Effect Of TSC2 Gene Expression Downregulation By Lentivirus Induced Rna Interference On U937 Cell Line And Its Mechanism

Objective:To investigate the influence on growth,proliferation,differentiation and apoptosis of leukemia cell lines and the mTORC1 pathway activity for down-regulated expression of TSC2 gene on U937 leukemia cells.Methods:Gene expression was down-regulated by Lentivirus induced RNA interference on TSC2 high expressed U937 cell line;the cell proliferation was detected using CCK-8 assay and clone forming assay;the cell cycle was detected by PI(propidium iodide)labeled flow cytometry;the cell differentiation was detected by flow cytometry;the cell apoptosis was detected by flow cytometry with Annexin V-PE/7-AAD staining;the gene expression level and protein kinase activity were detected by qRT-PCR and Western blot.Results:1.The resuts of real-time PCR and Western blot assay showed that the expression of TSC2 gene mRNA and protein were downregulated and the interference efficiency was 71.7%.2.Down-regulated expression of TSC2 gene promoted U937 cell proliferation and clone form ability(P<0.05).The proportion in G0/G1 phase of TSC2 low expression U937 cell was much lower than that of the control cells(P<0.05),the S phase and G2/M phase was remarkably higher than that of the control cells(P<0.05).there were no statistically significant differences in cell apoptosis and differentiation(P>0.05).3.TSC2 low expression up-regulated the activity of mTOR(Ser2448),4EBP1(Thr37/46)and P70S6K(Thr389),but did not influence the activity of AKT(Ser473);TSC2 low expression up-regulated the expression of cyclinD1、c-myc and PTEN,but did not influence the expression of P27kip1 and BCL-XL.Conclusions:These results indicated that low expression of TSC2 could promote the growth of U937 cell line through up-regulated the mTORC1 pathway activity,which will provide a new pathogenesis and therapeutic strategy for AL.