miR-34a靶向STAT1基因调控牙周膜细胞增殖及凋亡的分子机制

Molecular Mechanisms of MiR-34a Targeting STAT1 Gene Regulating Proliferation and Apoptosis of Periodontal Ligament Cells

目的:探讨miR-34a靶向STAT1基因调控牙周膜细胞增殖及凋亡的分子机制。方法:将PDLCs细胞分成Pm、Py、Pz三组,Pz组(PDLCs细胞不做任何处理),Pm组(转染miR-34a模拟物)、Py组(转染miR-34a抑制物),RT-PCR检测STAT1、miR-34a水平,Westernblot法检测STAT1蛋白表达,流式细胞仪、MTT法分别检测PDLCs细胞凋亡、增殖情况。结果:RTPCR检测STAT1、miR-34a水平结果显示,与Pz组对比,Py组STAT1、miR-34a水平有显著下降,Pm组STAT1、miR-34a水平最高,Pz组STAT1、miR-34a水平其次,Py组STAT1、miR-34a水平最低(均P<0.05)。Westernblot法检测STAT1蛋白发现,与Pz组对比,Py组STAT1蛋白较Pm组有显著下降,Pm组STAT1蛋白表达最高,Pz组蛋白表达其次,Py组STAT1蛋白表达最低(均P<0.05)。流式细胞术检测发现,与Pz组对比,Pm组PDLCs细胞凋亡明显增加,与Pm组对比,Py组细胞凋亡明显降低(均P<0.05),Pm组细胞凋亡呈三组最高,Pz组其次,Py组最低。MTT检测结果显示:Pz组PDLCs细胞在24h、48h、72h时均缓慢增长,Py组PDLCs细胞24h、48h增殖变化较平稳,72h时增殖速度明显加快,Pm组受miR-34a模拟物影响,PDLCs细胞数量增殖缓慢(均P<0.05)。结论:miR-34a低表达能够抑制PDLCs细胞凋亡,促进其生长,这一作用机制可能与降低STAT1蛋白表达有关。

Objective To investigate the molecular mechanism of the proliferation and apoptosis of periodontal ligament cells regulated by STAT1 gene targeting to miR-34a.Methods Human periodontal ligament cells were isolated,cultured and diluted.According to Lipofectamine 2000 instructions,PDLCs were divided into three groups:Pm group,Py group and Pz group.Pz group(PDLCs were not treated with any treatment),Pm group(transfected mimic of miR-34a),Py group(transfected microarray).miR-34a inhibitor,transfected for about 4 hours,discarded the upper liquid,replaced the medium,cultured for 48 hours.RT-PCR was used to detect the levels of STAT1 and miR-34a.Western blot was used to detect the expression of STAT1.Flow cytometry and MTT were used to detect the apoptosis and proliferation of PDLCs.Results RT-PCR detection of STAT1 and miR-34a levels showed that compared with Pz group,STAT1 and miR-34a levels in Py group decreased significantly.STAT1 and miR-34a levels in Pm group were the highest,followed by STAT1 and miR-34a levels in Pz group,and STAT1 and miR-34a levels in Py group were the lowest(P<0.05).Western blot analysis of STAT1 protein showed that compared with Pz group,the expression of STAT1 protein in Py group was significantly lower than that in Pm group.The expression of STAT1 protein in Pm group was the highest,followed by that in Pz group,and the expression of STAT1 protein in Py group was the lowest(P<0.05).Flow cytometry showed that compared with Pz group,the apoptosis of PDLCs in Pm group increased significantly,and decreased significantly in Py group(P<0.05).The apoptosis of PDLCs in Pm group was the highest in three groups,followed by Pz group and the lowest in Py group.MTT results showed that PDLCs in Pz group grew slowly at 24h,48h and 72h.PDLCs in Py group increased steadily at 24h and 48h,and increased significantly at 72h.The number of PDLCs in Pm group increased slowly under the influence of miR-34a(P<0.05).Conclusion The low expression of miR-34a can inhibit PDLCs cell apoptosis and promote its growth.This mechanism may be related to the reduction of STAT1 protein expression.