The Role And Molecular Mechanism Of MiR-195 In Cervical Cancer Carcinogenesis And Development

BackgroundCervical cancer is one of the most common female reproductive cancers worldwide. The incidence of cervical cancer ranks second in female cancers, which is next to breast cancer. Cervical cancer is the fifth leading cause of cancer death in female worldwide. According to statistics, there are about 50 million new cases of cervical cancer around the world each year, accounting for 5% of all new cases of cancers. Nearly 270,000 patients died of cervical cancer each year. New cases of cervical cancer are 13-15 million in China, which is roughly one-third of the global total incidence. In China, about 50,000 people died of cervical cancer. The main pathological types of cervical cancer are squamous cell carcinoma and adenocarcinoma. The progress of cervical cancer is a very complex process, which is regulated by deregulation of gene functions at multiple stages. However, the mechanism underlying the development of cervical cancer remains elusive. It is known that high-risk human papillomavirus (HPV) infection is the cause of cervical cancer. However, women infected with HPV will not necessarily develop cervical cancer. Further, not all patients with precancerous lesions or with cervical cancers finally develop into invasive cervical cancer. This indicates that the development of cervical cancer is a complex process. In addition to human HPV virus, there are other factors contribute.to the development of cervical cancer. Therefore, the molecular mechanisms underlying the progression of cervical cancer need to be studied.MicroRNAs (miRNA) are 20-25 nucleotides RNAs that regulate gene expression at post-transcriptional level by binding to its target mRNAs for translational repression or mRNA cleavage. It was reported that miRNAs play important roles in a series of biological processes including cell proliferation, differentiation, apoptosis, and development. Abnormal expression of miRNAs happens in diseases such as diabetes, cardiovascular diseases, and rheumatoid arthritis. Recent studies have shown that miRNAs are deregulated in a variety of human cancers, including cervical cancer, and play key roles in the processes of cancer initiation, progression, and metastasis. Some miRNAs act as oncogenes while others act as tumor suppressor genes. MiRNAs and their target genes work coordinately to regulate tumorigenesis and tumor development. It was also reported that abnormal expression of miRNAs in the plasma of cancer patients and in cancer tissue is tightly correlated with tumor carcinogenesis, development, metastasis, and drug tolerance. Therefore, miRNAs can act as biomarkers for cancer diagnosis. Studies have shown that microRNA-195 (miR-195) acts as a putative tumor suppressor in a variety of cancers such as non-small cell lung cancer, and breast cancer, etc. However, the function of miR-195 in cervical cancer development is unknown.To study the role of miR-195 in cervical cancer development, in the first part of the thesis, we detected the expression level of miR-195 in cervical cancer tissue, and analyzed its correlation with cervical cancer carcinogenesis, development and metastasis. Data demonstrated that miR-195 expression is negatively correlated with cervical cancer development and metastasis. Cell proliferation and metastasis are two key steps during the development of cervical cancer. The proliferation of cancer cells are closely related with tumor growth, while cancer cell invasion, migration determine cervical cancer metastasis. Therefore, in the second part of this thesis, we used in vitro cell culture model to detect the effect of miR-195 on the proliferation, invasion and migration of cervical cancer cell HeLa. After determining the effect of miR-195 on HeLa cell behavior, in the third part of this thesis, we explored the mechanism by identifying cyclinD2 (CCND2) and MYB as miR-195 target genes.Cell proliferation and cell cycle are closely related, and cyclinD2 (CCND2) is a very important cyclin. CCND2 forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. Studies have shown that CCND2 is abnormally expressed in human brain glioma, gastric cancer, and other tumors. MYB is a widely expressed transcription factor. It plays a major role in cell differentiation and proliferation through regulating gene expression. Studies have shown that MYB is highly expressed in neuroblastoma tissue and breast cancer tissue, and its expression is related with prognosis. Therefore, it is an important indicator of prognosis. It is also reported that MYB protein is closely related with tumor invasion and metastasis. Therefore, CCND2 and MYB are two important tumor-promoting protein. Our data showed that miR-195 binds to the 3'-UTRs of CCND2 and MYB gene and thereby inhibits their expression. There results elucidate the mechanism underlying how miR-195 inhibits HeLa cell proliferation, invasion and migration.The basic framework and main contents of this thesis are shown in figure 1. and the detailed flowchart and techniques of each part will be further elaborated in the paper.Part I:The expression level of miR-195 in cervical cancer tissue Objective:To detect the expression of miR-195 in cervical cancer tissues and adjacent normal