A Study On The Effect And Mechanism Of Recombinant Lentivirus Lenti-stat1/stat3-siRNA On The Apoptosis Of U251 Cells

Objective To investigate the effects of recombinant lentivirus lenti-stat1,lenti-stat3-siRNA and lenti-stat1/stat3-siRNA on the apoptosis of human glioma cell line U251 and the molecular mechanism.Methods The recombinant lentivirus lenti-stat1,lenti-stat3-siRNA and lenti-stat1/stat3-siRNA vectors were constructed and identified.The expression of STAT1 and STAT3 in human glioma U251 cells was detected by Real Time PCR and Western blot after infection with lentivirus expression vector.The proliferation of U251 cells was detected by CCK-8 method.The apoptosis and cell cycle of U251 cells were detected by flow cytometry.The effect of U251 cell migration ability was analyzed by cell scratch assay.The possible molecular mechanism was analyzed by RNA-Seq Results After infection with lenti-stat1 and lenti-stat1/stat3-siRNA,the mRNA and protein expression levels of STAT1 of U251 cells were significantly increased,After infection with lenti-stat3-siRNA and lenti-stat1/stat3-siRNA,the mRNA and protein expression levels of STAT3 of U251 cells were significantly decreased.The results showed that Stat1,Stat3-siRNA and its co-expressing Stat3-siRNA-Stat1 recombinant lentiviral vector were successfully established.Compared with the control group,after knocking out STAT3 and over-expressing STAT1,the proliferation and migration ability of U251 cells in each experimental group decreased significantly;the apoptotic rate and the number of G0-G1 cells were significantly increased.Among them,recombinant lentivirus lenti-stat1/stat3-siRNA had the most significant effect of U251 cells.RNA-Seq showed that recombinant lentivirus lenti-stat1,lenti-stat3-siRNA and lenti-stat1/stat3-siRNA could cause differential expression of U251 cells.Conclusion 1.Recombinant lentivirus lenti-stat1/stat3-siRNA can significantlyincrease the expression of STAT1 gene and inhibit the expression of STAT3 gene in U251 cells.2.Recombinant lentivirus lenti-stat1,lenti-stat3-siRNA and lenti-stat1/stat3-siRNA can effectively inhibit the proliferation and migration of U251 cells and induce apoptosis.The lenti-stat1/stat3-siRNA has the most pronounced effects,indicating that downregulation of STAT3 and upregulation of STAT1 have a significant synergistic effect against U251 cell growth.3.RNA sequencing analysis indicated that multiple genes of the FOXO signaling pathway were alterd by recombinant lentivirus lenti-stat1/stat3-siRNA,which could lead to the more effective apoptosis than monogenic lenti-stat1 and lenti-stat3-siRNA.Further research on the mechanism is warranted.