miR-141-3p通过靶向生长分化因子-6对RANKL诱导RAW264.7破骨分化的影响

Effect of miR-141-3p on RANKL-induced osteoclast differentiation of RAW264.7 by targeting growth differentiation factor-6

目的研究miR-141-3p通过靶向生长分化因子-6(growth differentiation factor 6,GDF-6)对RANKL诱导的RAW264.7细胞在破骨分化中的调控作用。方法RAW264.7细胞使用RANKL处理,诱导分化为破骨细胞,通过qRT-PCR和Western blot法检测GDF-6及抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)、组织蛋白酶K(cathepsin K,CTSK)的表达水平,同时qRT-PCR检测miR-141-3p的表达。实验分为未诱导组、RANKL诱导并转染载体对照组(RANKL+OE-NC)和RANKL诱导并转染GDF-6过表达载体组(RANKL+OE-GDF-6),对各组细胞进行TRAP染色,qRT-PCR和Western blot法检测破骨相关指标TRAP、CTSK、金属蛋白酶9(matrix metalloproteinase 9,MMP-9)、金属蛋白酶2(matrix metalloproteinase 2,MMP-2)及GDF-6的表达水平,同时qRT-PCR检测miR-141-3p的表达水平。实验分为过表达对照组(mimics NC)、过表达miR-141-3p组(mimics)、敲低对照组(inhibitor NC)、敲低miR-141-3p组(inhibitor)及过表达miR-141-3p转染过表达GDF-6载体组(miR-141-3p mimics+OE-GDF-6),检测方法同上,对各组细胞进行TRAP染色,qRT-PCR和Western blot法检测破骨相关指标TRAP、CTSK、MMP-9、MMP-2及GDF-6的表达水平,同时qRT-PCR检测miR-141-3p的表达水平。通过双荧光酶实验检测miR-141-3p和GDF-6的靶向关系。结果与未诱导组相比,RAW264.7诱导分化5 d后细胞内TRAP、CTSK、miR-141-3p表达水平升高(P<0.01),同时GDF-6表达水平降低(P<0.01)。转染OE-GDF-6载体后,RAW264.7向破骨细胞的分化受到抑制,TRAP、CTSK、MMP-9、MMP-2表达降低(P<0.01),TRAP染色阳性的破骨细胞数目减少。转染miR-141-3p mimics后,RAW264.7向破骨细胞分化受到促进,TRAP染色阳性的破骨细胞数量增多,破骨相关标志物TRAP、CTSK、MMP-9、MMP-2表达升高(P<0.01),GDF-6表达降低(P<0.01)。转染miR-141-3p抑制剂后,上述实验结果与模拟物组相反,RAW264.7向破骨细胞分化受到抑制。与miR-141-3p模拟物组相比,OE-GDF-6转染细胞后,可以在一定程度上拮抗miR-141-3p模拟物对破骨细胞分化的促进作用。双荧光素酶实验结果表明miR-141-3p可以与GDF-6-3′UTR-WT结合,抑制荧光素酶活性。结论miR-141-3p促进RANKL诱导RAW264.7向破骨细胞分化,其可能是通过靶向抑制GDF-6完成对破骨细胞分化的调控。

Objective To study the effects of miR-141-3p on osteoclast differentiation by targeting growth differentiation factor-6(GDF-6)in RANKL-induced RAW264.7 cells.Methods RAW264.7 cells were induced to osteoclasts with RANKL.qRT-PCR and Western blot were used to detect the expression levels of GDF-6 and osteoclast-related molecules tartrate resistant acid phosphatase(TRAP)and cathepsin K(CTSK).In addition,the expression level of miR-141-3p was detected by qRT-PCR.The experiment included three groups,the non-induced group,the RANKL-induced with transfected control vector group(RANKL+OE-NC),and the RANKL-induced with transfected GDF-6 overexpression vector group(RANKL+OE-GDF-6).TRAP staining was used to observe the morphology of osteoclasts.qRT-PCR and Western blot were used to detect the expression levels of osteoclast-related molecules TRAP,CTSK,matrix metalloproteinase-9(MMP-9),matrix metalloproteinase-2(MMP-2)as well as GDF-6.Meanwhile,the expression level of miR-141-3p was detected by qRT-PCR.Then the experiment was conducted with five groups,the mimics NC group,the miR-141-3p mimics group,the inhibitor NC group,the miR-141-3p inhibitor group,and the miR-141-3p mimics with GDF-6 overexpression group,while all of cells were induced with RANKL for five days.The targeting effect of miR-141-3p on GDF-6 was detected by dual luciferase assay.Results Compared to the non-induced group,RAW264.7 that was induced for five days expressed increasing levels of TRAP,CTSK,and miR-141-3p(P<0.01),while the expression level of GDF-6 decreased(P<0.01).The differentiation of the RAW264.7 in osteoclasts was inhibited after transfected with the GDF-6 overexpression vector,with decreasing levels of TRAP,CTSK,MMP-9,and MMP-2(P<0.01).TRAP staining results also showed that the number of TRAP positive multinucleated cells decreased.After transfected with miR-141-3p mimics,the differentiation of RAW264.7 into osteoclasts was promoted,which manifested as the increasing numbers of TRAP positive multinucleated cells and the increased expression levels of TRAP,CTSK,MMP-9,and MMP-2(P<0.01),the the expression of GDF-6 was decreased(P<0.01).However,all the above experimental results were opposite in the miR-141-3p inhibitor group,which showed that the differentiation of RAW264.7 into osteoclasts was inhibited.Compared with the miR-141-3p mimics group,the promoting effect of miR-141-3p mimics on osteoclast differentiation could be antagonized after transfected with OE-GDF-6.The results of luciferase assay showed that miR-141-3p could bind to GDF-6 mRNA 3′-UTR-WT and inhibit the activity of luciferase.Conclusion miR-141-3p promotes the differentiation of RAW264.7 induced with RANKL into osteoclasts,which may regulate osteoclast differentiation by targeting GDF-6.