miR-424靶向FBXW7对多发性骨髓瘤细胞增殖、凋亡的影响

Effect of miR-424 on Proliferation and Apoptosis of Multiple Myeloma Cells by Targeting FBXW7

目的探讨miR-424靶向F-box和WD重复结构域7(F-Box and WD repeat domain containing 7,FBXW7)轴对多发性骨髓瘤(multiple myeloma,MM)细胞增殖、凋亡的影响。方法以实时荧光定量PCR(RT-qPCR)实验检测人正常骨髓浆细胞与MM细胞系MM.1S、RPMI 8226、U266中miR-424及FBXW7表达。体外培养U266细胞并随机分为对照组、miR-424抑制剂组(转染miR-424抑制剂)、FBXW7过表达组(转染FBXW7过表达质粒)、阴性对照组(转染miR-424阴性对照和空载质粒)、miR-424抑制剂+FBXW7敲低组(转染miR-424抑制剂和FBXW7siRNA)。以RT-qPCR实验检测miR-424及FBXW7表达;以Edu染色、TUNEL染色分别检测增殖、凋亡;以蛋白质印迹实验检测增殖相关蛋白(Cyclin D1、PCNA)、凋亡相关蛋白(Bax、Bcl-2)与FBXW7蛋白表达。在裸鼠右腋皮下接种转染后的各组U266细胞来构建多发性骨髓瘤移植瘤裸鼠模型,检测其移植瘤生长情况并比较其21 d移植瘤体积。以双荧光素酶报告实验验证miR-424对U266细胞FBXW7的靶向调控作用。结果与人正常骨髓浆细胞相比,MM.1S、RPMI 8226、U266细胞miR-424表达升高(P<0.05),FBXW7 mRNA表达降低(P<0.05)。与对照组相比,miR-424抑制剂组、FBXW7过表达组细胞增殖率、Cyclin D1、PCNA及Bcl-2蛋白表达、21 d移植瘤体积降低(P<0.05),FBXW7mRNA表达、凋亡率、FBXW7和Bax蛋白表达升高(P<0.05);阴性对照组细胞各指标无显著差异(P>0.05)。与miR-424抑制剂组相比,miR-424抑制剂+FBXW7敲低组细胞增殖率、Cyclin D1、PCNA及Bcl-2蛋白表达、21 d移植瘤体积增大(P<0.05),FBXW7 mRNA表达、凋亡率、FBXW7和Bax蛋白表达降低(P<0.05)。miR-424可靶向下调U266细胞FBXW7表达。结论下调miR-424可通过上调FBXW7表达而抑制MM细胞增殖及在裸鼠体内生长,促使其凋亡。

Objective To investigate the effect of miR-424 on the proliferation and apoptosis of multiple myeloma(MM)cells by targeting the F-box and WD repeat domain containing 7(FBXW7).Methods Real-time fluorescence quantitative PCR(RT-qPCR)experiment was applied to detect the expression levels of miR-424 and FBXW7 in human normal bone marrow plasma cells and MM cell lines MM.1S,RPMI 8226,U266.U266 cells were cultured in vitro and randomly separated into 5 groups:control group,miR-424 inhibitor group,FBXW7 overexpression group,negative control group,miR-424 inhibitor+FBXW7 knockdown group.Edu staining and TUNEL staining were applied to detect the cell proliferation and apoptosis,respectively.Western blotting assay was applied to detect the expression levels of proliferation related proteins(Cyclin D1,PCNA),apoptosis related proteins(Bax,Bcl-2),and FBXW7 protein.A nude mouse model of multiple myeloma transplantation was constructed by subcutaneous inoculation of transfected U266 cells in the right axilla of nude mice,the growth of the transplanted tumors were detected and the transplanted tumor volume was compared on the 21st day.Dual luciferase reporter experiment was applied to verify the targeted regulatory effect of miR-424 on FBXW7 in U266 cells.Results In comparison with those in the normal human bone marrow plasma cells,the expression of miR-424 in the MM.1S,RPMI 8226,and U266 cells were increased(P<0.05),while the expression of FBXW7 mRNA were decreased(P<0.05).In addition,when compared with the control group,the miR-424 inhibitor group and the FBXW7 overexpression group showed decreased cell proliferation rate,lower protein expression levels of Cyclin D1,PCNA and Bcl-2,and decreased transplanted tumor volume on the 21st day(P<0.05),and increased apoptosis rate,FBXW7 mRNA and protein expression levels,and Bax protein expression level(P<0.05).There was no significant difference in the indicators in cells of the negative control group(P>0.05).Moreover,when compared with the miR-424 inhibitor group,the miR-424 inhibitor+FBXW7 knockdown group exhibited increased cell proliferation rate,Cyclin D1,PCNA and Bcl-2 protein expression,and transplanted tumor volume on the 21st day(P<0.05),and decreased apoptosis rate,FBXW7 mRNA and protein expression levels,and Bax protein expression level(P<0.05).It was also observed that miR-424 was able to targetly downregulate FBXW7 expression in U266 cells.Conclusion Down-regulation of miR-424 can inhibit MM cell proliferation and growth in nude mice,and promote the apoptosis by up-regulating FBXW7 expression.