Effects Of Lentiviral ShRNA Targeting Knockdown Mitochondrial Inner Membrane Protein(Mic19,Mic60)Expression On The Biological Effects Of Hepatocellular Carcinoma Cells

Objective The function of mitochondria depends on the proper organization of mitochondrial membranes.The morphology of the inner membrane is regulated by the recently identified mitochondrial contact site and crista organizing system(MICOS)complex.MICOS mutants exhibit alterations in crista formation,leading to mitochondrial dysfunction.The expression of MICOS(Mic19/CHCHD3 and Mic60/mitofilin)were detected by lentiviral shRNA targeting and the effects of knockdown mitochondrial inner membrane protein(Mic19,Mic60)on the biological function of hepatocarcinoma cells were studied.Methods The mitochondrial inner membrane proteins Micl9 and Mic60 sequences were searched by NCBI database.The recombinant lentivirus plasmid of shRNA Mic19-1?shRNA Mic 19-2 and shRNA Mic60-1?shRNA Mic60-2 were designed.Recombinant lentiviral plasmids were co-transfected into 293 T cells with packaging-supporting plasmids respectively,packaging caused virus,the virus packaged infected and HepG2 cells,the positive cells screened with puromycin were collected,the blank group and pLKO.1 lentivirus plasmid group were used as controls.The expression of Mic19 and Mic60 protein was detected by Western blotting to screen the stable cells with the highest efficiency.The effects of knockdown of Mic19 and Mic60 on the biological behavior of hepatocarcinoma cells were observed by MTT assay,Western blotting assay,Wound healing assay and Transwell assay.Results The shRNA Micl9-1(0.57910.042)?shRNA Mic19-2(0.524±0.111)were significantly lower than those in the blank group and the empty group,P<0.01.The shRNA Mic60-1(0.172±0.107)?shRNA Mic60-2(0.269±0.027)were significantly lower than the control groups,P<0.01.Western blotting showed that the relative expression of caspase3(1.34 ± 0.145)and caspase9(1.209 ± 0.173)protein in the shMicl9 was significantly higher than that in blank group(0.652±0.089)and empty group(0.671 ± 0.023),The difference was statistically significant,P<0.01;The relative expression of caspase3(1.453 ± 0.151)and caspase9(1.057 ± 0.11)protein in the shMic60 was significantly higher than that in blank group(0.45 ± 0.033)and empty group(0.471 ± 0.183),the difference was statistically significant,P<0.01.MTT assay showed that the absorbance(A)value of shMicl9 group was lower than the control groups at 36 h after infection,P<0.05;The A value of shMic60 group was significantly lower than the control groups at 12h,P<0.01.Wound healing assay showed that the migration distance of shMic19 and shMic60 group began to decrease from 24h,and the migration distance of shMicl9 group was(286.8 ± 38.97)?m at 48h,and the migration distance of shMic60 group was(214,6 ± 41.64)?m,compared with the control groups were statistically significant,P<0.01.In the Transwell assay with Matrigel,the number of transmembrane cells in the shMicl 9 was(440.4 ± 65.35)per low-power field,and the number of transmembrane cells in the shMic60 was(235.2 ± 21.3)were significantly lower than the control groups,P<0.01.In the Transwell assay without Matrigel,the number of transmembrane cells in the shMicl9 was(172.4 ± 15.5)per low-power field,and the number of transmembrane cells in the shMic60 was(118.2 ±25.53)were significantly lower than the control groups,P<0.01.Conclusion The recombinant lentiviral shRNA Micl9-2 and shRNA Mic60-1 could effectively knock down the expression of Mic19 and Mic60 in HepG2 cells.The expression of Mic19 and Mic60 could inhibit the proliferation,migration and invasion of HepG2 cells and promote the apoptotic ability of HepG2 cells,and provide a reference for the further study on the effect of mitochondrial membrane protein(Mic19,Mic60)on the development of hepatocellular carcinoma.