The Role Of MiR-26a In Uterine Leiomyoma And CpG Methylation Status Of Its Promoter Regulation Region

Background and objective: Uterine fibroids represent the most common benigntumors of the female genital common in30-50year and a frequent cause of abnormauterine bleeding,large pelvic masses, fibroids degeneration cause abdominal discomfortand recurrent pregnancy loss or infertility,that is a serious threat to the physical and mentalhealth of the majority of women.The estimate cumulative incidence rate of tumors is about20-30%,it reported that40-50year-old women with uterine fibroids incidence is as high as51.2%-60%. Uterine leimyoma flesh tumour evil change rate is from0.13%to1.39%.Because of its high incidence and, with the high number of incidence for this tumor,andyounger trend,management of uterine leimyomas and has place an enormous burden onthe health care system and cause a great impact on the economy and the long-termdevelopment of the society.However,the mechanism of its phthogenesis is not yet fullyunderstood.If we can have a new understanding of pathogenesis,it could provide new ideasfor therapy.Uterine fibroids are benign smooth muscle tumors which had been known to bedependent on ovarian steroid hormones, such as estrogen and progesterone, and drugtherapy mainly by inhibiting the expression of estrogen and progesterone receptors. In ourpreliminary experiments,we found that the expression level of miR-26a was significantlylower in uterine leiomyomas than myometrium tissues(P<0.05),and miR-26a coulddownregulate the expression of ERα，PRa.So we guess Whether the overexpression ofERα，PR a can enhance the biological effect of estrogen and progesterone in uterinefibroids by the abnormal downregulation of the miR-26a? Furthermore whether aberrantmethylation of the promoter CpG site cause miR-26a lower expression in uterine fibroidsthan normal myometrium? According to preliminary experimental results and elevationideas, we explore the pathogenesis of uterine fibroids by studying miR-26a gene effect onhormone and studying the methylation status in promoter CpG sites of miR-26a analysis inorder to get new insight about targeted therapy.Methods: We used collagenase typeⅠto digeste the uterine leomyoma tissue andobtained the uterine leomyoma cell by screen cloth separation,then identificated the purityby immunohistochemistry.Then we investigated the effects of miR-26a and hormone-based drug and theproliferation of uterine leiomyoma cell by Cell Counting Kit-8(CCK-8) assay afterculturing cells, transfecting cells and treating cells with17β-estradiol, progestrone, estrogen receptors α inhibitor and progesterone receptors a inhibitor.We found the miR-26a promoter sequences which the length was452bases had fourpotential methylation sites (30site,99site,283site and406site) by specific analysissoftware.Then we examined the hypermethylation status of four sites in6cases of uterinefibroid and6cases of normal myometrium by Bisulfate Sequencing PCR. We extractedDNA from the uterine fibroid and normal myometrium, treated DNA with bisulfate,purified after modifying DNA, then anaylyed10cloing sequences which are the productsof PCR to get the frequency of methylation. A total of12samples that were taken120clone sequenced120cycle to determine the CpG methylation frequency of the four sites.Results: The uterine leiomyoma cells could be obtained by using collagenasedissection,and the success rate of the method had reached up89%.Becauseimmunohistochemistry staining with ɑ-Actin demonstrated these cells were positive, Wedetermined that the purity of the isolated uterine leiomyoma cell was over96%.We found the transfection efficiency above75%when we transfected NC-mimic-FAM(Carboxyl fluorescein)into uterine leiomyoma cells.It show that the cell proliferationsof miR-26a mimic transfected uterine leiomyoma cells was slower than the control cellssignificantly (P<0.05),and the same as the cell that was treated by the estrogen receptorinhibitors or the progesterone receptor inhibitors; the cell proliferations of the miR-26ainhibitor transfected was promoted compared with the control cells,and the effect wasenhanced after treating with17β-estradiol or progestrone obviously (P<0.05). Takentogether,our findings shown that the low expression of miR-26a can strengthen the effectof17β-estradiol or progestrone on promoting the proliferation of uterine leiomyoma cells.It demonstrated that the bisulfite conversion efficiency is more than98%by BSP clonesequencing test,and the methylation status of Uterine fibroids no diffenrence with normalmyometrium. Furthermore we found the frequency of methylation in the uterine fibroidsand normal myometrium were73.33%±23.38,58.33%±24.83;68.33%±14.72,56.67%±19.66;70%±8.94,70%±12.65;51.67%±7.53,73.33%±16.33,by detecting the fourCG sites in the miR-26a promoter sequence, and in406site we significantly found adifference between uterine fibroids and normal myometrium which the frequency ofmethylation in uterine fibroids was lower(P<0.05).Conclusion: The human leiomyoma cell can be got through collagenase digestiontechnique and explants culture technique,which could apply the cells for study in vitro.Itdemonstrated that the low expression of miR-26a gene can strengthen the biological effects of17β-estradiol or progestrone on promoting the growth of uterine leiomyoma cells whichwere closely related to the chance that the low expression of the miR-26a gene canenhance the expression of ERα,PR a.The methylation analysis showed that the methylationstatus of the406site plays important in the occurrence of uterine leiomyoma,maybe it canprovide evidence to the pathogenyesis.