摘要
目的探讨微小RNA-212(miR-212)对转化因子-β1(TGFβ1)干预A549细胞诱导肺纤维化及上皮-间质转化(EMT)的影响及可能的相关机制。方法体外培养A549细胞,实验分组:PBS组(常规培养)、TGFβ1组(含有5 ng/mL TGFβ1培养液培养)、TGFβ1+anti-miR-con组(转染anti-miR-con后使用含有5 ng/mL TGFβ1培养液培养)、TGFβ1+anti-miR-212组(转染antimiR-212后使用含有5 ng/mL TGFβ1培养液培养)、TGFβ1+anti-miR-212+si-con组(共转染anti-miR-212与si-con后使用含有5ng/mL TGFβ1培养液培养)、TGFβ1+anti-miR-212+si-TOB2组(共转染anti-miR-212与si-TOB2后使用含有5 ng/mL TGFβ1培养液培养)。实时荧光定量聚合酶链反应(qRT-PCR)与蛋白免疫印迹法(Western blot)检测细胞中miR-212与TOB2的表达水平;甲基噻唑基四唑(MTT)检测干扰miR-212的表达对TGFβ1诱导A549细胞增殖的抑制作用,以及下调TOB2的表达对TGFβ1诱导A549细胞增殖的促进作用;双荧光素酶报告基因检测验证miR-212与TOB2的靶向关系;Western blot检测干扰miR-212的表达或下调TOB2的表达对细胞周期蛋白1(CyclinD1)、波形蛋白(Vimentin)、α平滑肌肌动蛋白(α-SMA)、I型胶原a1(COL1a1)、上皮钙黏附素(E-cadherin)表达的影响。结果与PBS组比较,TGFβ1组细胞增殖率显著升高[(100.23±5.64)%比(218.34±16.38)%,P<0.05],miR-212[(1.00±0.14)比(6.07±0.86)]、CyclinD1[(0.54±0.06)比(0.89±0.07)]、Vimentin[(0.26±0.04)比(1.09±0.13)]、α-SMA[(0.43±0.05)比(0.86±0.07)]、COL1a1[(0.57±0.06)比(1.17±0.13)]的表达水平显著升高(P<0.05),而TOB2[(1.02±0.12)比(0.44±0.14)]、E-cadherin[(0.93±0.09)比(0.47±0.06)]的表达水平显著降低(P<0.05);干扰miR-212的表达可显著抑制TGFβ1诱导A549细胞增殖(P<0.05),抑制CyclinD1、Vimentin、α-SMA、COL1a1表达(P<0.05),促进Ecadherin表达(P<0.05);共转染WT-TOB2的miR-212组荧光素酶活性显著低于miR-con组(P<0.05),共转染MUT-TOB2的miR-212组与miR-con组荧光素酶活性比较差异无统计学意义;相较于TGFβ1+anti-miR-212+si-con组,TGFβ1+anti-miR-212+si-TOB2组A549细胞增殖率显著升高[(69.29±8.04)%比(88.27±9.26)%,P<0.05],CyclinD1[(0.59±0.08)比(0.75±0.11)]、Vimentin[(0.42±0.05)比(0.56±0.07)]、α-SMA[(0.38±0.06)比(0.53±0.06)]、COL1a1蛋白[(0.41±0.05)比(0.55±0.04)]表达水平显著升高(P<0.05),E-cadherin蛋白表达水平显著降低[(0.82±0.08)比(0.45±0.06),P<0.05]。结论干扰miR-212的表达可通过上调TOB2的表达而抑制TGFβ1诱导的A549细胞增殖及EMT进而发挥抗肺纤维化作用。
Objective To investigate the effect of microRNA-212(miR-212)on transforming factor-β1(TGFβ1)in the induction of pulmonary fibrosis and epithelial-mesenchymal transition(EMT)in A549 cells and its possible related mechanisms.Methods A549 cells were cultured in vitro,and the experimental group was assigned into PBS group(conventional culture),TGFβ1 group(containing 5 ng/mL TGFβ1 medium culture),TGFβ1+anti-miR-con group(used after transfection of anti-miR-con containing 5 ng/mL TGFβ1 culture medium),TGFβ1+anti-miR-212 group(transfected with anti-miR-212 was cultured with 5 ng/mL TGFβ1 medium),TGFβ1+antimiR-212+si-con group(co-transfected anti-miR-212 and si-con were cultured with 5 ng/mL TGFβ1 medium),TGFβ1+anti-miR-212+si-TOB2 group(co-transfected with anti-miR-212 and si-TOB2 were cultured with 5 ng/mL TGFβ1 medium).The expression levels of miR-212 and TOB2 in cells were detected by qRT-PCR and Western blot.MTT assay interfered with the expression of miR-212 and inhibited the proliferation of A549 cells induced by TGFβ1,and down-regulated the expression of TOB2 on the proliferation of A549 cells induced by TGFβ1.Dual luciferase reporter assays verified the targeting relationship between miR-212 and TOB2.Western blot was used to detect the effect of miR-212 expression or down-regulation of TOB2 expression on the expression of CyclinD1,vimentin,α-SMA,COLA1 and E-cadherin.Results Compared with PBS group,the cell proliferation rate of TGFβ1 group was significantly increased[(100.23±5.64)%vs.(218.34±16.38)%,P<0.05],and the expression levels of miR-212[(1.00±0.14)vs.(6.07±0.86)],CyclinD1[(0.54±0.06)vs.(0.89±0.07)],Vimentin[(0.26±0.04)vs.(1.09±0.13)],α-SMA[(0.43±0.05)vs.(0.86±0.07)]and COL1 a1[(0.57±0.06)vs.(1.17±0.13)]were significantly increased(P<0.05),while TOB2[(1.02±0.12)vs.(0.44±0.14)]and E-cadherin[(0.93±0.09)vs.(0.47±0.06)]were significantly increased.The expression level was significantly lower(P<0.05).Interfering with the expression of miR-212 significantly inhibited the proliferation of A549 cells induced by TGFB1(P<0.05),inhibited the expression of CyclinDl,Vimentin,α-SMA and COL1 a1(P<0.05),and promoted the expression of E-cadherin(P<0.05).The luciferase activity of miR-212 group co-transfected with WT-TOB2 was significantly lower than that of miR-con group(P<0.05).The difference of luciferase activity between miR-212 group and miR-con group co-transfected with MUT-TOB2 was not statistically significant.Compared with TGFβ1+anti-miR-212+si-con group,the proliferation rate of A549 cells in TGFβ1+anti-miR-212+si-TOB2 group was significantly increased[(69.29±8.04)%vs.(88.27±9.26)%,P<0.05],CyclinD1[(0.59±0.08)vs.(0.75±0.11)],Vimentin[(0.42±0.05)vs.(0.56±0.07)],α-SMA[(0.38±0.06)vs.(0.53±0.06)]and COL1 a1 protein[(0.41±0.05)vs.(0.55±0.04)]expression.The level was significantly increased(P<0.05),and the expression level of E-cadherin protein was significantly decreased[(0.82±0.08)vs.(0.45±0.06),P<0.05].Conclusion Interfering with the expression of miR-212 can inhibit TGFβ1-induced A549 cells proliferation and EMT by up-regulating the expression of TOB2 and then exert anti-pulmonary fibrosis.
作者
靳立振
赵永华
于巧青
JIN Lizhen;ZHAO Yonghua;YU Qiaoqing(Department of Severe Medicine,People's Hospital of Langfang,Langfang,Hebei 065000,China)
出处
《安徽医药》
CAS
2021年第2期330-335,共6页
Anhui Medical and Pharmaceutical Journal
