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miR-34a靶向STAT1基因调控牙周膜细胞增殖及凋亡的分子机制

来源:用户上传      作者:吴雪 段少宇 梁萍 刘欣

[摘要]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:RT-PCR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。

[关键词]miR-34a;STAT1;牙周膜细胞;牙周炎;增殖;凋亡

[中圖分类号]R329.2+8 [文献标志码]A [文章编号]1008-6455(2021)04-0115-04

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

WU Xue,DUAN Shao-yu,LIANG Ping,LIU Xin

(Department of Stomatology,Beijing Electric Power Hospital,Beijing 100073,China)

Abstract: 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.

Key words: miR-34a; STAT1; periodontal ligament cells; periodontitis; proliferation; apoptosis

牙周炎是牙周组织发生慢性炎症病变,患者可能会出现牙龈出血、牙槽骨破坏和牙周袋等症状,随着病情的发展还会发展为牙齿松动和脱落,是现在成年人牙齿缺失的主要原因[1]。牙周炎是常见的口腔疾病,研究表明,牙周膜细胞(PDLCs)生长失衡是导致牙周炎出现的主要原因[2],PDLCs细胞凋亡会引起其他疾病,严重威胁人们的身体健康[3]。近些年研究发现牙周炎中的致病菌主要对患者体内的炎症反应和免疫反应起作用,PDLCs细胞作为牙周支持组织必不可少的一部分,具有效应性,会受到炎性因子影响增长受限或过剩,牙体出现疾病也与PDLCs细胞有关[4-5]。miR-34a是微小RNA之一,有相关研究证明,miR-34a会激活恶性生物活性和恶性特质,加重疾病进程,PDLCs细胞在维持牙周功能正常运转有重要作用,在牙周炎组织中miR-34a表达水平上升,PDLCs凋亡细胞明显增加,而当miR-34a水平下降时,能有效改善牙周组织的损伤,说明miR-34a与PDLCs凋亡程度呈正相关[6-7]。STAT1信号分布在机体多个组织细胞内,多种因子参与和调控PDLCs细胞分化,抑制STAT1信号表达可以明显减少PDLCs细胞凋亡,说明STAT1信号在牙周炎发展过程中起重要作用[8]。由此,本文研究通过建立实验分组,探讨miR-34a靶向STAT1基因调控PDLCs细胞增殖及凋亡的分子机制。

1 材料和方法

1.1 主要试剂和仪器:RT-PCR试剂盒(上海Superchip technology),Trizol试剂(浙江AMEKO),逆转录试剂盒(武汉 赛维尔),RT-PCR试剂盒(上海 优予),miR-34a-mimics、miR-34a-inhibitions(上海 吉玛),无龋坏牙体组织。

1.2 人PDLCs细胞分离:生理盐水清洗牙体组织,放入10%胎牛血清的α-MEM培育液再次清洗,刮取下根部组织剪碎,加入消化液混匀,放置在准备好的培养箱中静置12~14h,37℃下培养,消化后取出移至EP管中,经800r/min离心后弃去上清,移入培养箱中加入1ml培养液进行培养,对细胞生长状况进行观察。

1.3 细胞培养传代:初次接种细胞呈圆形,悬浮状态,1d后逐渐贴壁,2d后贴壁细胞呈梭形或多边形,7d后細胞密度生长至80%~90%,传代培养弃去培养液,0.25%胰蛋白酶消化,用吸管进行吹打,制作细胞悬液,移植新培养瓶中,继续培养收集第5代细胞,将PDLCs细胞稀释至1×105/ml,平铺到16孔板中,待PDLCs细胞密度生长为60%~80%时进行转染操作。

1.4 细胞转染和分组:将稀释的PDLCs细胞加入培养皿中进行转染,按照Lipofectamine 2000说明书操作进行,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),转染4h左右,弃去上层液体,更换培养基,培养48h。

1.5 RT-PCR检测STAT1、miR-34a水平:PDLCs细胞总RNA运用Trizol法进行提取,总RNA提取反转录成cDNA,按说明书进行实验,用DNA荧光染料SYBR GreenⅠ对STAT1、miR-34a表达水平进行检测,内参采用β-actin、60℃、10min,95℃、72℃、各30s,95℃、5min,循环次数以40为准,实验次数至少3次。在细胞培养液中加入氯仿,摇荡,离心,4℃ 1 200转离心,加异丙醇、75%乙醇,离心,干燥,-80℃保存。总RNA提取反转录成cDNA,内参采用U6,65℃,15min、95℃、72℃,各45s、95℃,15min,以40次为准,实验次数至少3次。用相对定量2-ΔΔCT计算STAT1、miR-34a表达。

1.6 Westernblot法检测STAT1蛋白表达:在6孔板中加入100μg的胰蛋白酶提取液,再注入2ml的培养基,将转染后的细胞放入EP管中,并与胰蛋白酶提取液按照1:100进行混合,再放入冰箱中冷冻10min,使细胞完全裂变,成为E溶液;在EP管中按照1:100比例加入胰蛋白酶提取液2ml,使细胞完全裂变成为F溶液,把E和F溶液以80:1的体积进行摇匀,配制成工作液,放入37.5℃的保温箱中20min,冷却后计算STAT1蛋白浓度。

