摘要
目的 探讨长链非编码RNA(lncRNA)HOX转录反义RNA(HOTAIR)对人牙周膜干细胞增殖和骨向分化的影响.方法 分离培养人牙周膜干细胞(hPDLSCs),并诱导成骨分化(诱导组),随后分为pcDNA 组、pcDNA-lncRNA HOTAIR 组、anti-miR-NC 组、anti-miR-1-3p 组、pcDNA-lncRNA HOTAIR+miR-NC组和pcDNA-lncRNA HOTAIR+miR-1-3p组,正常培养的hPDLSCs设为对照组.定量实时PCR检测lncRNA HOTAIR、miR-1-3p、骨钙素(OCN)、骨桥蛋白(OPN)、碱性磷酸酶(ALP)的mRNA表达,MTT法检测细胞增殖,Western blotting检测OCN、OPN、ALP蛋白表达,荧光素酶报告基因检测lncRNA HOTAIR和miR-1-3p的靶向结合.结果 与对照组比较,诱导组lncRNA HOTAIR表达量降低,miR-1-3p表达量增加(P<0.05).与pcDNA组、anti-miR-NC组比较,pcDNA-lncRNA HOTAIR组、anti-miR-1-3p组细胞活力、OCN、OPN、ALP的mRNA蛋白表达水平降低(P<0.05).lncRNA HOTAIR靶向下调miR-1-3p表达(P<0.05).与pcDNA-lncRNA HOTAIR+miR-NC组比较,pcDNA-lncRNA HOTAIR+miR-1-3p组miR-1-3p表达量、细胞活力及OCN、OPN、ALP的mRNA水平和蛋白水平均升高(P<0.05).结论 lncRNA HOTAIR通过降低miR-1-3p表达抑制hPDLSCs的增殖和骨向分化.
Abstract
Objective To investigate the effects of long non-coding RNA(lncRNA)HOX transcription antisense RNA(HOTAIR)on the proliferation and osteogenic differentiation of human periodontal ligament stem cells.Methods Human periodontal ligament stem cells(hPDLSCs)were isolated and cultured,and osteogenic differentiation was induced(induction group).They were then divided into pcDNA group,pcDNA-lncRNA HOTAIR group,anti-miR-NC group,anti-miR-1-3p group,pcDNA-lncRNA HOTAIR+miR-NC group and pcDNA-lncRNA HOTAIR+miR-1-3p group,normal culture hPDLSCs served as control group.Quantitative real-time PCR(qRT-PCR)was used to detect the expression of lncRNA HOTAIR,miR-1-3p,and the mRNA expression of osteocalcin(OCN),osteopontin(OPN)and alkaline phosphatase(ALP).MTT method was used to detect cell proliferation.The protein expressions of OCN,OPN and ALP were detected by western blotting,and the targeted binding of lncRNA HOTAIR and miR-1-3p was detected by luciferase reporter gene.Results Compared with the control group,the expression of lncRNA HOTAIR in hPDLSCs in the induction group was decreased,and the expression of miR-1-3p was increased(P<0.05).Compared with pcDNA group and anti-miR-NC group,cell viability of hPDLSCs at 24 h,48 h,and 72 h,the mRNA levels and protein expression levels of OCN,OPN,and ALP in pcDNA-lncRNA HOTAIR group and anti-miR-1-3p group were decreased(P<0.05).Compared with pcDNA-lncRNA HOTAIR+miR-NC group,the expression of miR-1-3p,cell viability and 72 h,and the mRNA and protein levels of osteogenesis-related genes OCN,OPN,and ALP in hPDLSCs in pcDNA-lncRNA HOTAIR+miR-1-3p group were increased(P<0.05).Conclusions LncRNA HOTATR inhibits proliferation and osteogenic differentiation of hPDLSCs by reducing miR-1-3p expression.
基金项目
2020年市级应用技术研究与开发资金项目(20YFZJ0012)
NETL
NSTL
万方数据