The Study Of MiRNA And Its Target On HPV16

IntroductionHuman Papilloma Virus are a group of small non-enveloped double-stranded circular DNA virus. Basic research and epidemiological evidence demonstrate that the infection of HPV, especially high-risk HPV is necessary for the occurrence of cervical carcinoma. Cervical cancer is one of the most common life-threatened cancers among women worldwide, with a morbidity of490thousand and a mortality of270thousand per year. At present, over200the genotypes of HPV have been discovered. And according to the tumorigenic ablity, they can be divided into3categories:high-risk types, potential types and low-risk types. Through a large number of clinical epidemiology surveys reveal that more than99%samples of cervical cancer contain HPV DNA. Among them, HPV16is the most closely associated to the tumor incidence with the strongest tumorigenic ability, as a main type to result in cervical squamous cell carcinoma.MicroRNAs are a group of endogenously expressed non-coding small RNA with a length of19-25nucleotides. Being an important component of eukaryotic gene regulation, miRNA exists in plants, nematodes and human cells. MiRNA regulates diverse cellular pathways and participates in a series of life activities, including growth, proliferation, apoptosis, cell metabolism and hormone secretion. Evidence in recent studies shows that miRNA is involved in the pathogenesis of malignant tumor. Besides, the abnormal expression of miRNA is found in various tumor tissues. Through analysis of the tumor related gene sequences,52.5%miRNAs are found located near the tumor related regions or fragile sites, which indicates that miRNA plays an important role in the mechanism of human tumors. Some scholars hold the opinion that the function of miRNAs with abnormal expression might be similar to oncogenes or tumor inhibitor genes. These miRNAs regulate the expression of the genes which are related to cell cycle progression, differentiation and apoptosis. In that way, they play vital roles in tumor angiogenesis, proliferation, apoptosis, invasion and metastasis.Large-scale studies in recent years show that miRNA played an important role in breast cancer, lung cancer, gastric cancer, liver cancer, prostate cancer, pancreatic cancer, colon cancer and some other malignant tumors. The present research also show that abnormal expression of miRNA exists in cervical cancer especially in HPV related cervical cancer. In HPV positive cervical cancer cell lines, the miRNAs with abnormal expression can affect the cell proliferation, apoptosis and protein expression if they are up-regulated or down-regulated. Sequence analysis reveals that miRNA appears near the HPV integration site much more frequently than near the non-integration sites. And this phenomenon is more common in HPV16.The relationship between miRNA and virus gradually attract the attention of people, which result in further studies on the function of miRNAs in the infection process.In chronic infection of HCV and HIV, it has been confirmed that miRNA can derectly combine with the noncoding regions of the virus which leads to proliferation and permanent existence of the virus. In hepatitis B, miRNA has been proved to regulate the expression of HBV gene as well as lead to the occurrence of liver cirrhosis and hepatocellular carcinoma.It is supposed whether there is an miRNA which can directly combine with the target in HPV16or not.In the early stage of our experiment, we obtain the complete sequences of HPV16and human endogenous miRNA sequence from the database, and predict the combinging target through bioinformatics technique. We use MiRanda, TargetScan, RNAhybrid and some other software to predict the probable binding site between HPV16and miRNAs. In addition, we focus on the alternative splicing region of HPV16, the most valuable site. The results prove that there are four miRNAs, hsa-miR-576-5p, hsa-miR-875-5p, hsa-miR-3144-3p and hsa-miR-199a can combine with the alternative splicing region of HPV16E6.Our study mainly screens the miRNA predicted by bioinformatics analysis and test their target by using the Luciferase Report System. The miRNAs with positive result are transfected into HPV16positive cervical cancer cell line