Differential Expression Of Apoptosis-associated Genes In Jurkat T Cells Treated With Anisomycin And Its Identification

Background: Anisomycin is a kind of protein synthesis inhibitors which can induceapoptosis of various cells. Our group has previously found that anisomycin can inhibitcell proliferation, block cell cycle and induce cell apoptosis of Jurkat T cells, andactivation of JNK signal pathway is related to their apoptosis. And miRNA let-7c maybe involved in the activation of JNK signal pathway. This shows its potential to beapplied to clinical treatment of T lymphocytic leukemia. Therefore, it has importantscientific significance to delineate the mechanism of anisomycin to induce cellapoptosis of Jurkat T cells. Currently, many genes associated with cell apoptosis havebeen found in mammalian cells. Which genes related to cell apoptosis exactly wereinvolved in the process of anisomycin to induce the cell apoptosis of Jurkat T cells?Are they related to JNK/miRNA let-7c? This study is the first systematic analysis on adifferential expression profile of genes induced by anisomycin using a qPCR Arraytechnique, in order that we can further elucidate the mechanism of anisomycin toinduce cell apoptosis of Jurkat T cells to lay a foundation for its clinical application.Objective: A qPCR Array technique was used to screen a differential expressionprofile of genes in Jurkat T cell apoptosis by anisomycin, and then it was focused toconfirm whether differential expression gene E2F2is related to cell apoptosis ofJurkat T cells induced by anisomycin, and whether E2F2is related to miRNA let-7cmediating anisomycin to induce Jurkat T cell apoptosis.Methods: Size and number of colony formation of Jurkat T cells treated withdifferent doses of anisomycin were observed under an inverted microscope, andchromatin morphology of Jurkat T cells were also done under a fluorescencemicroscope. Apoptotic rate of Jurkat T cells treated with different concentrations ofanisomysin were analyzed with flow cytometry.13genes with significantlydifferential expressions were screened out from88genes related to apoptosis through qPCR Array. Their differential expressions were further confirmed using RT-PCRand qPCR, and then E2F2was selected to confirm its function. After Jurkat T cellswere transfected respectively with miRNA let-7c mimic and miRNA let-7c inhibitor,flow cytometry detects rate of the cell apoptosis, qPCR or/and Western Blot detectedlevels of miRNA let-7c and E2F2to confirm whether miRNA and E2F2is related toJurkat T cell apoptosis. After Jurkat T cells were transfected with E2F2siRNA, flowcytometry detected the rate of the cell apoptosis, content changes of miRNA let-7andE2F2were detected through qPCR or/and Western Blot to further confirm whetherE2F2and miRNA let-7c is related to mediate anisomycin to induce the apoptosis ofJurkat T cells.Results: The size and number of colony formation of Jurkat T cells were obviouslydecreased and reduced with the increasing anisomycin concentration. Jurkat T cellnuclear shrinkage, formation of apoptotic bodies, the percentage of apoptotic cellswere increased with anisomycin. The qPCR Array analysis shows that1h after thetreatment of anisomycin, the expressions of total29genes, including AIFM2, CAS5,CD226, CD27, CIDEA, DAPK2, EGFR, ERBB3, FADD, FASLG, IFNA2, IFNB1,IGF1, IGF1R, IKBKG, IL10, IL1A, IL1B, IL6R, IL7, MAP3K1, MAP3K10,MAP3K14, NTRK1, PDCD1, PPP3CC, STAT1, STAT5B and TNFRSF10A, wereup-regulated. The expressions of12genes, including AKT2, AKT3, CAS4, CD24,E2F2, EndoG, IKBKB, IL4, MAP3K5, PIK3R2, TNF and TNFRSF10B, weredown-regulated. Among them, the expressions of CD226, FADD and IL1B wereup-regulated to20~45fold, and AKT2, CD24, E2F2, EndoG, IL4and PIK3R weredown-regulated to10~20fold.6h after the treatment of anisomycin, the expressionsof total15genes, including APAF1, API5, CAS7, CAS9, CD226, CD27, E2F2,ERBB3, IFNB1, IGF1R, IL2, RIPK1, STAT1, TNFRSF10A and XIAP, wereup-regulated, and the expressions of5genes, including DAPK3, FASLG, IKBKG,IL6R and IRAK1, were down-regulated. Among them, the expressions of CAS7,CAS9, CD226, IL2and TNFRSF10A were up-regulated to10~20fold, and theexpressions of FASLG and IL6R were down-regulated about15fold. Screened13genes with differences over10fold, namely CD226, CD24, E2F2, FASLG,TNFRSF10A, CAS7, CAS9, PIK3R2, IKBKB, AKT2, AKT3, FADD and EndoG were verified using qPCR, showing that changes of E2F2, CD24, IKBKB, AKT2,CAS9, CD226, and TNFRSF10A were consistent with those of the qPCR array.miRNA let-7c mimic and miRNA inhibitor showed that miRNA let-7c promotedapoptosis of Jurkat T cells, qPCR and Western Blot showed that there was arelationship between miRNA let-7c and E2F2. Flow cytometry showed that theknockdown of E2F2gene inhibited the cell apoptosis induced with anisomycin. qPCRand Western Blot showed the relation between E2F2and let-7c was confirmed.Conclusions: Anisomycin can induce the obvious apoptosis of Jurkat T cells, whichmay be related to E2F2, CD24、 PIK3R2、 IKBKB、 CAS9, CD226andTNFRSF10A genes. In the process of apoptosis of Jurkat T cells induced byanisomycin, E2F2is correlated with miRNA let-7c to function.