Effects Of Lentivirus-mediated Knockdown Of MCM7 Gene On Human Leukemia Cells And Hepatocellular Carcinoma Cells

PART 1 Effects of Lentivirus-mediated knockdown of MCM7 gene on human leukemia K562 cellsBackground:MCM7 is one of the subunits of the MCM2-7 complex that are essential for DNA replication licensing and control of cell cycle progression. It also participates in mRNA transcription and the regulation of DNA damage. It has been reported that MCM7 was highly expressed in many cancer cells including leukemia cells. Thus, lentivirus-mediated siRNA targeting MCM7 was used to suppress its endogenous expression in K562 cells and develop a novel therapeutic strategy for leukemia.Method:Lentivirus-mediated siRNA targeting MCM7 was designed and infected into K562 cells.The level of tr MCM7 mRNA transcription and protein expression in K562 cells was determined by Real-time PCR and Western blot analysis. The proliferation of the transfected K562 cells was assessed by CCK8 assay. Cell cycle progression and apoptosis were calculated by Flow cytometry. The levels of related protein expression in the transfected K562 cells were detected by Western blot. Moreover, the stably transfected K562 cells were injected into nude mice. The transplanted tumors were measured every 4 days, then Western blot and Immunocytochemistry were used to evaluate the expression of the proteins for mechanism.Results:The mRNA transcription and protein expression of MCM7 in the stably transfected K562 cells decreased with the percentage of (92.3±1.88) % and (91.18±0.93) %respectively. Compared with control groups, the proliferative rate of K562 cells was significantly reduced after LV-MCM7-RNAi infection for 48h.The cell cycle progression of K562 cells was blocked, with a massive increase in the percentage of cells in the G0/G1 phase and a decrease in S phase. The apoptosis rate of transfected K562 cells was significantly higher.PLK1 and pPLK1 protein were down-regulated. P73 and bax protein were up-regulated simultaneously. In vivo, the tumor size and weight dramatically decreased in the MCM7-RNAi group than those in negative control group. Down-regulation of PLK1 and pPLK1 protein and up-regulation of P73 and bax protein were also detected in the transplanted tumor tissues .Conclusions:Lentivirus-mediated MCM7 shRNA has effectively down regulated the levels of MCM7 mRNA transcription and protein expression. Moreover, the silence of MCM7 has led to the significant decrease in proliferation, blocking the cell cycle progression of K562 cells in vitro.Simultaneity, the apoptosis of leukemia cells was enhanced. Both of the functions above cause the inhibition of tumor growth in nude mice. Overall, these results suggest that the proliferation and cell cycle of K562 cells could be regulated by silencing MCM7 gene which may be a promising gene therapeutic method to treat gastric cancer.PART2 Effect of MCM7 Gene Silencing by Lentivirus-shRNA on Human hepatocellular carcinoma MHCC-97H cellsObjectiveTo investigate the inhibition effect of silencing MCM7 Gene by RNAi on the biological behavior of MHCC-97H cells in vitro.MethodsLentivirus for RNAi of MCM7 containing a sequence of human MCM7 mRNA coding region were constructed, the other Lentivirus for RNAi of MCM7 containing a sequence of no significant and homology to human gene sequences was constructed, and transfected into MHCC-97H cells, respectively. There were control group, LV-NC-RNAi group and LV-MCM7-RNAi group in our study. The expression of MCM7 in the cells was detected by Realtime-PCR and Western blot after transfection . The effects of MHCC-97H cells proliferation were measured using MTT assay.The invasive ability was measured by Transwell experiment. The locomotion ability was measured byscrape assay.ResultsThe Lentivirus RNAi vector of MCM7 was constructed successfully.Compared with LV-NC-RNAi group and Control group cells, MCM7 mRNA and protein expression in LV- MCM7-RNAi group cells were down-regulated (P<0.05). MHCC-97H cells proliferation was inhibited including growth reduction, cell cycle arrest in G0/G1 phase and induction of apoptosis. In cell migration and cell invasion assay, compared with Control group and LV-NC-RNAi group, the interference number of MHCC-97H cells was significantly reduced(P<0. 05).ConclusionsBlocking the expression of_MCM7 gene can inhibit cell proliferation and decrease the cell invasiveness in MHCC-97H cell line. GO / G1 phase arrest and induction of apoptosis could be the underlying mechanism. MCM7 gene could be a potential therapeutic target to treat hepatoma.