miR-552靶向调控Wnt抑制因子-1促进MG-63骨肉瘤细胞增殖、侵袭、迁移的实验研究

Effect of miR-552 on proliferation, migration and invasion of osteosarcoma cell lines MG-63 via targeting WIF1

目的 探讨miR-552对Wnt抑制因子-1(WIF1)的靶向调控作用以及对人骨肉瘤MG-63细胞增殖、侵袭和迁移活性的影响。方法 体外培养人骨肉瘤细胞MG-63和人成骨细胞hFOB1.19,采用实时荧光定量PCR(QPCR)检测miR-552的表达水平。分别采用miR-552 inhibitor和shWIF1转染MG-63细胞,实验分为空白对照组、anti-阴性对照(anti-NC)组、anti-552组、miR-552 inhibitor+pcDNA3.1vector共转染阴性对照(anti-552-NC)组、miR-552 inhibitor+pcDNA3.1-shWIF1(共转染)组。采用MTT法、Transwell法检测细胞增殖、侵袭和迁移活性的变化。采用双荧光素酶报告实验验证WIF1与miR-552的靶向关系。采用Western blotting检测各组β-catenin、CyclinD1、APC的表达水平。结果 MG-63细胞中miR-552的表达水平为1.48±0.21,明显高于hFOB1.19细胞的1.00±0.07(P<0.05)。anti-552组miR-552的表达水平为0.51±0.12,低于空白对照组(P<0.05);WIF1表达水平为1.45±0.23,高于空白对照组(P<0.05)。共转染组WIF1表达水平为0.68±0.13,低于空白对照组和anti-552组(P<0.05)。培养24、48、72和96h后,anti-552组MG-63细胞增殖率分别为(83.14±4.25)%、(67.58±3.26)%、(53.41±3.25)%和(48.29±2.86)%,低于anti-NC组(P<0.05)。anti-552组MG-63侵袭、迁移细胞数分别为264±32和489±44,均少于空白对照组和anti-NC组(P<0.05)。双荧光素酶报告实验证实WIF1是miR-552的下游靶基因。anti-552组β-catenin、Cyclin D1、APC蛋白表达量分别为0.84±0.15、0.83±0.16和0.59±0.12,均低于空白对照组和anti-NC组(P<0.05)。而共转染组β-catenin、CyclinD1、APC蛋白表达量分别为2.86±0.45、1.93±0.34和1.40±0.28,明显高于anti-552组(P<0.05)。结论 miR-552在骨肉瘤中表达上调,可通过靶向抑制WIF1的表达,异常激活Wnt/β-catenin信号,进而促使骨肉瘤细胞的增殖、侵袭和迁移。

Objective To investigate the effect of miR-552 on Wnt inhibitor-1 (WIF1) targeting regulation and proliferation,invasion and migration of human osteosarcoma MG-63 cell line.Methods Human osteosarcoma cell line MG-63 and human osteoblast line hFOB1.19 were cultured in vitro .Real-time fluorescence quantitative PCR (QPCR) was used to detect the expression of miR-552.MG-63 cells were transfected with miR-552 inhibitor and shWIF1,respectively.The experiment was divided into blank control group,anti-NC group,anti-552 group,anti-552 inhibitor + pcDNA3.1 vector co-transfection negative control (anti-552-NC) group and miR-552 inhibitor + pcDNA3.1-shWIF1 (co-transfection) group.MTT assay and Transwell assay were used to detect the changes of cell proliferation,invasion and migration activity.The targeting relationship between WIF1 and miR-552 was validated by double luciferase reporter assay.The β-catenin,Cyclin D1,adenomatous polyposis coli (APC) proteins of MG-63 cells were detected by Western blotting.Results The expression level of miR-552 in MG-63 cells was 1.48±0.21,which was significantly higher than that in hFOB1.19 cells (P<0.05).The expression level of miR-552 in anti-552 group was 0.51±0.12,lower than that in blank control group (P<0.05);the expression level of WIF1 was 1.45±0.23,higher than that in blank control group (P<0.05).The expression level of WIF1 in co-transfection group was 0.68±0.13,lower than that in blank control group and anti-552 group (P<0.05).After 24,48,72 and 96 hours of culture,the proliferation rates of MG-63 cells in anti-552 group were (83.14±4.25)%,(67.58±3.26)%,(53.41±3.25)% and (48.29±2.86)%,respectively,lower than those in anti-NC group (P<0.05).The number of invasive and migratory cells of MG-63 in anti-552 group was 264±32 and 489±44,respectively,which were less than that in blank control group and anti-NC group (P<0.05).The double luciferase gene reporting experiment confirmed that WIF1 was the downstream target gene of miR-552.The expression levels ofβ-catenin,Cyclin D1 and APC in anti-552 group were 0.84±0.15,0.83 ±0.16 and 0.59±0.12 respectively,which were lower than those in blank control group and anti-NC group (P<0.05).β-catenin,Cyclin D1,APC proteins of MG-63 cells in anti-552+shWIF1 group were 2.86±0.45,1.93±0.34 and 1.40±0.28,which were higher than those in anti-552 group (P<0.05).Conclusion miR-552 significantly up-regulated in osteosarcoma cell lines MG-63,and promote the proliferation,migration and invasion of MG-63 cells by activating Wnt/β-catenin signaling via targeting inhibition of WIF1.