the status of estrogen receptor, progestrone receptor and lymphatic metastasis were also analyzed and provides preliminary theory basis for clinical significance of SOX17and ECRG4aberrant methylation. Materials and Methods1. SubjectsThirty-six paired clinical samples of BC-including tumor issues and adjacent non-cancerous tissues were collected from breast center of Nanfang Hospital.2. Quantitative DNA methylation detectionGenomic DNA was extracted from and clinical samples using DNA extraction kit. Genomic DNA was modified with sodium bisulfite. Quantitative methylation analysis of36clinical samples-including tumor tissues and adjacent non-cancerous tissues and were determined by the MassArray(?)EpiTYPER platform (sequenom).3. Quantitative mRNA deteetion We used Real-time PCR to evaluate the mRNA expression levels of SOXl7and ECRG4in36clinical samples-ineluding tumor tissues and adjacent non-cancerous and. Relative levels of SOX17and ECRG4mRNA were calculated using2△△Ct. β-actin was used as an internal control. Data were expressed from three separate experiments.4. Correlation analysis between methylation levels and transcription levelsThe relationship between methylation levels and transcription levels was analyzed using the Spearman test. All tests were two-sided; significant level was assigned at a=0.05. All the data was analyzed by SPSS13.0.5. Methylation levels analysis in different groupsThe methylation levels of SOX17and ECRG4in tumor tissues and adjacent non-cancerous tissues were compared by Wilcox rank sum test. The methylation levels in differenit groups of age, the status of estrogen receptor and progestrone receptor were compared by Wilcox rank sum test. The methylation levels in three different groups of age, the status of lymphatic metastasis were compared by Kruskal-Wallis test. All tests were two-sided; significant level was assigned at a=0.05. All the data was analyzed by SPSS13.0.Results1.1CpG island methylation levels and mRNA levels of SOX17in BC tumor tissues and adjacent non-cancerous tissues1.1.1CpG island methylation of SOX17in BC tumor tissues and adjacent Non-cancerous tissues50%(18/36) of the tumor tissues exhibited higher methylation levels of SOX17CpG island compared to adjacent non-cancerous tissues. The average methylation levels of each CG unit increased from16%to28%in tumor tissues as compared to adjacent non-cancerous tissues.41.7%(15/36)of patients exhibited similar methylation levels in tumor tissues and adjacent non-cancerous tissues.1.1.2SOX17mRNA levels in BC tumor tissues and adjacent non-cancerous tissues69.4%(25/36) of the tumor tissues showed lower SOX17mRNA levels than adjacent non-cancerous tissues.8.3%(3/36) of the tumor tissues showed similar mRNA levels with adjacent non-cancerous tissues and5.22.2%(8/36) of the tumor tissues exhibited higher mRNA levels than adjacent non-cancerous tissues.1.1.3The relationship between the CPG island methylation and mRNA expression of SOX17in tumor tissues and adjacent non-cancerous tissuesAmong the25tumor tissues with lower mRNA levels than adjacent non-cancerous tissues,18exhibited higher methylation levels and a negative association was found between the CPG island methylation and mRNA expression (rs=-0.446, P=0.025);7tumor tissues had similar methylation levels with adjacent non-cancerous tissues. Among8tumor tissues with higher mRNA level,5had similar methylation level with the adjacent non-cancerous tissue;3had lower methylation level than adjacent non-cancerous tissues.3tumor tissues had no significant difference in mRNA level and methylation level compared with adjacent non-cancerous tissues.1.2Analysis of SOX17CpG island methylation level in the different clinical data groupsThe methylation level of SOX17CpG island were higher in>40years old,≤60years old group than≤40years old and>60years old group, which there was statistical significance in four CG units; there was no significant difference in estrogen receptor positive/negative groups and progesterone receptor positive/negative groups; there was no statistical difference in the different states of lymph node metastasis groups;2.1CpG island methylation levels and mRNA levels of ECRG4in BC tumor tissues and adjacent non-cancerous tissues2.1.1CpG island methylation of ECRG4in BC tumor tissues and adjacent non-cancerous tissues55.6%(20/36) of the tumor tissues exhibited higher methylation levels of ECRG4CpG island compared to adjacent non-cancerous tissues. The average methylation levels of each CG unit increased from5%to44%in