miR-144靶向Bcl-2抑制儿童急性淋巴细胞白血病细胞增殖并促进凋亡的分子机制研究

Molecular Mechanism of miR-144 Inhibiting Proliferation and Promoting Apoptosis in Pediatric Acute Lymphoblastic Leukemia Cells by Targeting Bcl-2

目的:观察miR-144对儿童急性淋巴细胞白血病(ALL)CEM/C1细胞增殖和凋亡的影响,并探讨其作用机制。方法:运用qRT-PCR法和Western blot法检测非恶性血液病患儿脊髓组织(Normal组)、ALL患儿脊髓组织(ALL组)、CEM/C1细胞(CEM/C1组)、ALL癌细胞珠MOLT-4(MOLT-4组)和CCRF-CEM(CCRF-CEM组)中miR-144、Bcl-2mRNA和Bcl-2蛋白表达水平并比较组间差异;将不同质粒以脂质体法转染至CEM/C1细胞,分为miR-NC组(转染miR-NC)、miR-144组(转染miR-144mimics)、inhibitor NC组(转染inhibitor NC)、miR-144inhibitor组(转染miR-144inhibitor)、si-NC组(转染si-NC)、si-Bcl-2组(转染si-Bcl-2)、miR-144+Vector组(miR-144mimics和pcDNA 3.1共转染)、miR-144+Bcl-2组(miR-144mimics和pcDNA 3.1-Bcl-2共转染),Western blot检测各组细胞中Bcl-2蛋白表达;MTT法检测各组细胞增殖;流式细胞术检测各组细胞凋亡;双荧光素酶报告基因实验检测各组细胞的荧光素酶活性。结果:与Normal组相比,ALL组miR-144表达显著降低,Bcl-2mRNA和蛋白表达均显著升高(P<0.01)。与CEM/C1组比较,MOLT-4组和CCRF-CEM组miR-144表达显著升高,Bcl-2mRNA和蛋白表达均显著降低(P<0.01)。与miR-NC组相比,miR-144组细胞增殖率降低,凋亡率升高,Bcl-2(WT)细胞的荧光活性降低(P<0.01)。与si-NC组相比,si-Bcl-2组细胞增殖率降低,凋亡率升高(P<0.01)。与miR-144+Vector组相比,miR-144+Bcl-2组细胞增殖率降低,凋亡率升高(P<0.01)。结论:miR-144可抑制儿童ALL癌细胞增殖,促进其凋亡,可能与其能靶向Bcl-2有关,或可为儿童ALL治疗提供新的靶点。

Objective:To investigate the effect of miR-144on cell proliferation and apoptosis of CEM/C1in children with acute lymphoblastic leukemia(ALL)and to explore its mechanism.Method:qRT-PCR was used to detect normal tissues,children's ALL tissues(ALL group),human T cell leukemia cells CEM/C1(CEM/C1 group),ALL cells MOLT-4(MOLT-4group),CCRF-CEM(expression of miR-144,Bcl-2mRNA and Bcl-2protein in CCRF)and compared differences between groups;miR-NC group(transfected miR-NC),miR-144group(transfected miR-144mimics),inhibitor NC group(transfected inhibitor NC),miR-144inhibitor group(transfected miR-144inhibitor),si-NC group(transfected si-NC),si-Bcl-2group(transfected si-Bcl-2),miR-144+Vector group(co-transfected miR-144mimics and pcDNA 3.1),miR-144+Bcl-2group(co-transfected miR-144mimics and pcDNA 3.1-Bcl-2)were transfected into CEM/C1cells by liposome method.The protein expression of Bcl-2in each group was detected by Western blot;the cell proliferation in each group was detected by MTT assay.The apoptosis rate in each group was detected by flow cytometry.The luciferase activity of each group was detected by dual luciferase reporter gene assay.Results:Compared with Normal group,the expression of miR-144was significantly decreased and the expression of Bcl-2mRNA and protein was significantly increased in ALL group(P<0.01).Compared with CEM/C1group,the expression of miR-144was decreased and the expression of Bcl-2mRNA and protein was increased in the cells of CCRF-CEM group(P<0.01).Compared with miR-NC group,the proliferation of miR-144cells was decreased and apoptosis was increased.Compared with the si-NC group,the proliferation of cells in the si-Bcl-2group was decreased,and the apoptosis was increased.Compared with the miR-NC group,the fluorescence activity of the wild-type Bcl-2cells in the miR-144group was decreased;Compared with the miR-144+Vector group,the cell proliferation was decreased and the apoptosis was increased in the miR-144+Bcl-2group.Conclusion:miR-144could inhibit the cell proliferation in pediatric acute lymphoblastic leukemia cells and promote its apoptosis,which may be related to the targeting of Bcl-2,which will provide a new target for targeted therapy of pediatric acute lymphoblastic leukemia cells.