LncRNA HIF1A-AS2对人骨髓间充质干细胞的影响△

Effects of LncRNA HIF1A-AS2 on human bone marrow mesenchymal stem cells

摘要:

［目的］ 探讨 LncRNA HIF1A-AS2 对骨髓间充质干细胞成骨分化的作用及机制。［方法］ 人骨髓间充质干细胞 （hu- man bone marrow mesenchymal stem cells, hMSCs） 分为 6 组，为空白组、骨诱导组、AS2 NC 过表达组、AS2 过表达组、AS2 NC 干扰组、AS2 干扰组。后 4 组分别体外转染相应的 LncRNA HIF1A-AS2 的慢病毒载体，再将除空白组外的 hMSCs 进行成骨诱导培养。检测细胞凋亡、细胞增殖和细胞钙化沉积情况。检测 HIF1A-AS2、MMP2 和 VEGF165 mRNA 的表达水平及 MMP2 和 VEGF165 蛋白的表达水平。［结果］RT-PCR 结果显示，成骨诱导分化最强时间点为 7 d。细胞凋亡率由高至低依次为，空白组 >AS2 干扰组>AS2 NC 过表达组>AS2 NC 干扰组>骨诱导组>AS2 过表达组，整体差异有统计学意义（P<0.05）。细胞增殖 OD 值由低至高依次为：空白组P<0.05）。AS2 过表达组的红色钙化结节沉积较为显著，AS2 干扰组的红色钙化结节沉积数量相对较少。AS2 过表达组的 MMP2 和 VEGF165 的 mRNA 和蛋白表达水平显著高于 AS2 干扰组 （P<0.05）。［结论］ LncRNA HIF1A-AS2 过表达会促进细胞增殖，抑制细胞凋亡，增加 MMP2 和 VEGF165 的表达水平，促进 hMSCs 细胞分化为成骨细胞。

Abstract:

[Objective] To investigate the effect of LncRNA HIF1A-AS2 on osteogenic differentiation of human bone marrow mesen- chymal stem cells (hMSCs) . [Methods] The hMSCs were divided into six groups, including blank group, osteoinduction group, AS2 NC overexpression group, AS2 overexpression group, AS2 NC interference group and AS2 interference group. The later four groups were respec- tively transfected with the corresponding LncRNA HIF1A-AS2 lentivirus vector in vitro, and then the hMSCs except the blank group were cultured for osteogenic induction. The cell apoptosis, cell proliferation and cell calcification were detected, in addition, the mRNA expres- sion levels of HIF1A-AS2, MMP2 and VEGF165 and the protein expression levels of MMP2 and VEGF165 were detected. [Results] RTPCR showed that the time of maximum osteogenic differentiation was 7 d after the culture. The apoptosis rate was ranked from high to low as blank group > AS2 interference group > AS2 NC overexpression group > AS2 NC interference group > osteoinduction group > AS2 over- expression group, with statistically significant differences among them (P<0.05) . The OD value of cell proliferation was arranged from low to high as blank group < AS2 interference group < AS2 NC interference group < osteoinduction group < AS2 NC overexpression group < AS2 overexpression group, with the statistically significant overall difference (P<0.05) . The deposition of calcified nodules revealed by aliz- arin red staining was more significant in the AS2 overexpression group, whereas which was less in the AS2 interference group. The mRNA and protein expression levels of MMP2 and VEGF165 in the AS2 overexpression group were significantly higher than those in the AS2 inter- ference group (P<0.05) . [Conclusion] The LncRNA HIF1A- AS2 overexpression promotes cell proliferation, inhibit cell apoptosis, in- crease the expression levels of MMP2 and VEGF165, and promote the differentiation of hMSCs into osteoblasts.