| 靳立振,赵永华,于巧青.微小 RNA-212调控 TOB2蛋白表达对转化因子 -β1干预 A549细胞诱导肺纤维化及上皮 -间质转化的影响[J].安徽医药,2021,25(2):330-335. |
| 微小 RNA-212调控 TOB2蛋白表达对转化因子 -β1干预 A549细胞诱导肺纤维化及上皮 -间质转化的影响 |
| miR-212 affects TGFβ1 intervention in A549 cells to induce pulmonary fibrosis and EMT by regulating TOB2 expression |
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| DOI：10.3969/j.issn.1009-6469.2021.02.029. |
| 中文关键词: 肺纤维化 细胞增殖 微小 RNA-212 TOB2 转化因子 -β1 A549细胞 上皮 -间质转化 |
| 英文关键词: Pulmonary fibrosis Cell proliferation miR-212 TOB2 TGFβ1 A549 cells EMT |
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| 中文摘要: |
| 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 5 ng/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［(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 miR212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GFβ1+ anti-miR-212+ 的表，光素，T,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。 |
| 英文摘要: |
| 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 5ng/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 A549cells 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,αSMA,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 COL1a1 [(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 TGFβ1 (P < 0.05), inhibited the expression of CyclinD1, Vimentin, α-SMA and COL1a1 (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 COL1a1 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. |
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