tissues, and to analyze the correlation between miR-195 with cervical cancer carcinogenesis and metastasis.Methods:To establish the association of miR-195 and cervical cancer development, we first assessed the miR-195 expression levels in 69 paired samples of cervical cancers and their adjacent samples. RNA was extracted from cancer tissue and normal tissue. and miR-195 expression level was detected by real-time fluorescent quantitative PCR. The relationship between the expression of miR-195 and cervical cancer development and lymph node metastasis was then analyzed.Results:1. The results showed that miR-195 expression was significantly down-regulated in cervical cancer tissue compared to adjacent non-tumor tissue. These data suggested that miR-195 expression is negatively correlated with cervical cancer development.2. The results showed that miR-195 expression was significantly down-regulated in lymph node metastatic cervical cancer compared to non-metastatic cervical cancer. These data suggested that miR-195 expression is negatively correlated with cervical cancer metastasis.Conclusion:1. The miR-195 expression was significantly down-regulated in cervical cancer tissue compared to adjacent non-tumor tissue.2. The miR-195 expression was significantly down-regulated in lymph node metastatic cervical cancer compared to non-metastatic cervical cancer.Part ?:The effect of miR-195 on the proliferation, migration and invasion of HeLa cellsObjective:To study the effect of miR-195 on the proliferation, migration, and invasion of HeLa cells.Methods:In this study, mir-195 and control RNA were transfected into HeLa cells and cell proliferation was detected. Cell cycle was further detected by flow cytometry. Wound healing model was used to detect the effect of miR-195 on HeLa cell migration. Matrigel-based transwell study was used to detect the effect of miR-195 on HeLa cell invasion.Results:1. Compared with the control group, the proliferation rate was significantly decreased in the cells transfected with miR-195. At day5, the cell number of control group is (14.6±0.8)×105, and the cell number of miR-195 group is (10.4±1.0)×105, which shows significant difference (P< 0.05). There data indicated that miR-195 inhibits HeLa cell proliferation.2. Cell cycle distribution was detected using flow cytometry. The data showed that the HeLa cells transfected with miR-195 had increased numbers of cells in the G0/G1 phase (49% in control group vs 60% in miR-195 group) and decreased numbers of cells in the S phase (41% in control group vs 30% in miR-195 group). These data suggested that miR-195 induces G0/G1 arrest in HeLa cells.3. The data showed that the migration capacity was significantly decreased in the miR-195-expressing cells compared to control cells.4. The Matrigel invasion assays showed that the HeLa cells transfected with miR-195 displayed a markedly decreased invasion ability compared with control cells.Conclusions:1. miR-195 is a tumor suppressor miRNA in cervical cancer, which inhibits the proliferation, migration, and invasion of HeLa cells.2. miR-195 could be a new potentisl target for cervical cancer therapy.Part ?:Identification of CCND2 and MYB as miR-195 functional target genesObjective:To identify miR-195 target genes in HeLa cells, thereby to clarify the mechanism of miR-195 role in regulating HeLa behaviors.Methods:Using online bioinformatics tools, including TargetScan, DIANA microT, and PicTar, it was found that a series of 3'-UTRs of human genes contained potential miR-195 binding sequences. Two of them, CCND2 and MYB, were closely associated with the cell cycle and cell migration. Therefore, they were picked for further analysis. To determine whether CCND2 and MYB are miR-195 target genes in HeLa cells, the HeLa cells were transfected with miR-195 or control RNA, and the expression of CCND2 and MYB was detected by quantitative PCR and immunoblotting. Further, the 3'-UTRs of the CCND2 and MYB genes were cloned downstream of luciferase gene and the luciferase activity was detected in the presence and absence of miR-195. Finally, CCND2 and MYB were expressed in miR-195 expressing cells and cell proliferation, migration and invasion were detected.Results:1. Using bioinformatics analysis and literature search, we finally selected CCND2 and MYB genes for validation.2. Real-time qPCR data showed that CCND2 and MYB mRNA significantly decreased after miR-195 transfection in HeLa cells. The immunoblotting data showed that the protein levels of CCND2 and MYB protein significantly decreased after miR-195 transfection. The results showed that miR-195, but not the control RNA, specifically decreased the luciferase levels from the luciferase reporters with 3'-UTRs of the CCND2 and MYB genes, suggesting that miR-195 regulates the expression of CCND2 and MYB directly.3. In miR-195 expressing cell, overexpression of CCND2 significantly rescued cell proliferation but not cell migration and invasion.4. In miR-195 expressing cells, overexpression of MYB significantly rescued cell proliferation, migration and invasion.Conclusion:1. miR-195 directly binds to the 3'-UTRs of CCND2 and MYB genes, and inhibits their expression.2. miR-195 inhibits cervical cancer proliferation, migration, and invasion by suppressing the expression of CCND2 and MYB genes.