Study On The Function And Mechanism Of MicroRNAs In Cervical Intraepithelial Lesion Progression To Carcinoma

Cervical cancer is the second common gynecological tumors in the world, it is far higher incidence of cervical cancer in developing countries than that in others. It will progress through mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ and infiltrating carcinoma process from the normal tissue to cervical cancer. However, it is not inevitable that cervical intraepithelial lesion progression to carcinoma, only about 15-45% of cervical intraepithelial lesion will become cancerous,other will resume. The cervical intraepithelial lesion has long been excessive treatment clinically that due to a lack of marker for intraepithelial lesions of malignant transformation. Therefore, it is an important scientific problem of the research of cervical cancer that to identify and select the marker for malignant transformation.MicroRNAs(mi RNAs) are endogenous small non-coding RNA molecules, which functioned in many biologic processes through negatively regulating target mRNAs via resulting mRNA cleavage or repressing mRNA translation. It has been demonstrated that miRNAs playregulatory roles in diverse biological processes and the aberrant expression of miRNAs is found in many diseases especially in cancer. miRNAs have been reported to act as oncogenes or tumor suppressors in a variety of cancers, such as breast, pancreatic, lung, hepaticcancer and gastric cancer.Several innate properties of miRNAs, including small and stable against degradation and easily detected make them attractive as potential biomarkers. MiRNAs are also detectable in bodily fluids including plasma,serum, urine, saliva and tears. It is suitable to establish simple and quick diagnosis methods. It is also safer and easier to cure disease than other drugs as its small molecules and easy synthesis. MiRNAs help us open a new vision of understanding cancer progression and have huge potential as therapy targets and biomarkers.Firstly, we examined the miRNA expression profiles in 2 cervical cancer and normal cervical tissues using miRNA microarray and identified many aberrant expressed miRNAs in cervical cancer, in which there are many known oncogenic or tumor suppressive miRNAs. According to microarray results, we further examined the expression of 10 differentially expressed miRNAs, including miR-10 b, miR-193 b, miR-23 b, miR-29 a,miR-375, miR-320 a, miR-21, miR-9, miR-15 a, miR-16 in 40 cervical cancer and 30 normal cervical tissues using Taqman probe based q-PCR.The results showed that the expression of miR-10 b, miR-193 b and miR-375 was decreased clearly in cervical cancer tissues. The expressionof miR-320 a, miR-21, miR-15 a and miR-16 was dramatically up-regulated in cervical cancer tissues. To further explore the possible role of these miRNAs during cervical cancer progression from cervical intraepithelial lesion, we examined the expression of the above seven miRNAs in 30 mild dysplasia, 32 moderate dysplasia, 37 severe dysplasia, 35 carcinoma in situ,40 cervical cancer and also 30 normal cervical tissues. The results showed that miR-10 b decreased gradually during cervical cancer progression significantly, while miR-21 increased during this process, suggesting that miR-10 b and miR-21 played important role in cervical carcinogenesis.Moreover, the lower level of miR-10 b was positive correlation with malignant transformation, hinting its potential application in cervical cancer early diagnosis.To investigate the role of miR-10 b in cervical carcinogenesis, we re-introduced miR-10 b mimics into two cervical cancer cell lines, Hela and SiHa. As a result, the overexpression of miR-10 b in both of the two cervical cancer cell lines can inhibit cell proliferation, migration and invasion significantly, while promote cell apoptosis. These results indicated that miR-10 b played important roles in cervical carcinogenesis as a tumor suppressor. Meanwhile, we introduced miR-10 b inhibitors into the above two cervical cancer cell lines to suppress the endogenous miR-10 b expression. As a result, the suppression of miR-10 b in both of the two cervical cancer cell lines can promote cell proliferation, migration andinvasion significantly, while suppress cell apoptosis. These results demonstrated that miR-10 b functioned as a tumor suppressor in cervical carcinogenesis and down-regulation of miR-10 b might be important risk of cervical carcinogenesis.We further dissected the mechanism by which miR-10 b functioned as a tumor suppressor in cervical cancer. miR-10 b was predicted to bind to the3′-UTR region of HOXA1 using two algorithms Pic Tar and Target Scan. To validate the negative regulation of miR-10 b on HOXA1, we cloned the 3′-UTR of HOXA1 into a luciferase reporter construct. The luciferase assay showed that miR-10 b mimics repressed the luciferase activity of HOXA13′-UTR significantly and the repression was dependent on the miRNA binding site. Consistent with the reporter assay, we observed an evident decrease of HOXA1 protein in presence of miR-10 b mimics compared to scramble control in both of Hela and SiHa cells. Knockdown of HOXA1 can significantly inhibit cervical cancer cell proliferation, migration and invasion and the rescue experiments also improved that miR-10 b played its tumor suppressive role in cervical cancer through targeting HOXA1. The investigation of HOXA1 in cervical cancer clinical samples indicated that HOXA1 was significantly up-regulated in cervical cancer tissues compared with the normal controls. These results indicated that HOXA1 might play important roles in cervical cancer as an oncogene. The aberrant down-regulation of miR-10 b in cervical cancer cell can give rise toup-regulation of HOXA1 and further to promote cervical cancer progression.Then we asked that what give rise to the down-regulation of miR-10 b in cervical cancer? DNA methylation was the main reason of gene silencing.So we analyzed DNA methylation level of the promoter of miR-10 b and found that the methylation level was clearly higher in cervical cancer tissues than that in normal controls. In addition, demethylation drug treatment led to up-regulation of miR-10 b in cervical cancer cells,suggesting that hypermethylation of miR-10 b promoter is the main reason of its down-regulation in cervical cancer. So demethylation drugs can re-activate a group of tumor suppressive miRNAs, and then suppress cancer progression.Taken together, we identified many miRNAs participating in cervical cancer progression and revealed the function and mechanism of miR-10 b in suppressing cervical carcinogenesis. Our work provides new thought and theory basis for monitoring and intervention for intraepithelial lesions of malignant transformation.