Effect Of Transgelin On The Biological Behavior Of HBV-positive Hepatocellular Carcinoma Cells And Its Mechanism

Objective:To explore the effects of transgelin(TAGLN)on the biological behavior of HBV-positive hepatocellular carcinoma cells and its possible mechanism.Methods:Part I:1.Western blot was used to detect the difference of TAGLN expression between HBV positive hepatocellular carcinoma cells(HepG2.2.15)and HBV negative hepatocellular carcinoma cells(HepG2);2.Immunohistochemistry was used to detect the expression of TAGLN in HBV-positive and HBV-negative hepatocellular carcinomas.Part?:1.The shTAGLN lentivirus plasmid was constructed by three plasmid systems and sequenced;The plasmid was divided into control group shNC and the target genomes were shTAGLN1~#and shTAGLN2~#.2.293T cells were co-transfected with shNC,shTAGLN1~#and shTAGLN2~#plasmids and auxiliary plasmids.After 72 hours of culture,a large number of uneven green fluorescence was observed under inverted fluorescence microscopy.Cell supernatants were collected and packaged.Viral titers of shNC and shTAGLN lentiviral solution were determined by dilution counting method.3.HepG2.2.15 cells were infected with shTAGLN lentivirus.After 72 hours of culture,the expression of fluorescence was observed under inverted fluorescence microscopy to determine the optimum plural infection(MOI)of HepG2.2.15 cells infected with shTAGLN.4.HepG2.2.15 cells were infected with shTAGLN lentivirus.After 12 hours,they were replaced with fresh complete medium and cultured for 72 hours;shNC-2.15,shTAGLN1~#-2.15 and shTAGLN2~#-2.15 cells were screened for 2-3 times in a complete medium containing 10ug/mL puromycin.The expression of TAGLN in the cells of each group was detected by Western blot after 15 days.Part?:1.The effects of shTAGLN on the proliferation of HepG2.2.15 cells were detected by CCK-8 method and clone formation assay;2.Transwell assay was used to detect the effect of shTAGLN on the migration and invasion of HepG2.2.15 cells;3.Western blot was used to detect the effect of shTAGLN on PI3K,p-PI3K,AKT and p-AKT protein expression in HepG2.2.15 cells.Results:1.The expression of TAGLN in human HBV-positive hepatocellular carcinoma tissues and cells was higher than that in HBV-negative hepatocellular carcinoma tissues and cells(P<0.01);2.Sequencing results showed that the TAGLN sequence of the recombinant plasmid was identical with that of human TAGLN sequence in GenBank,suggesting that shTAGLN lentivirus plasmid was successfully constructed;Lentiviral titer test results:shNC virus titer was 1?10~9 TU/mL,shTAGLN1~#and shTAGLN2~#virus titer was 8?10~8 TU/mL;The optimum MOI value of HepG2.2.15 cells infected with shNC was 80,and that of HepG2.2.15 cells infected with shTAGLN1~#and shTAGLN2~#was 60;The expression of TAGLN in shTAGLN1~#-2.15 and shTAGLN2~#-2.15 cell lines was inhibited(P<0.01)?3.shTAGLN inhibited the proliferation and cloning of HepG2.2.15 cells,and reduced their migration and invasion ability(P<0.01),shTAGLN inhibited the expression of AKT,p-AKT,PI3K and p-PI3K proteins in HepG2.2.15 cells(P<0.01).Conclusion:1.Compared with HBV-negative hepatocellular carcinoma,TAGLN expression increased in HBV-positive hepatocellular carcinoma tissues and cells.2.Interference with TAGLN expression can inhibit the proliferation of HepG2.2.15cells and weaken their ability of migration and invasion.The mechanism may be related to the decreased expression of AKT,p-AKT,PI3K and p-PI3K proteins.