雷公藤红素通过miR-17-5p和miR-155-5p靶向抑制cyclin D1阻滞A549细胞周期的研究

目的:研究雷公藤红素对人肺腺癌A549细胞周期的影响,并探讨其机制。方法:梯度浓度的雷公藤红素作用于人肺腺癌A549细胞后,MTT法检测细胞活力的变化,流式细胞术检测细胞凋亡,筛选出雷公藤红素的半数致死浓度;而后用半数致死浓度的雷公藤红素作用于A549细胞,流式细胞术检测细胞周期的变化,Western blot检测细胞周期蛋白D1(cyclin D1)的表达水平;real-time PCR检测微小RNA(miR)-17-5p和miR-155-5p表达的变化;生物学信息软件预测miR-17-5p和miR-155-5p与cyclin D1的相关性;将miR-17-5p mimics和miR-155-5p mimics以及各自的突变体mutant-miR-17-5p和mutant-miR-155-5p分别与pcDNA-GFP-cyclin D1-3’UTR共转染至A549细胞后荧光显微镜和流式细胞术检测GFP表达;最后将miR-17-5p mimics和miR-155-5p mimics转染至A549细胞后,real-time PCR检测miR-17-5p和miR-155-5p表达水平的变化,Western blot检测cyclin D1的表达水平。结果:随雷公藤红素作用浓度的增大,A549细胞的活力抑制率逐渐增高,细胞凋亡率逐渐增加(P<0.01),提示雷公藤红素可以有效抑制A549细胞的生长,诱导其凋亡,其中3μmol/L最接近半数致死浓度;应用该浓度雷公藤红素作用于A549细胞后,细胞被阻滞在G 1期,cyclin D1表达水平显著降低(P<0.01),miR-17-5p和miR-155-5p的表达水平显著升高(P<0.01)。生物学信息软件预测提示cyclin D1的3’UTR存在miR-17-5p和miR-155-5p的结合位点。GFP检测结果显示,miR-17-5p mimics/miR-155-5p mimics和pcDNA-GFP-cyclin D1-3’UTR共转染至A549细胞后,GFP的表达水平降低(P<0.05),提示miR-17-5p和miR-155-5p能直接靶向cyclin D1的3’UTR发挥作用;将miR-17-5p mimics/miR-155-5p mimics转染A549细胞后,miR-17-5p/miR-155-5p的表达水平显著升高(P<0.01),cyclin D1表达水平均显著降低(P<0.01)。结论:雷公藤红素可阻滞A549细胞于G 1期,进一步导致了细胞生长抑制和凋亡增加,其机制可能是通过上调miR-17-5p和miR-155-5p的表达而导致了cyclin D1的靶向抑制。本研究为雷公藤红素作为临床非小细胞肺癌的治疗药物提供新的理论依据。

Celastrol blocks cell cycle of A549 cells by regulating miR-17-5p/miR-155-5p and targeting cyclin D1

AIM:To investigate the effect of celastrol on the cell cycle of human lung adenocarcinoma A549 cells and to probe into its mechanisms.METHODS:A549 cells were exposed to celastrol at gradient concentrations.The cell viability and apoptosis were detected by MTT assay and flow cytometry,respectively,and the median lethal concentration(LC 50)of celastrol was screened.The A549 cells were treated with celastrol at LC 50,and the cell cycle was detected by flow cytometry.The expression of cyclin D1 was determined by Western blot,and the expression of microRNA(miR)-17-5p and miR-155-5p was detected by real-time PCR.The correlation between cyclin D1 and miR-17-5p or miR-155-5p was predicted by bioinformatics software.After miR-17-5p mimics/miR-155-5p mimics/mutant-miR-17-5p/mutant-miR-155-5p and pcDNA-GFP-cyclin D1-3’UTR were cotransfected into the A549 cells,the changes of GFP expression were evaluated by fluorescence microscopy and flow cytometry.Finally,after miR-17-5p mimics or miR-155-5p mimics were transfeced into the A549 cells,the expression of miR-17-5p and miR-155-5p was detected by real-time PCR,and the protein level of cyclin D1 was determined by Western blot.RESULTS:With the increasing concentration of celastrol,the viability inhibition rate and apoptotic rate of the A549 cells were increased,indicating that celastrol effectively inhibited the growth of A549 cells and induced apoptosis.The LC 50 of celastrol was almost 3μmol/L.After treatment with celastrol at LC 50,the A549 cell cycle was arrested at G 1 phase,the protein expression of cyclin D1 was down-regulated(P<0.01),and the expression levels of miR-17-5p and miR-155-5p were significantly increased(P<0.01).The results of bioinformatics software prediction indicated that there were binding sites for miR-17-5p and miR-155-5p in the 3’-UTR of cyclin D1.After cotransfected with miR-17-5p or miR-155-5p and pcDNA-GFP-cyclin D1-3’UTR into the A549 cells,the expression of GFP declined(P<0.05).After miR-17-5p or miR-155-5p mimics were transfected into A549 cells,the results of real-time PCR showed this treatment significantly increased the miRNA expression(P<0.01),and the results of Western blot showed the transfection inhibited cyclin D1 expression(P<0.01).CONCLUSION:Celastrol blocks the A549 cells at G1 phase,inhibits the viability and induces apoptosis,which may be caused by up-regulating the expression of miR-17-5p and miR-155-5p,and then down-regulating cyclin D1 expression.This study provides a new theoretical basis for the treatment of non-small-cell lung cancer with celastrol.