长链非编码RNA CCAT1在支气管哮喘中的表达及对气道平滑肌细胞的影响

Expression of long non-coding RNA CCAT1 in bronchial asthma and its effects on airway smooth muscle cells

目的探究长链非编码RNA(lncRNA)结肠癌相关转录因子1(CCAT1)对气道平滑肌细胞的影响。方法选取2016年10月至2018年10月邯郸市中心医院收治的56例支气管哮喘急性发作期患儿,及56例同期健康体检儿童。实时荧光定量PCR(qRT-PCR)检测支气管哮喘患儿和健康儿童血浆lncRNA CCAT1表达水平。Lipofectamine 2000^(TM)法构建lncRNA CCAT1过表达、低表达人气管平滑肌细胞系(分别记为pc-CCAT1组、si-CCAT1组),同时设置相应阴性对照(分别为pc-NC组、si-NC组),使用qRT-PCR进行验证;CCK-8实验、BrdU实验分别检测细胞活力、增殖能力,划痕实验检测细胞迁移能力,Western blot检测细胞中B细胞淋巴瘤2(Bcl-2)和Bcl-2相关X蛋白(Bax)表达。结果哮喘组儿童血浆lncRNA CCAT1表达高于健康组(P<0.001),pc-CCAT1组细胞中lncRNA CCAT1表达水平高于pc-NC组(P<0.001),si-CCAT1组细胞中lncRNA CCAT1的表达水平低于si-NC组(P<0.05)。48、72、96小时,pc-CCAT1组细胞的OD值高于pc-NC组(P<0.001),si-CCAT1组细胞的OD值低于si-NC组(P<0.001)。与pc-NC组相比,pc-CCAT1组BrdU阳性细胞百分比、细胞迁移率、Bcl-2蛋白表达升高(P<0.05),Bax蛋白表达降低(P<0.05);与si-NC组相比,si-CCAT1组BrdU阳性细胞百分比、细胞迁移率、Bcl-2蛋白表达降低(P<0.05),Bax蛋白表达升高(P<0.001)。结论支气管哮喘患儿血浆CCAT1表达上调,lncRNA CCAT1参与气道平滑肌细胞的增殖、迁移和凋亡过程。

Objective To explore the effects of long non-coding RNA(lncRNA)colon cancer-associated transcription 1(CCAT1)on airway smooth muscle cells.Methods A total of 56 children with acute bronchial asthma admitted to Handan Central Hospital from October 2016 to October 2018,as well as 56 children with physical examination at the same time,were selected.The expression level of plasma lncRNA CCAT1 in children with bronchial asthma and healthy children was detected by real-time quantitative PCR(qRT-PCR).The human tracheal smooth muscle cell lines with lncRNA CCAT1 overexpression and low-expression were constructed by Lipofectamine 2000^(TM) method(pc-CCAT1 group and si-CCAT1 group).And corresponding negative controls(pc-NC group and si-NC group)were set up.The verification was conducted by qRT-PCR.The cells activity and proliferation ability were detected by CCK-8 and BrdU assay.The migration ability of cells was detected by scratch assay.The expressions of B-cell lymphoma 2(Bcl-2)and Bcl-2 associated X protein(Bax)were detected by Western Blot.Results The expression of plasma lncRNA CCAT1 in asthma group was higher than that in healthy group(P<0.001).The expression level of lncRNA CCAT1 in cells in pc-CCAT1 group was higher than that in pc-NC group(P<0.001),which was lower in si-CCAT1 group than si-NC group(P<0.05).At 48 h,72 h and 96 h,OD value of cells in pc-CCAT1 group was greater than that in pc-NC group(P<0.001),which was lower in si-CCAT1 group than si-NC group(P<0.001).Compared with pc-NC group,percentage of BrdU-positive cells,cell migration rateand Bcl-2 expression increased(P<0.05),while Bax decreased in pc-CCAT1 group(P<0.05).Compared with si-NC group,percentage of BrdU-positive cells,cell migration rateand Bcl-2 expression were reduced(P<0.05),while Bax was reduced in si-CCAT1 group(P<0.001).Conclusions The expression of plasma CCAT1 is up-regulated in children with bronchial asthma.lncRNA CCAT1 is involved in the proliferation,migration and apoptosis of airway smooth muscle cells.