Xu Min, Gao Wen-Wan, Luo Yu-Jie, Wang Yi, Tao Kun
Department of Immunology, College of Basic Medical Science ,Chongqing Medical University, Chongqing 400016, China.
Department of Immunology, College of Basic Medical Science ,Chongqing Medical University, Chongqing 400016, China,E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2019 Apr;27(2):396-402. doi: 10.19746/j.cnki.issn.1009-2137.2019.02.014.
To investigate the pro-apoptotic effect and mechanism of miR-30a overexpression on chronic myeloid leukemia K562 cells.
The k562 cells were transfected with the recombinant plasmid pEGFP-pre-miR-30a, the real-time quantitative PCR was used to detect the level of miR-30a and BCR/ABL, and then the cell apoptosis was assessed by flow cytometry with AnnexinV-FITC/PI double staining. Western blot was used to detect the expression of BCR/ABL protein,apoptosis-related protein BCL-2 and BAX, PTEN, AKT and p-AKT.
Sequencing and digestion map indicated that the recombinant plasmid was constructed successfully. Compared with 2 control groups, the miR-30a expression in k562 cells transfected with recombinant plasmid pEGFP-pre-miR-30a was obviously up-regulated. The expression of BCR/ABL mRNA and BCR/ABL protein was both significantly down-regulated. Apoptotic rate was significantly enhanced (both P<0.05),and the expression of anti-apoptotic protein BCL-2 was down-regulated while the expression of pro-apoptotic protein BAX was up-regulated. The level of PTEN was significantly up-regulated in omparison with control groups,no variation was found in total AKT, but the expression of p-AKT was down-regulated.
The overexpression of miR-30a is abled to down-regulate the level of BCR/ABL mRNA and BCR/ABL protein, and increase apoptotic rate, its mechanism may be related with inhibition of the activity of BCR/ABL-PTEN/AKT signaling pathway.
探讨miR-30a过表达对慢性髓性白血病K562细胞的促凋亡作用及机制。
将重组质粒pEGFP-pre-miR-30a转染K562细胞，采用实时定量PCR检测miR-30a和BCR/ABL水平，然后用AnnexinV-FITC/PI双染流式细胞术评估细胞凋亡。采用蛋白质免疫印迹法检测BCR/ABL蛋白、凋亡相关蛋白BCL-2和BAX、PTEN、AKT及p-AKT的表达。
测序及酶切图谱表明重组质粒构建成功。与2个对照组相比，转染重组质粒pEGFP-pre-miR-30a的K562细胞中miR-30a表达明显上调。BCR/ABL mRNA和BCR/ABL蛋白表达均显著下调。凋亡率显著提高（均P<0.05），抗凋亡蛋白BCL-2表达下调，促凋亡蛋白BAX表达上调。与对照组相比，PTEN水平显著上调，总AKT无变化，但p-AKT表达下调。
miR-30a过表达能够下调BCR/ABL mRNA和BCR/ABL蛋白水平，增加凋亡率，其机制可能与抑制BCR/ABL-PTEN/AKT信号通路活性有关。
