Let-7c Mediates Anisomycin-caused Apoptosis Of Jurkat T Cells By Connecting JNK To AP-1/STAT1/STAT3 Signaling

Background: micro RNAs(mi RNAs) are short(~22 nucleotides(nt) in length) non-coding RNAs(nc RNAs) that post-transcriptionally regulate gene expression by binding to specific m RNA targets and promoting their degradation and/or translational inhibition. Recent researches have demonstrated that mi RNAs are key regulators of many cell processes, including apoptosis. Anisomycin is a protein synthesis inhibitor, secreted by Streptomyces griseolus, which can bind with the 60 S ribosomal subunit and prevent peptide bond formation to result in block of peptide elongation and degradation of polyribosome. Our group has previously found that anisomycin can induce apoptosis of Jurkat T cells, a human T cell lymphoma cell line, and up-regulate the levels of mi RNA let-7c. However,it is unclear that how the let-7c mediates anisomycin-induced apoptosis in Jurkat T cells and if there exists any association between let-7c and JNK.Objective: To further explore the potential mechanism of let-7c on apoptosis of Jurkat T cells induced by anisomycin, which will lay a theoretical foundation and experimental evidence for its clinical application to treatment of leukemia.Methods: In vitro model of anisomycin to induce the apoptosis of Jurkat T cells was built for study. Jurkat T cells were pretreated with JNK inhibitor SP600125 for 2 hours before co-cultured with anisomycin,and the levels of mi RNA let-7c were detected by q PCR to confirm if there exists any association between JNK and let-7c. In order to manipulate levels of let-7c, the let-7c-related mimics, inhibitors and its overexpressional vector were transfected into Jurkat T cells. After anisomycin treatment, Flow cytometry was used to evaluate the role of let-7c in apoptosis of Jurkat T cells induced by anisomycin. Using Western Blot, we examined influence of let-7c on the expressions of apoptosis-associated transcription factor AP-1, P-AP-1, STAT1, P-STAT1, STAT3, P-STAT3 and apoptosis-related proteins Bim, P-Bim, Bcl-x L, P-Bcl-x L. Furthermore, the m RNA levels of Bim and Bcl-x L were detected by both RT-PCR and q PCR. After Jurkat T cells were transfected with Bim si RNA, q PCR and Western Blot were employed to determine alterations of Bim m RNA and protein, respectively. Flow cytometry was performed to analyze Bim si RNA on apoptosis of Jurkat T cells induced by anisomycin. Finally, Western Blot and in situ immunofluorescence staining was adopted to detect the expression and distribution of active Bak and active Bax in Jurkat T cells to clarify the downstream signal molecules of Bim and their functions.Results: The level of mi R let-7c was remarkably increased in the cells treated by anisomycin, which could be rescued using SP600125 to inhibit JNK signaling before the treatment of anisomycin. The Flow cytometry analysis showed that anisomycin could induce the obvious apoptosis of Jurkat T cells and there was the consistent tendency for the apoptosis induced by let-7c-mimic and anisomycin, whereas the anisomycin-induced the apoptosis could be reversed if mi R let-7c inhibitor was transfected before the addition of anisomycin. The expressions of both phosphorylated AP-1 and phosphorylated STAT1 were significantly up-regulated by anisomycin or the transfection of let-7c mimics or its overexpressional vector. On the contrary, the phosphorylation of STAT3 was inhibited by anisomycin or let-7c mimics or its overexpressional vector. Simultaneously, the levels of unphosphorylated AP-1, STAT1 and STAT3 were not changed significantly. RT-PCR, q PCR and Western Blot results together showed that Bim m RNA, protein and activity were increased by anisomycin or let-7c mimics,whereas Bcl-x L m RNA and protein but not activity were reduced by anisomycin or let-7c mimics. Anisomycin promoted the expression of Bim m RNA, but this change could be reversed by knocking down Bim gene in the cells. There was the consistent tendency for the alterations of Bim, active Bak and active Bax proteins. Knockdown of Bim gene could impede anisomycin to boost the apoptosis of the cells.Conclusion:The present study shows that anisomycin can activate Bak/Bax through breaking the balance between an anti-apoptotic Bcl-2 family member and a proapoptotic BH3-Only protein by JNK/let-7c/AP-1/STAT1/STAT3 signaling, finally resulting in the apoptosis of Jurkat T cells.