LncRNA XIST调节miR-545-3p/SMAD5轴对人成骨细胞增殖、迁移及成骨分化的影响

Influences of LncRNA XIST on proliferation,migration and osteogenic differentiation of human osteoblasts by regulating miR-545-3p/SMAD5 axis

目的探讨长链非编码RNA X染色体失活特异转录本(lncRNA XIST)调控miR-545-3p/SMAD5轴对人成骨细胞(hOB)增殖、迁移及成骨分化的影响。方法将hOB细胞分为ctrl组、pcDNA组、pcDNA-XIST组、pcDNA-XIST+miR-NC组、pcDNA-XIST+miR-545-3p mimic组;qRT-PCR检测细胞中XIST、miR-545-3p表达;CCK-8法检测细胞增殖;划痕实验检测细胞迁移;Western blot检测细胞中SMAD5、碱性磷酸酶(ALP)、Runt相关转录因子2(Runx2)、骨钙素(OCN)蛋白及细胞核中p-SMAD5蛋白表达;茜素红S染色检测矿化结节形成;双荧光素酶报告基因实验分别验证XIST和miR-545-3p、miR-545-3p和SMAD5的关系。结果与ctrl组、pcDNA组比较,pcDNA-XIST组hOB细胞中XIST表达、OD_(450)值(48、72 h)、划痕愈合率、SMAD5、ALP、Runx2、OCN蛋白及核p-SMAD5蛋白表达升高,miR-545-3p表达降低,矿化结节形成增多(P<0.05);上调miR-545-3p减弱了过表达XIST对hOB细胞增殖、迁移及成骨分化的促进作用;XIST靶向负调控miR-545-3p表达,miR-545-3p靶向调控SMAD5表达。结论过表达XIST可能通过海绵化miR-545-3p来上调SMAD5表达,进而促进hOB细胞增殖、迁移及成骨分化。

Objective To explore effects of long non-coding RNA X chromosome inactivation specific transcript(lncRNA XIST)on the proliferation,migration and osteogenic differentiation of human osteoblasts(hOB)by regulating miR-45-3p/SMAD5 axis.Methods HOB cells were divided into the ctrl group,pcDNA group,pcDNA XIST group,pcDNA XIST+miR NC group and pcDNA XIST+miR-545-3p mimic group.The expression of XIST and miR-545-3p in cells was detected by quantitative real-time PCR(qRT-PCR),the cell proliferation was determined using a Cell Counting Kit-8(CCK-8)assay cell migration was detected by scratch assay.The cell proliferation was detected by Cell Counting Kit-8(CCK-8)assay,cell migration was detected by scratch assay.Western blot was performed to detect the expression of SMAD5,alkaline phosphatase(ALP),Runt-related transcription factor 2(Runx2),osteocalcin(OCN)protein and p-SMAD5 protein in cell nucleuses.Alizarin red S(ARS)was conducted to detect the formation of mineralized nodules,and dual-luciferase reporter gene assay was applied to verify relationship between XIST and miR-545-3p,miR-545-3p and SMAD5.Results Compared to the ctrl group and pcDNA group,the expression of XIST,OD(450)value(48,72 h),scratch healing rate,SMAD5,ALP,Runx2,OCN protein and nuclear p-SMAD5 protein expression in the pcDNA XIST group was lowered,the expression of miR-545-3p was elevated,and the formation of mineralized nodules increased(P<0.05).The up-regulated miR-545-3p attenuated the over expression of XIST on the proliferation,migration and osteogenic differentiation,and XIST negatively regulated the expression of miR-545-3p,and miR-545-3p targeted the expression of SMAD5.Conclusion Over expression of XIST may up-regulate the expression of SMAD5 by sponging miR-545-3p,thereby promoting the proliferation,migration and osteogenic differentiation of hOB cells.