miR-31-5p对牙髓干细胞HIF-1α/BNIP3信号通路及成骨相关因子表达的影响

Effects of miR-31-5p on HIF-1α/BNIP3 signaling pathway and the expression of osteoblast-related factors of dental pulp stem cells

目的 :探讨微小RNA-31-5p(miR-31-5p)对牙髓干细胞(DPSCs)低氧诱导因子1α(HIF-1α)/Bcl-2/腺病毒E1B 19-kDa相互作用蛋白3(BNIP3)信号通路及成骨相关因子表达的影响。方法 :体外培养人DPSCs,分为对照组(不转染)、mimic NC组(转染negative control-miR-31-5p)、miR-31-5p mimic组(转染hsa-miR-31-5p mimic)、si RNA NC组(转染nonsense siRNA)和miR-31-5p siRNA组(转染miR-31-5p siRNA)。实时荧光定量PCR(qRT-PCR)检测各组DPSCs细胞中miR-31-5p、HIF-1α、BNIP3、碱性磷酸酶(ALP)、Runt相关转录子2(Runx2)mRNA的表达情况,MTT法检测各组DPSCs细胞增殖情况,ALP活性测定试剂盒检测各组DPSCs细胞ALP活性,蛋白印迹(WB)法检测各组DPSCs细胞中HIF-1α、BNIP3、Runx2蛋白的表达情况。采用SPSS 24.0软件包对数据进行统计学分析。结果:与对照组、mimic NC组相比,miR-31-5p mimic组DPSCs细胞A值、ALP mRNA表达水平及活性、Runx2 mRNA及蛋白表达水平显著降低(P<0.05),ALP染色明显减弱,miR-31-5p mRNA、HIF-1α、BNIP3 mRNA及HIF-1α、BNIP3、Beclin1蛋白表达水平显著升高(P<0.05)。与对照组、siRNA NC组相比,miR-31-5p siRNA组DPSCs细胞A值、ALP mRNA表达水平及活性、Runx2 mRNA及蛋白水平显著升高(P<0.05),ALP染色明显增强,miR-31-5p mRNA、HIF-1α、BNIP3 mRNA及HIF-1α、BNIP3、Beclin1蛋白表达水平显著降低(P<0.05)。结论 :miR-31-5p可以激活HIF-1α/BNIP3信号通路,抑制DPSCs细胞的成骨相关因子表达。

PURPOSE: To investigate the effects of microRNA-31-5p(miR-31-5p) on the signal pathway of hypoxia inducible factor-1α(HIF-1α)/Bcl-2/adenovirus E1B 19-kDa-interacting protein 3(BNIP3) and the expression of osteoblastrelated factors of dental pulp stem cells(DPSCs). METHODS: Human dental pulp stem cells(DPSCs) were cultured in vitro and divided into the control group(no transfection), mimic NC group(transfected with negative control-miR-31-5p),miR-31-5p mimic group(transfected with hsa-miR-31-5p mimic), siRNA NC group(transfected with nonsense siRNA)and miR-31-5p siRNA group(transfected with miR-31-5p siRNA).The expressions of miR-31-5p, HIF-1α, BNIP3, alkaline phosphatase(ALP) and Runt-related transcription factor-2(Runx2) mRNA in DPSCs were detected by real-time fluorescence quantitative PCR;the proliferation of DPSCs was detected by MTT;ALP activity of DPSCs was detected by ALP activity test kit;and the protein expressions of HIF-1α, BNIP3 and Runx2 in DPSCs were detected by Western blot. Statistical analysis was carried out with SPSS 24.0 software package. RESULTS: Compared with the control group and mimic NC group, the A value, ALP mRNA expression level and activity, Runx2 mRNA and protein expression levels of DPSCs in miR-31-5p mimic group were significantly lower(P<0.05), ALP staining decreased significantly, and the expression levels of miR-31-5p mRNA, HIF-1α, BNIP3 mRNA and HIF-1α, BNIP3, Beclin1 protein were significantly higher(P<0.05).Compared with the control group and siRNA NC group, the A value, ALP mRNA expression level and activity, Runx2 mRNA and protein expression levels of DPSCs in miR-31-5p siRNA group were significantly higher(P<0.05), ALP staining enhanced significantly, and the expression levels of miR-31-5p mRNA, HIF-1α, BNIP3 m RNA and HIF-1α, BNIP3,Beclin1 protein were significantly lower(P<0.05). CONCLUSIONS: MiR-31-5p may inhibit the expression of osteoblastrelated factors of DPSCs, and activating HIF-1α/BNIP3 signaling pathway.