Establishment Of Methylation Detection Methods Of MiR-200c Gene And Their Application In Breast Cancer Specimens

MicroRNAs(mi RNAs)are a class of small non-coding single-stranded RNAs,which post transcriptionally regulate gene expression through binding onto the 3' untranslated region of target m RNAs and causing their degradation or translational inhibition.A large number of studies have shown that the expression of mi RNA gene is regulated by epigenetic modification,such as DNA methylation in promoter region,as well as the protein coding genes.mi R-200 c is one of a mi RNA closely related to the breast cancer metastasis and promoter hypermethylation of mi R-200 c gene has been correlated with various tumor to date.Due to the controversial results about the expression responsive CpG sites of mi R-200 c promoter,as well as the methods employed to detect mi R-200 c gene methylation,application of mi R-200 c gene methylation detection in tissue samples has not yet been widely carried out.Objective:To identify the expression responsive CpG loci of mi R-200 c gene and to establish and use qualitative and quantitative MSP methods for mi R-200 c gene methylation to analyze the breast cancer tissue samples.Methods:In situ hybridization method was employed for the detection of mi R-200 c expression in breast lesions;Demethylating drug 5-Aza-Cd R was applied to treat four breast cancer cell lines and expression of mi R-200 c was quantitated with qRT-PCR method;Online software was used to predict CpG island of mi R-200c;11 CpG sites in mi R-200 c CpG island were detected on 5-Aza-Cd R treated breast cancer cell line with bisulfite clone sequencing;MSP and DHPLC methods were constructed to determine mi R-200 c CpG island methylation level in breast cancer cell lines;mi R-200 c gene methylation of breast cancer tissues and paired paracancerous tissues were detected with qualitative and quantitative MSP methods.Results:1.In situ hybridization showed that the expression of mi R-200 c has no statistically significant expression difference between breast cancer and the normal breast tissue and benign breast lesions;2.The result of qRT-PCR showed that compared with the control group which was treated with 0?mol/L 5-Aza-Cd R,the mi R-200 c expression level in the 5?mol/L of 5-Aza-Cd R treated group was significantly upregulated in BT549 and MDA-MB-231 cell lines while there is little change in MCF7 and T47 D cell lines;3.The CpG island prediction result shows that there is a 147 bp length of CpG island 316~170bp upstream of mi R-200 c coding gene,which contains 11 CpG sites;4.Sodium bisulfite clone sequencing results showed that the total methylation rate of CpG island of mi R-200 c gene in MCF7 and T47 D cell lines,which have higher mi R-200 c expression level,was lower than those of the BT549 and MDA-MB-231 cell line which have lower expression of mi R-200c;5.The constructed MSP and d HPLC methods verify that the CpG island of mi R-200 c in MDA-MB-231 cell line is hypermethylated,while in MCF7 cell line is hypomethylated;6.Qualitative and quantitative MSP results showed that the methylation rate of mi R-200 c gene has no significant difference between breast cancer tissues and paired paracancerous tissues.Conclusions:1.A length of 147 bp CpG island locating at 316~170bp upstream of mi R-200 c coding gene,is the expression responsive region for mi R-200 c.2.The establishment of qualitative MSP and DHPLC methods,which could distinguish mi R-200 c gene CpG island methylation status,provides convenience for the detection of subsequent breast cancer tissue specimens.