hsa-miR-23a-3p靶向TGFβ2对急性淋巴细胞白血病CEM/C1细胞增殖、凋亡、侵袭及细胞骨架重组的影响

Effects of hsa-miR-23a-3p targeting TGFβ2 on the proliferation,apoptosis,invasion and cy⁃toskeleton recombination of acute lymphoblastic leukemia CEM/C1 cells

目的:探究hsa-miR-23a-3p靶向转化生长因子β2(TGFβ2)对急性淋巴细胞白血病细胞株CEM/C1增殖、凋亡、侵袭及细胞骨架重组的影响。方法:将CEM/C1细胞随机分为4组:Control组、mimic组、pcDNA-TGFβ2组和mimic+TGFβ2组,采用Lipofectamine 2000分别转染mimic对照、miR-23a-3p mimic、TGFβ2过表达质粒及共转染miR-23a-3p mimic+TGFβ2过表达质粒。实时荧光定量PCR(RT-qPCR)法检测miR-23a-3p、TGFβ2表达;荧光素酶报告实验验证miR-23a-3p与TGFβ2的靶向关系;克隆形成实验检测细胞增殖;流式细胞仪检测细胞凋亡;Transwell检测细胞侵袭;微管形成实验检测细胞微管形成的结节数;Western blotting法检测Ki67、Survivin、Caspase-3、Bax、Bcl-2、VEGF蛋白表达。结果:与Control组相比,mimic组miR-23a-3p表达、细胞凋亡率、Caspase-3蛋白表达量和Bax/Bcl-2比值升高,TGFβ2 mRNA表达、克隆形成率、微管形成结节数及Ki67、Survivin、VEGF蛋白表达量降低,侵袭细胞数减少(均P<0.05),pcDNA-TGFβ2组与mimic组各指标变化相反(均P<0.05);与pcDNA-TGFβ2组比较,mimic+TGFβ2组克隆形成率、微管形成结节数和Ki67、Survivin、VEGF蛋白表达量降低,细胞凋亡率和Caspase-3蛋白表达量及Bax/Bcl-2比值升高,侵袭细胞数减少(均P<0.05)。荧光素酶报告实验证实miR-23a-3p和TGFβ2存在靶向关系。结论:hsa-miR-23a-3p通过靶向下调TGFβ2表达抑制人白血病细胞增殖、侵袭、细胞骨架重组,促进细胞凋亡。

Objective:To investigate the effect of hsa-miR-23a-3p-targeting transformation growth factor beta 2(TGFβ2)on the proliferation,apoptosis,invasion and cytoskeleton recombination of acute lymphoblastic leukemia cell line CEM/C1.Methods:CEM/C1 cells were divided into four groups:Control group,mimic group,pcDNA-TGFβ2 group and mimic+TGFβ2 group.The cells were transfected with mimic negative control,miR-23a-3p mimic,or TGFβ2 overexpressed plasmid,and co-transfected with miR-23a-3p mimic and TGFβ2 overexpressed plasmid.The luciferase reporter assay was used to verify the targeting relationship between miR-23a-3p and TGFβ2.The expression levels of miR-23a-3p and TGFβ2 were detected by RT-qPCR.Cell proliferation,invasion and apoptosis were detected by clonal formation assay,Transwell assay and flow cytometry,respectively.The angiogenesis of CEM/C1 cells was assessed by using a tube formation assay.The protein expressions of Ki67,Survivin,caspase-3,Bax,Bcl-2,and VEGF were determined by western blooting.Results:Compared with Control group,miR-23a-3p expression,cell apoptosis rate,caspase-3 protein expression and Bax/Bcl-2 ratio were increased,while the TGFβ2 mRNA expression,clonal formation rate,microtubule nodal number,the protein expressions of Ki67,Survivin and VEGF,and the num-ber of invasive cells were decreased in mimic group(P<0.05).The changes of pcDNA-TGFβ2 group were opposite to those in mimic group(P<0.05).Compared with pcDNA-TGFβ2 group,the microtubule nodular formation,the protein expressions of Ki67,Survivin and VEGF and the number of invasive cells in mimic+TGFβ2 group were decreased,whereas the cell apoptosis rate,caspase-3 protein expression and Bax/Bcl-2 ratio were increased(P<0.05).Luciferase reporter assay confirmed the targeting relationship between miR-23a-3p and TGFβ2.Conclusion:hsa-miR-23a-3p can inhibit cell proliferation,invasion and cytoskeletal recombination,and promote apoptosis in human leukemia cell line by targeting TGFβ2.