Experimental Study On The Effect Of RNA Interfering MEX3A Gene Expression On The Proliferation And Apoptosis Of Bladder Cancer Cells

Objective:MEX3A gene is a newly discovered tumor-related factor,and its relationship with bladder cancer has not yet been clarified.The purpose of this study is to investigate the effect of inhibiting the expression of MEX3A gene on the proliferation and apoptosis of bladder cancer cells by lentivirus-mediated RNA interference.Methods:1.Select 5637 and T24 two kinds of bladder cancer cell lines,real-time quantitative PCR MEX3A gene expression in two cell lines,high expression for subsequent experiments.2.According to the design principles of RNA interference sequence design and select the appropriate RNA interference target sequence.3.Synthesis of interfering sequence containing single-stranded DNA oligo annealed to generate double-stranded DNA;followed by digested site directly connected to the digested lentiviral vector;the connection into the product of the prepared E.coli State cells,PCR identification of positive recombinant,sent sequencing validation,sequencing of the correct clones for plasmid extraction applications.4.After identified and sequenced,293T cells were co-transfected with the packaging vector to produce lentivirus particles.After the titer of the virus was determined,transfected into bladder cancer cells.MEX3A-shRNA lentivirus was transfected into the experimental group and negative control lentivirus was transfected into the control group.Antibiotic screening to establish stable expression of siRNA cell lines.5.Collect the cells of experimental group and control group and extract the total RNA,reverse transcription,real-time quantitative PCR detection MEX3A gene knockdown efficiency.6.Three days after lentiviral transfection,the experimental group and the control group were seeded in 96-well plates for 5 consecutive days,and the cells were counted and photographed daily using the FOV analyzer.7.Five days after lentiviral transfection,the cells in the control group and the experimental group were respectively collected.After induced by apoptosis,the cells were stained with Annexin V-APC,and the apoptotic cells were detected by flow cytometry.Results:1.MEX3A gene in bladder cancer 5637 and T24 cells are expressed,and the expression of 5637 is higher than T24,5637 cells were selected for follow-up experiments.2.The result of gel electrophoresis showed that the size of the positive cloned PCR fragment was 341bp and the size of empty vector cloned PCR fragment was 307bp,which was consistent with the expected value.Sequencing results showed that the recombined lentiviral vector was consistent with the designed targeting strand and the lentiviral vector was constructed successfully.3.By hole gradient dilution method to determine the virus titer of 2×10~9TU/ml,the virus packaging success.4.Real-time PCR results showed that the efficiency of knockdown was 74%.The lentiviral vector transfected 5637 cells could stably inhibit the expression of MEX3A gene.5.Whole-cell scanning analyzer cell count test showed that compared with the control group,the experimental group 5637 cell number and proliferation rate were significantly inhibited.6.Flow cytometry showed that compared with the control group,5637 cells in the experimental group apoptosis increased significantly.Conclusion:1.The application of lentivirus-mediated RNA interference technology can successfully establish a stable cell line MEX3A gene expression.2.Inhibition of MEX3A gene expression will reduce the number of bladder cancer cell proliferation and increase the number of bladder cancer cells,indicating that MEX3A gene positively regulates the proliferation of bladder cancer cells and negatively regulates the apoptosis of bladder cancer cells.