SiHa cell, so that the following cytological experiments can be carried on. We observe the influence caused by miRNA in SiHa cell on the proliferation, cell apoptosis and the expression of oncogene, in order to explore the possible mechanisms involved.Part Ⅰ The establishment of miRNA plasmid and the luciferase reporter plasmid of the target sequenceObjective To establish the miRNA plasmid and the, luciferase reporter plasmid vector of the target sequence as a preparation for the following investigation.Method Select pSilencer4.1-CMV, the stable and efficient expression plasmid for miRNA, as the vector. Select psi-CHECK-2to construct the target vector. Amplify both miRNA and the target sequence, recombine plasmid DNA, transfect them into competent cells, culture transformed strains, and extract the plasmid.Result The sequencing result of miRNA extract plasmid revealed the insert sequence was correct.Conclusion The expression plasmids and the luciferase reporter plasmid were successfully constructed, which contains ther miRNA and its target, respectively.Part Ⅱ Verification of the combination between miRNA and the target by Dual Luciferase Report SystemObjective To test whether the four miRNAs could be able to combine with the relevant target.Method Transfect the miRNA and the target plasmid into HPV negative cervical cancer cell line C-33A cell. Use Dual Luciferase Report Assay System E1910to test the Luciferase activity of the cell24hours after transfection.Result After PSILENCER4.1-miR199a-CMV and PSILENCER4.1-miR576-CMV are transfected into C-33A cells together with their target luciferase report plasmid PSICHECK-2-HPV16-5, PSICHECK-2-HPV16-6, PSICHECK-2-HPV16-2, and PSICHECK-2-HPV16-3, the luciferase activity in test group didn’t significantly decrease (P>0.05) compared with the control groups; after PSILENCER4.1-miR875-CMV and PSILENCER4.1-miR3144-CMV are transfected into C-33A cells together with their target luciferase report plasmid PSICHECK-2-HPV16-4and PSICHECK-2-HPV16-1, the luciferase activity in test group significantly decreased (P<0.001) compared with the control groups.Conclusion Among the four miRNAs predicted by bioinformatics, miR-875and miR-3144were testified to combine with their target, HPV16-4and HPV16-1, respectively.Part Ⅲ Effects of miR-875and miR-3144on the cell apoptosis, proliferation and the expression of oncogenes in SiHa cellObjective To investigate whether the up-regulation of miR-875and miR-3144will affect the proliferation, apoptosis and the expression of oncogenes in HPV16positive cell strain SiHa cell.Method Culture HPV16positive cervical cancer cell line SiHa cells in vitro and transfect PSILENCER4.1-miR875-CMV and PSILENCER4.1-miR3144-CMV into SiHa cells. Observe the morphological changes of the cells24hours after transfection. Observe the apoptosis rate by using Annexin V-FITC/PI double staining through flow cytometer and Hoechst straining. Detect the cell proliferation and draw the cell growth curve using Roche xCELLigence RTCA DP cell analyzer. Extract total RNA of SiHa cells after transfection, and detecte the expression of E6mRNA by RT-PCR.Result Compared with the control group and the negative control group, the apoptosis rate of the test group which was transfected PSILENCER4.1-miR875-CMV and PSILENCER4.1-miR3144-CMV significantly increased in Annexin V-FITC/PI double staining through flow cytometer (P<0.05) and Hoechst straining result(P<0.001) respectively. In the detection of the cell proliferation by Roche xCELLigence RTCA DP cell analyzer, the Cell Index of the test group decreased significantly (P<0.05) compared with the control group and negative control group while the cell growth curve is obviously inhibited in test group. The results of RT-PCR display, the expression of E6mRNA decreased significantly in test group (P<0.05).Conclusion Up-regulation of miR-875and miR-3144can promote the cell apoptosis SiHa cell, inhibit the cell proliferation, and reduce the expression of E6mRNA.Conclusions1. MiR-875and miR-3144are proved to be able to combine with their respective target, HPV16-4and HPV16-1.2. Up-regulation of miR-875and miR-3144can promote the cell apoptosis SiHa cell, inhibit the cell proliferation, and reduce the expression of E6mRNA.