miR-218-5p靶向B细胞淋巴瘤因子3表达影响白血病K562细胞的增殖和凋亡

Effect of miR-218-5p on the proliferation and apoptosis of leukemia K562 cells by targeting BCL3

目的探讨微小RNA-218-5p(miR-218-5p)靶向B细胞淋巴瘤因子3(BCL3)基因对白血病细胞增殖和凋亡的影响。方法反转录及荧光定量PCR(RT-qPCR)和蛋白质印迹法(Westernblotting)检测正常人骨髓细胞和白血病骨髓细胞中miR-218-5p和BCL3的表达水平。以白血病细胞K562为研究对象,分别构建过表达miR-218-5p或沉默BCL3的K562细胞株,噻唑蓝(MTT)法检测细胞增殖,流式细胞仪检测细胞凋亡,Westernblotting检测细胞增殖抗原(Ki67)、细胞周期蛋白D1(CyclinD1)和活化的半胱氨酸天冬氨酸蛋白酶(CleavedCaspase-3)的表达水平。双荧光素酶报告基因实验和Westernblotting验证miR-218-5p和BCL3的靶向调控关系。结果与正常人骨髓细胞相比,白血病骨髓细胞中miR-218-5p的表达水平(1.00±0.13)比(0.44±0.18)显著降低,BCL3的表达水平(1.04±0.32)比(4.76±1.38)、(0.26±0.05)比(0.78±0.13)显著升高。过表达miR-218-5p或沉默BCL3均可显著抑制K562细胞CyclinD1(0.69±0.08)比(0.34±0.05)、(0.65±0.07)比(0.18±0.04)和Ki67表达(0.68±0.07)比(0.23±0.04)、(0.54±0.06)比(0.24±0.04),促进Cleaved-Caspase-3表达(0.38±0.04)比(0.89±0.11)、(0.17±0.04)比(0.56±0.07),抑制细胞增殖[(98.72±12.38)%比(69.79±7.62)%]、[(97.48±11.49)%比(65.73±6.86)%],促进细胞凋亡[(7.26±0.85)%比(24.46±2.67)%]、[(6.63±0.85)%比(26.46±2.81)%]。BCL3是miR-218-5p的靶基因,miR-218-5p可负性调控BCL3的表达。过表达BCL3可部分逆转miR-218-5p对K562细胞增殖和凋亡的影响。结论miR-218-5p通过靶向BCL3抑制白血病细胞增殖,促进细胞凋亡。

Objective To investigate the effect of micro-218-5p (mi R-218-5p) on the proliferation and apoptosis of leukemia cells by targeting B-cell lymphoma factor 3 (BCL3) gene.Methods The expression levels of mi R-218-5p and BCL3 in normal human bone marrow cells and leukemia bone marrow cells were detected by q RT-PCR and Western blot.The K562 cell lines over-expressing mi R-218-5p or silencing BCL3 were constructed.Cell proliferation was detected by MTT assay,apoptosis was detected by flow cytometry,the expression levels of Ki67,Cyclin D1 and Cleaved Caspase-3 were detected by Western blot.The dual luciferase reporter gene assay and Western blot were used to verify the targeted and regulatory relationship between mi R-218-5p and BCL3.Results Compared with normal human bone marrow cells,the expression level of mi R-218-5p (1.00±0.13) vs.(0.44±0.18) in leukemia bone marrow cells was significantly decreased,while the expression level of BCL3 (1.04±0.32) vs.(4.76±1.38),(0.26±0.05) vs.(0.78±0.13) was significantly increased.Over-expression of mi R-218-5p or silencing BCL3 significantly inhibited the expression level of Cyclin D1 (0.69±0.08 vs.0.34±0.05),(0.65±0.07 vs.0.18±0.04) and Ki67 (0.68±0.07) vs.(0.23±0.04),(0.54±0.06) vs.(0.24±0.04) in K562 cells,promoted the expression of Cleaved-Caspase-3(0.38±0.04) vs.(0.89±0.11),(0.17±0.04) vs (0.56±0.07),inhibited cell proliferation[(98.72±12.38)%vs.(69.79±7.62)%],[(97.48±11.49)%vs.(65.73±6.86)%]and promoted apoptosis[(7.26±0.85)%vs.(24.46±2.67)%],[(6.63±0.85)%vs.(26.46±2.81)%].BCL3 was a target gene of mi R-218-5p,and mi R-218-5p negatively regulated the expression of BCL3.The over-expression of BCL3 partially reversed the effect of mi R-218-5p on proliferation and apoptosis of K562 cells.Conclusion mi R-218-5p inhibits leukemia cell proliferation and promotes apoptosis by targeting BCL3.