The Role Of DNA Methylation Of MiR-375 Gene Mediated By Folic Acid In Cervical Cancer

Objective:Cervical cancer is a malignant tumor, which cause serious damage to the women’s health. In our country,cervical cancer takes the second place in the ranking, and there is the high incidence in Shanxi Province. Cervical cancer is a process to happen,and cervical intraepithelial neoplasias is the precancerous lesions.So how to stop CIN go to cancer become a problem to be solved at present. As is known to all. the occurrence of cervical cancer is influenced by many factors, at present the latest research shows that microRNAs and folate deficiency can lead to cervical cancer, and they may be the coordination of HPV infection.But how they can lead to cervical cancer.there is not a conclusion.So the purpose of this study is to:1. To detect the expression levels of miR-375 in cervical intraepithelial neoplasia tissue and cervical cancer tissue, and discuss its role in the development of cervical lesions.2. Under different concentration of folic acid intervene,we detect the expression levels of miR-375 and study the relationship of folic acid and the expression levels of miR-375. Further more,we discuss its role in the development of cervical cancer.3. Under different concentration of folic acid intervene,we detect the DNA methylation states of miR-375 gene and study the relationship of folic acid and the DNA methylation states of miR-375 gene and to explore the molecular mechanisms of cervical cancer.This study provide the new idea for the drug treatment of the cervical cancer.Methods:1. Source of specimen We randomly collected patients, cervical tissues who diagnosed CIN(30cases) and cervical cancer (30cases) from May 2013 to January 2014 at The Second Hospital of Shanxi Medical University.When we take the cervical tissues in vitro.we wash the blood using normal saline immediately.then put them in freezing tubes. When specimen stored in liquid nitrogen tank were more than 1 hour.we take them out and store them into-80℃ refrigerator. In addition,we collected 30 patients, cervical tissues who diagnosed chronic cervicitis as matched group. All patients were signed informed consent.2. Detection the expression levels of miR-375 Depending on the pathological results, we divide the collected patients’cervical tissues into the CIN group, cervical cancer group and matched group. Total RNA was extracted from these specimen and the expression levels of miR-375 was detected by real-time PCR technology.3. Detection the DNA methylation states of miR-375 We cultured cervical cancer cell CaSki in vitro and intervened it with different concentration of folic acid,then extracted DNA and detected the DNA methylation states of miR-375 by methylation specific PCR.4. Results determined 10μL PCR products were obtained in 2% agarose gel electro-phoresis, Ethidium Brmide dyes, imager and camera observation. If methylation products were obtained, no matter whether non-methylated products were obtained or not, they were judged to be methylation-positive; If the products appear non-methylated only, when the result was non-methylated; If obtained neither the methylation products nor the non-methylated products, it marks the experiment was failed.5. Statistical analysis Using SPSS16.0software package for the chi-square test, P<0.05 for the difference was statistically significant.Result:1. miR-375 is significantly down-regulated in CIN tissue and cervical cancer tissue comparing to normal tissue. There was remarkable difference in terms of the expression levels of miR-375 between them (P<0.05)2. In cervical cancer cell.the positive rate of DNA methylation states of miR-375 were56.67%(17/30),56.67%(17/30),53.33%(16/30).46.67%(14/30), 36.67%(11/50),20.00%(6/30),6.67%(2/30) in each group,and showed a trend of decreasing with the concentration of folic acid increasing.3. In cervical cancer cell,the expression levels of miR-375 were increasing gradually with the concentration of folic acid increasing.4. In cervical cancer cell, the expression levels of miR-375 and miR-375 gene methylation states were negatively correlated.Conclusion:1. miR-375 is significantly down-regulated in CIN tissue and cervical cancer tissue comparing to normal tissue.2. DNA abnormal hypermethylation of miR-375 is important event during the development of cervical cancer. To reverse miR-375 hypermethylation status may be preventing cervical cancer.