1.7 流式细胞仪检测PDLCs细胞凋亡程度:用PBS溶液洗涤转染2d后的细胞2次,离心5min,重悬细胞在100μl结合缓冲液中进行,用5μl标记FITC的AnnexinⅤ与5μl PI染色混匀,避光孵育15min后加入400μl结合缓冲液混匀,洗涤3次对细胞凋亡情况进行检测。

1.8 MTT检测PDLCs细胞增殖能力:PDLCs细胞离心转染3~5min,在96孔板中接种细胞进行培养,每孔200μl,室温37℃,5% CO2,培养时长不少于24h,不大于72h,加入MTT培养不少于3h,不大于4h,加入150μl的DMSO混匀,测光密度用酶标仪进行测定(OD490nm),对细胞活力进行分析。

1.9 统计学分析:用SPSS 19.0软件对STAT1、miR-34a水平变化、STAT1蛋白表达、细胞增殖调亡情况进行统计和分析,用均值±标准差表示计算出的结果,组间两两比对,用χ2或t检验,P<0.05为差异有统计学意义。

2 结果

2.1 STAT1、miR-34a水平变化:RT-PCR检测STAT1、miR-34a水平结果显示,与Pz组比对,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,组间比较差异有统计学意义(均P<0.05)。

2.2 STAT1蛋白变化情况:Western blot法检测STAT1蛋白发现,Pz、Pm、Py三组细胞STAT1蛋白表达量分别为1.06±0.23、1.51±0.48、0.74±0.13,与Pz组比对,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,组间比较差异有统计学意义(均P<0.05)。

2.3 PDLCs细胞调亡情况:流式细胞术检测发现,与Pz组相比,Pm组PDLCs细胞凋亡明显增加,Py组细胞凋亡明显降低,Pm组细胞凋亡呈三组最高,Pz组其次,Py组最低,组间比较差异有统计学意义(均P<0.05)。

2.4 MTT检测PDLCs细胞增殖能力: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)。

3 讨论

牙周炎是一种口腔科的慢性疾病,主要表现在牙龈炎症和牙槽骨病理性吸收,引起牙周炎的原因有很多种,例如:牙周膜细胞分化异常,免疫疾病和遗传等因素[9]。有大数据表明,我国有90%以上的人群都患有牙龈炎症,临床上表现为患者咀嚼功能降低,牙周炎的发生也有可能与患者的心血管疾病和糖尿病紧密相关,对人们的生活造成严重不便[10]。研究表明牙周炎患者体内STAT1水平出现失衡,宿主免疫、PDLCs细胞功能受到损伤,这些原因已经成为许多学者研究牙周炎机制的重点[11]。miR-34a广泛存在于哺乳动物中,具有组织特异性,同样广泛存在人体组织中,参与组织细胞的生长,但在一定程度上会抑制正常细胞的再生,甚至会加速促进PDLCs细胞的凋亡[12]。

通过观察STAT1、miR-34a水平,STAT1蛋白表达发现,与Pz组比对,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。与Pz组比对,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。有研究证实,miR-34b和miR-34c都能对细胞增殖、凋亡和调控以及纤维化产生影响,近些年来,与牙周炎相关的miRNA不断被发现,miR-34a在牙周膜细胞分化过程中的调控机制也逐漸被证实[13-14]。有研究证实,STAT1近年来被发现成为与细胞组织分化密切相关的信号通路分子[15]。STAT3是属于STAT家族中的转录活化因子蛋白,当STAT3发生磷酸化的时候形成二聚体,并转移进细胞核与染色体上靶基因启动子进行结合,诱导位于其下游的基因即靶基因转录激活。同时磷酸化的STAT3可以调控细胞G1/S期及G2/M期的调控因子使细胞生存周期延长,进而促进细胞的恶性转变。有研究发现miR-34a通过PI3K/Akt/Bad途径靶向GAS1促进甲状腺乳头状癌细胞增殖和抑制凋亡,而PI3K/AKT/mTOR抑制剂能反馈激活STAT3激酶活性,因此,推测MiR-34a调控STAT3蛋白可能与PI3K/Akt信号通路有关[16-17]。

通过流式细胞术检测发现,与Pz组比对,Pm组PDLCs细胞凋亡明显增加,Py组细胞凋亡明显降低,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。本实验在预测可能调控STAT1信号通路的miRNA中发现有miR-34a的结合位点,用MTT与流式细胞术检测得出,STAT1的基因活性受到miR-34a的影响可以对其表达进行调控[18-19]。同样证实,miR-34a的表达量上升,细胞再生过程中STAT1可能起到抑制作用。而有研究表明,STAT1是miR-34a的靶基因,STAT1与PDLCs细胞的增殖和凋亡有密切关系[20]。另研究发现,STAT1对PDLCs细胞起关键性作用。miR-34a和STAT1二者在调控细胞凋亡中发挥重要作用[21]。这与本文的研究结果一致。Xue Jin研究证实,促进PDLCs细胞分化可通过下调miR-34a的表达来实现,让PDLCs细胞恢复功能活性,这说明miR-34a可以有效反映PDLCs细胞凋亡水平[22],这与本文研究相符。

综上所述,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。

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[收稿日期]2019-10-21

本文引用格式:吴雪,段少宇,梁萍,等.miR-34a靶向STAT1基因调控牙周膜细胞增殖及凋亡的分子机制[J].中国美容医学,2021,30(4): 115-118.


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