Exploration Of The Role Of MiRNA Let-7c On Jurkat T Cell Apoptosis Induced By Anisomycin

Background: It has been reported that anisomycin can induce apoptosis of several types ofcancer cells in vitro. Our team previous work confirmed anisomycin could inhibit theproliferation of various cancer cells originated from different organs and induce the apoptosis ofJurkat T cells which was mediated by JNK signal pathway. But until now, there is no report whatapoptosis-related molecules are touched by downstream of JNK signaling pathway and whatmiRNAs regulate these molecule expressions. Obviously, secreesing out related miRNAs andillustrating their potential action mechanism has an important biolodical significantce, which willassist to comprehend the mechanism of anisomycin-induced Jurkat T cell apoptosis and to enrichthe cell apoptosis theory known.Objective: To explore the potential mechanism of let-7c on apoptosis of Jurkat T cells inducedby anisomycin.Methods: Flow cytometry was used to investigate Jurkat T cell apoptosis induced by differentconcentrations of anisomycin for6h and24h, respectively and the reverse effect of SP600125and PD98059on it. Using Western blot, we examined influence of40ng/ml anisomycin on theexpression of apoptosis-related protein P-BCL-XL and P-Bim with interference of SP600125and PD98059. The activity of apoptosis-associated transcription factor was verified by a TFreporter array. EMSA was used to detect the DNA-binding activity of AP-1. And miRNA arraywas adopted to screen the profile of miRNA expression in Jurkat T cells treated with40.0ng/mlanisomycin, which was confirmed by qPCR. We chose miRNA let-7c with the differentiallystable expression and transfected the let-7c related mimic and inhibitors in order to detect itsbiologic function via Flow Cytometry,Western Blot and qPCR.Result: The apoptosis of Jurkat T cells treated by anisomycin was in a does-dependent manner.As the increasing concentration of anisomycin, the number of double-dyed cells remarkably wasincreased. The JNK pathway specific-inhibitor SP600125could reverse this trend, but ERKpathway specific-inhibitor PD98059could not. Compared with the control, the expression ofP-Bim and P-BCL-XL proteins was significantly enhanced in anisomycin treated group. SP600125inhibited the expression of P-Bim and P-BCL-X, but PD98059could not.Transcriptional factor AP-1, NF-Κb and P53were increased, while HIF，STAT-1and STAT-3were decreased and ISRE showed no regularity. The EMSA result showed that anisomycin couldup-regulated the DNA binding activity of AP-1in a dose dependent manner and that SP600125inhibited the activity of AP-1, which had coincidence with the result acquired from TF reporterarray. Compared with the control, the expressions of16miRNAs were up-regulated and31miRNAs were down-regulated. Further the expressions of the significantly changed miRNAs oflet-7c, miR-26, miR-133b, miR-144, miR-193a, miR-296and miR-337were identified byquantitative real-time (qPCR). The up-regulation of let-7c and down-regulation of miR-133bwere verified by quantitative real-time PCR. It was showed via transfecting let-7c-mimic andlet-7c-inhibitor that let-7c could up-regulate the apoptosis of Jurkat T cells, which was tightlyrelated with the up-regulation of P-BCL-XL and P-Bim.Conclusion: Anisomycin can up-regulate the levels of let-7c, miR-26, miR-144, miR-193a andmiR-337in Jurkat T cells and down-regulate the expressions of miR-296and miR-133b.Anisomycin can induce Jurkat T cell apoptosis through let-7c regulating the expression ofP-BCL-XL and P-Bim protein.