tumor tissues as compared to adjacent non-cancerous tissues.19.4%(7/36)of patients exhibited similar methylation levels in tumor tissues and adjacent non-cancerous tissues.2.1.2ECRG4mRNA levels in BC tumor tissues and adjacent non-cancerous tissues66.7%(24/36) of the tumor tissues showed lower ECRG4mRNA levels than adjacent non-cancerous tissues.19.4%(7/36) of the tumor tissues showed similar mRNA levels with adjacent non-cancerous tissues and5.13.9%(5/36) of the tumor tissues exhibited higher mRNA levels than adjacent non-cancerous tissues.2.1.3The relationship between the CPG island methylation and mRNA expression of ECRG4in tumor tissues and adjacent non-cancerous tissuesAmong the25tumor tissues with lower mRNA levels than adjacent non-cancerous tissues,14exhibited higher methylation levels and a negative association was found between the CPG island methylation and mRNA expression (rs=-0.642, P=0.013);5tumor tissues had similar methylation levels with adjacent non-cancerous tissues;6exhibited lower methylation levels than adjacent non-cancerous tissue.2tumor tissues with higher mRNA level had similar methylation level with the adjacent non-caneerous tissue.3tumor tissues with higher mRNA levels exhibited higher methylation levels than adjacent non-cancerous tissue. Among6tumor tissues with no significant difference in mRNA level,3exhibited higher methylation levels than adjacent non-cancerous tissue;3exhibited lower methylation levels than adjacent non-cancerous tissue.2.2Analysis of ECRG4CpG island methylation level in the different clinical data groupsThere was no difference in different age groups about methylation level of ECRG4CpG island; the methylaton level in estrogen/progesterone receptor negative group exhibited higher than estrogen/progesterone receptor positive group with significant differences; there was no statistical difference in the different states of lymph node metastasis groups;Conclusions1. Among36samples,18cancer tissues were detected higher methylation levels of SOX17CpG island than the adjacent tissues. mRNA expression levels in18cancer tissues were lower than the adjacent tissues. It exhibited a negative association between the CpG island methylation and mRNA expression (rs=-0.446, P=0.025). The results suggested that SOX17may regulate the expression of the mRNA by changing the gene promoter CpG island methylation status.2. Among36samples,20cancer tissues were detected higher methylation levels of ECRG4CpG island than the adjacent tissues. mRNA expression levels in25cancer tissues were lower than the adjacent tissues. It exhibited a negative association between the CpG island methylation and mRNA expression(rs=-0.642, P=0.013). The high methylation levels of ECRG4CpG island were association with ER-and PR-. These results suggested that DNA methylation may be ECRG4breast cancer an important transcriptional regulatity mechanisms. BACKGROUND:Breast cancer has been a disease with the highest incidence of malignant tumors in the Chinese women. However, the incidence of breast cancer is different in the people in the same living condition, suggesting that different genetic susceptibility may palys an important role.To investigate whether the single nucleotide polymorphism (SNP) in TRIM31gene is associated with susceptibility of breast cancer in Guangdong women of Han Nationality. METHODS:The polymorphism of SNP rs1116221in TRIM31gene was inspected in216breast cancer patients and216healthy controls that collected coinstantaneously from Nanfang hospital, Southern Medical University, via MassARRAY(?)-IPLEX platform. Data was analyzed by Chi-square test between case and control groups. Odds ratio (OR) and95%confidence interval (95%CI) were calculated by unconditional logistic regression to analyze the associations between the susceptibility of breast cancer and genotypes, further analysis was carried based on the immunohistochemical results of estrogen receptor (ER) and progesterone receptor (PR). RESULTS:There were three genotypes for rs1116221in Guangdong women of Han nationality, CC CT and TT. The genotype frequencies were not statistically different between case and control groups(P>0.05). But in ER (+/-) groups, heterozygous genotype CT was related to breast cancer with ER(+)(OR=2.67,95%CI:1.02-7.02, P=0.034). CONCLUSION: There is no significant association between the polymorphism of rsl116221in gene TRIM31and susceptibility of breast cancer in Guangdong women of Han Nationality, however the heterozygous genotype CT is associated with ER(+).