Lentivirus-Mediated Lower Expression Of MRPL35 On The Effects Of Colorectal Cancer Cell HCT116 And Its Molecular Mechanisms

Objective:To investigate the effects of lentivirus-mediated lower expression of mitochondrial ribosomal protein large subunit L35（MRPL35）on the effect of colorectal cancer cells and its molecular mechanisms.Methods:1.Lentiviral infection of colorectal cancer cells.Infecting the human colorectal cancer cells HCT116 with shRNA-Control（NC）lentivirus and shRNA-MRPL35 lentivirus.Then collecting cell precipitates to extract RNA and protein.The expression of MRPL35 was detected by quantitative RT-PCR and the Western blot.2.Effects of knockdown the expression of MRPL35 on the function of colorectal cancer cells.The cells apoptosis was detected by using flow cytometry.And the expressions of cleaved-caspase 3,cleaved-caspase 9,Bax and cleaved-PARP which related to cell apoptosis were detected by the Western blot.The abilities of proliferation,invasion and migration of HCT116 cell were detected by MTT,Matrigel invasion and Wound healing assays.3.Molecular mechanisms of apoptosis of HCT116 cell by knockdown of MRPL35.The changes of mitochondrial reactive oxygen species（ROS）and mitochondrial membrane potential were observed by fluorescence staining and flow cytometry,the expression of JNK,ATM and P53 related to apoptosis was detected by Western blot.The expression of P53 was detected by using the plasmid of GV147/MRPL35^+/+and the vector GV147 transfected cell lines.Results:1.The expression levels of RNA and protein of MRPL35 were obviously lowered in comparison with the control cell group.2.The apoptosis rate obviously increased in cells of shRNA-MRPL35 group compared with the control group,while the abilities of invasion and migration weren’t changed.3.Cells of shRNA-MRPL35 group showed an increasing trend of mitochondrial ROS and the decreased drift of mitochondrial membrane potential.JNK and ATM were activated and the expression level of P-P53 was also significantly increased.While the expression of P-P53 was rescued by transfecting the plasmid of GV147/MRPL35^+/+.Conclusion:Downregulated expression of MRPL35 could increase mitochondrial ROS and decrease the mitochondrial membrane potential.Then excess ROS activated c-Jun N-terminal kinase（JNK）and resulted in DNA damage accumulation to activate ATM,which in turn activated P53 transcription factor to initiate the mitochondrial apoptosis pathway to promote colorectal cancer cell apoptosis,but had no effect on the ability of invasion and migration.