下调miR-19b靶向TP53INP1对骨肉瘤细胞侵袭迁移和凋亡的影响

Effect of down-regulation of miR-19b targeting TP53INP1 on invasion,migration and apoptosis of osteosarcoma cells

目的研究miR-19b靶向调控TP53INP1对骨肉瘤细胞侵袭迁移和凋亡的影响。方法将骨肉瘤MG63细胞分为:对照组、抑制物阴性对照组(转染抑制物阴性对照)、miR-19b抑制物组(转染miR-19b抑制物)、siRNA阴性对照+miR-19b抑制物组(转染siRNA阴性对照和miR-19b抑制物)、TP53INP1 siRNA+miR-19b抑制物组(转染TP53INP1 siRNA和miR-19b抑制物)、模拟物阴性对照组(转染模拟物阴性对照)、miR-19b模拟物组(转染miR-19b模拟物)。以qRT-PCR测定转染效果,Transwell小室测定细胞侵袭和迁移,流式细胞术测定细胞凋亡。靶基因预测软件预测TP53INP1为miR-19b的靶基因,双荧光素酶报告载体鉴定靶向关系。以Western blot测定上调或下调miR-19b对TP53INP1表达的影响。以miR-19b抑制物和TP53INP1 siRNA共转染至MG63细胞中,以流式细胞术和Transwell小室测定细胞凋亡和侵袭迁移变化。结果 miR-19b抑制物MG63细胞中miR-19b表达水平下降,侵袭和迁移能力下降,细胞凋亡率升高,与对照组、抑制物阴性对照组比较,差异有统计学意义(P <0. 05)。miR-19b与TP53INP1mRNA的3,UTR有结合位点,荧光素酶报告载体鉴定TP53INP1为miR-19b的靶基因。与抑制物阴性对照组比较,miR-19b抑制物组上调细胞中TP53INP1蛋白表达水平,与模拟物阴性对照组比较,miR-19b模拟物组下调细胞中蛋白表达水平。与siRNA阴性对照+miR-19b抑制物组比较,TP53INP1 siRNA+miR-19b抑制物组能够上调MG63细胞的侵袭和迁移能力并减少细胞凋亡(P <0. 05)。结论下调miR-19b通过靶向调控TP53INP1可抑制骨肉瘤细胞侵袭迁移并诱导细胞凋亡。

Objective To study the effect of down regulation of miR-19b targeting TP53INP1 on invasion,migration and apoptosis of osteosarcoma cells.Methods In osteosarcoma MG63 cells were divided into control group,inhibitor-negative control(transfection inhibitor negative control),miR-19b inhibitor group,siRNA-NC+Anti-miR-19b(transfection of siRNA negative control and mir-19b inhibitor),TP53INP1 siRNA+Anti-miR-19b(transfection of TP53INP1 siRNA and miR-19b inhibitor),NC(transfection of mimetic negative control).miR-19b(transfected mimic of miR-19b).Transfection efficiency was detected by qRT-PCR.Cell invasion and migration were measured by Transwell chamber.Apoptosis was measured by flow cytometry.The target gene prediction software predicted that TP53INP1 is the target gene of miR-19b,double luciferase reporter vector was identified.Western blot was used to detect the effect of up-or down-regulation of miR-19b on TP53INP1 expression.miR-19binhibitors and TP53INP1 siRNA were co-transfected into MG63 cells.Apoptosis,invasion and migration were measured by flow cytometry and Transwell chamber.Results The expression level of miR-19b in MG63 cells decreased with invasion and migration ability decreased and apoptotic rate increased compared with control group and negative control group(P<0.05).There were binding sites between miR-19b and TP53INP1(3'UTR).Luciferase reporter vector identified TP53INP1 as the target gene of miR-19b.Compared with negative control,the expression of TP53INP1 protein was up-regulated by miR-19b inhibitor,and down-regulated by the mimic.TP53INP1 siRNA+miR-19b inhibitor could increase the invasive and migratory ability of MG63 cells and reduce cell apoptosis compared with siRNA negative control+miR-19b inhibitor group(P<0.05).Conclusion Down-regulation of miR-19b can inhibit the invasion and migration of osteosarcoma cells and induce apoptosis by targeting TP53INP1.