miR-640调控Wnt信号通路对腰椎间盘突出患者髓核细胞衰老及凋亡的影响

Effects of miR-640 regulating Wnt signaling pathway on senile and apoptosis of nucleus pulposus cells in patients with lumbar disc herniation

目的探讨miR-640调控Wnt信号通路对腰椎间盘突出患者髓核细胞衰老及凋亡的影响及其机制。方法选取我院32例腰椎间盘突出患者的腰椎间盘组织作为观察组,另选取32例外伤患者未病变的腰椎间盘组织作为对照组,qPCR检测2组miR-640表达水平。分离培养人髓核细胞,培养后将原代培养髓核细胞分为Control组、NC组和miR-640组,NC组和miR-640组分别转染NC质粒和miR-640质粒,通过SA-β-gal实验检测各组细胞衰老水平,流式细胞仪检测各组细胞凋亡水平,免疫荧光检测β-连环蛋白(β-catenin)的表达水平和定位,双荧光素酶报告基因实验检测miR-640和β-catenin的靶向调控关系。结果与对照组比较,观察组腰椎间盘组织miR-640表达水平显著升高(P<0.01)。与Control组比较,miR-640组细胞衰老水平增加,细胞凋亡率增加,差异均具有统计学意义(P<0.01),NC组与Control组上述指标比较差异无统计学意义(P>0.05)。免疫荧光证实miR-640组细胞β-catenin的表达降低,β-catenin入核受到抑制,差异具有统计学意义(P<0.01)。双荧光素酶报告基因实验证实miR-640和β-catenin具有靶向关系,差异具有统计学意义(P<0.01)。结论腰椎间盘突出患者中miR-640表达水平升高,miR-640可以促进髓核细胞衰老和凋亡,其机制可能与Wnt信号通路的调控有关。

Objective To investigate the effects and mechanisms of miR-640 regulating Wnt signaling pathway on senile and apoptosis of nucleus pulposus cells in patients with lumbar disc herniation.Methods The lumbar disc tissues of 32 patients with lumbar disc herniation in our hospital were selected as the observation group,and the lumbar disc tissues of 32 patients with trauma without lesions were selected as the control group.The expression level of miR-640 of the two groups was detected by qPCR.The human nucleus pulposus cells were isolated and cultured,and the primary cultured nucleus pulposus cells were divided into the Control group,the NC group and the miR-640 group.The NC group and the miR-640 group were transfected with NC plasmid and miR-640 plasmid,respectively.SA-β-Gal experiment was used to detect the cell senile level in each group,flow cytometry was used to detect the apoptosis level of cells in each group,immunofluorescence was used to detect the expression level and localization ofβ-catenin.Dual luciferase reporter gene experiment was used to detect the targeted regulation relationship between miR-640 andβ-catenin.Results Compared with the control group,the expression level of miR-640 of the lumbar disc tissue in the observation group increased significantly(P<0.01).Compared with the Control group,the cell senile level and the cell apoptosis rate of the miR-640 group increased,and the differences were statistically significant(P<0.01).There was no statisticallysignificant difference in the above indicators between the NC group and the Control group(P>0.05).Immunofluo‐rescence confirmed that the expression ofβ-catenin decreased and the nuclear entry ofβ-catenin was inhibited in the miR-640 group,and the differences were statistically significant(P<0.01).Dual luciferase reporter gene experiment confirmed that miR-640 andβ-catenin had a targeted relationship,and the difference was statistically significant(P<0.01).Conclusion The expression level of miR-640 in patients with lumbar disc herniation is increased.miR-640 can promote the senile and apoptosis of nucleus pulposus cells,and its mechanism may be related to the regulation of Wnt signaling pathway.