Fu Hong-Hai, Sun Le-Gang, Ding Chang-Cheng, Ma Xiang-Rui, Huang Yu-Mei
Binzhou Medical College. Binzhou 256603, Shandong Province, China. E-mail:
Shanghai Kou Qiang Yi Xue. 2022 Jun;31(3):237-242.
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 osteoblast-related factors of dental pulp stem cells(DPSCs).
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.
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 mRNA and HIF-1α, BNIP3, Beclin1 protein were significantly lower(P＜0.05).
MiR-31-5p may inhibit the expression of osteoblast-related factors of DPSCs, and activating HIF-1α/BNIP3 signaling pathway.
探讨微小RNA-31-5p（miR-31-5p）对缺氧诱导因子-1α（HIF-1α）/Bcl-2/腺病毒E1B 19-kDa相互作用蛋白3（BNIP3）信号通路及牙髓干细胞（DPSCs）成骨相关因子表达的影响。
体外培养人牙髓干细胞（DPSCs），分为对照组（未转染）、模拟物阴性对照组（转染阴性对照-miR-31-5p）、miR-31-5p模拟物组（转染hsa-miR-31-5p模拟物）、小干扰RNA阴性对照组（转染无义小干扰RNA）和miR-31-5p小干扰RNA组（转染miR-31-5p小干扰RNA）。采用实时荧光定量PCR检测DPSCs中miR-31-5p、HIF-1α、BNIP3、碱性磷酸酶（ALP）和Runx相关转录因子2（Runx2）mRNA的表达；采用MTT法检测DPSCs的增殖；采用ALP活性检测试剂盒检测DPSCs的ALP活性；采用蛋白质免疫印迹法检测DPSCs中HIF-1α、BNIP3和Runx2的蛋白表达。使用SPSS 24.0软件包进行统计分析。
与对照组和模拟物阴性对照组相比，miR-31-5p模拟物组DPSCs的A值、ALP mRNA表达水平及活性、Runx2 mRNA和蛋白表达水平均显著降低（P＜0.05），ALP染色明显减弱，miR-31-5p mRNA、HIF-1α、BNIP3 mRNA及HIF-1α、BNIP3、Beclin1蛋白表达水平均显著升高（P＜0.05）。与对照组和小干扰RNA阴性对照组相比，miR-31-5p小干扰RNA组DPSCs的A值、ALP mRNA表达水平及活性、Runx2 mRNA和蛋白表达水平均显著升高（P＜0.05），ALP染色明显增强，miR-31-5p mRNA、HIF-1α、BNIP3 mRNA及HIF-1α、BNIP3、Beclin1蛋白表达水平均显著降低（P＜0.05）。
MiR-31-5p可能抑制DPSCs成骨相关因子的表达，并激活HIF-1α/BNIP3信号通路。
