The Biological Function And Molecular Mechanism Of Long Non-coding MALAT1 In Cervical Caicinogenesis

Cervical cancer is one of the most widely attention gynecological malignancies in the world.The incidence and mortality of cervical cancer are always high and increasing in young women in developing countries.The persistent infection of high-risk human papilloma virus（HR-HPV）is the most important factor,but the occurrence of cervical cancer is still a multi-factor,multi-step complex process of gene dysregulation.The early-gene of HPV E6 and E7 have been confirmed to promote the oncogenesis of cervical cancer.It’s one of the mechanisms of cervical cancer that E6 binding to the cellular protein p53 impedes cell apoptosis and promotes cell immortalization.E6 has also been shown to activate telomerase,which is most closely related to immortalization,and maintain its expression.Human telomerase reverse transcriptase（h TERT）is the catalytic subunit of the telomerase complex and is the major limiting factor for telomerase.In recent years,along with the development of high-throughput sequencing chip technology,long non coding RNA（lnc RNA）is a hot topic.Many studies have confirmed that lnc RNAs are involved the normal and abnormal pathophysiological processes of our body.Lnc RNA is closely related to a variety of diseases including tumors and function on the progress and prognosis of cancers through epigenetics,transcription,and post-transcriptional regulation.Among the selected lnc RNAs,metastasis-associated lung adenocarcinoma transcript 1（MALAT1）was first discovered in non-small cell lung cancer.Later,it was also confirmed that MALAT1 is highly expressed in many tumor tissues,which is including cervical cancer.However,the study of MALAT1 in cervical cancer is rare and its mechanism is still unclear.Therefore,using E6 as an entry point,this study will focus on the interaction of MALAT1 among molecular mechanism in the carcinogenesis of cervical cancer.We used small interference RNA technology to down-regulate the expression of MALAT1,then construct a plasmid carrying MALAT1 and package it with lentivirus,then transfect He La/Ca Ski cell and in the end harvest the cells that are stable and persistently low expression MALAT1 gene.The expression of h TERT and E6 at the level of m RNA and protein were detected by real-time fluorescence quantitative PCR and Western blot.Then we explored the possible molecular mechanisms among MALAT1,E6 and h TERT in cervical cancer and validated the influence of cell biological function in cervical cancer with vitro experiments.The study includes the following two parts:Part Ⅰ Construction and Identification of a plasmid expressing small hairpin RNA against MALAT1 gene in He La/Ca Ski CellsObjective: To construct plasmid expressing small hairpin RNA（shRNA）to knockdown expression of metastasis-associated lung adenocarcinoma transcript1（MALAT1）,and to identify the expression of MALAT1 and observe its effects among molecular biology.Methods: The MALAT1 gene sequence（Gene ID:378938 serial number: NR₀02819）was searched in Gen Bank and a specific sequence of sh RNA targeting MALAT1 was designed.The sh RNA-MALAT1 was cloned into the lentivirus vector plasmid,then the accuracy of sh RNA-MALAT1 was validated by DNA Sequencing.The plasmid was packaged,the resulting lentivirus including the recombinant lentivirus and two other helper plasmid was transfected into He La and Ca Ski cervical cancer cells.Cells were divided into blank control group（He La/Ca Ski cells not infected with virus）,negative control group（He La/Ca Ski cells infected with empty virus sh NC）and MALAT1 interference group（He La/Ca Ski cells infected with sh RNA-MALAT1 lentivirus）.The expression of green fluorescent protein（GFP）was used to estimate the efficiency of transfection.Real-time fluorescent quantitative PCR was used to verify the expression level of MALAT1 m RNA after transfection.The m RNA and protein levels of h TERT and E6 in cervical cancer cells were detected by RT-PCR and Western blot after successfully established si-MALAT1 cell model.Results: The plasmid expressing sh RNA targeting MALAT1 was successfully constructed and used to generate recombinant lentivirus.The m RNA level of MALAT1 in He La and Ca Ski cells transfected with sh RNA MALAT1 recombinant lentivirus were significantly lower than other two controls（p<0.01）.The difference between the two control groups was not statistically significant（p>0.05）.The expression of h TERT and E6 m RNA in He La/Ca Ski cells of si-MALAT1 group were also significantly lower than that of the two control groups（p<0.01）.So as the protein level of h TERT and E6 detected by Western blot in three groups.Conclusion: Stable lentiviral vector-mediated si RNA silencing of MALAT1 in He La and Ca Ski cell lines were successfully constructed,which could be a foundation for further investigating the potential function of MALAT1 in the pathogenesis of cervical cancer.MALAT1,E6 and h TERT may interact with each other and play a vital role in the carcinogenic mechanism for cervical cancer.Part Ⅱ Biological effect of silencing MALAT1 gene by lentivirus-mediated sh RNA in He La/Ca Ski cellsObjective: To explore the effect of silencing MALAT1 expression on the biological function of cervical cancer He La/Ca Ski cells.Methods: Based on the first part,the experiments were divided into blank control group（He La/Ca Ski cells not infected with virus）,negative control group（He La/Ca Ski cells infected with empty virus sh NC）and si-MALAT1 group（He La/Ca Ski cells infected with si-MALAT1 lentivirus）.CCK-8 cell proliferation assay,colony formation assay,wound healing assays,Transwell assay were performed to detect the effects on cell growth,proliferation,migration and invasion in vitro after downregulate the expression of MALAT1.Flow cytometry analysis was used to observe the change of cell cycle and cell apoptosis.Results: The cell growth,proliferation,migration and invasion of He La/Ca Ski cells in si-MALAT1 experiment group were all significantly lower than those in the negative control group and the blank group（p<0.05）.The difference between the two control groups was not statistically significant（p>0.05）.The ratio of G0/G1 phase cells in the si-MALAT1 experimental group was higher than that in the negative control group and the blank group（p<0.05）;the ratio of S phase cells in the si-MALAT1 experimental group was significantly lower than those of two controls（p <0.05）.The apoptotic rate of He La/Ca Ski cells in si-MALAT1 experiment group was significantly higher than that of the negative control group and the blank group,and the difference was statistically significant（p<0.05）.Conclusion: MALAT1 is involved in the regulation of biological behavior of human cervical cancer He La/Ca Ski cells in vitro: silencing MALAT1 could down-regulate the expression levels of m RNA and protein of h TERT and E6 genes in He La/Ca Ski cells,inhibit cell growth and proliferation,weaken migration and invasion ability of cells,arrest the cell cycle in the G0/G1 phase and induce apoptosis in cancer cells.