转化生长因子-β1对肺成纤维细胞中lncRNA-ATB和纤维化因子mRNA水平的影响
Effects of transforming growth factor-β1 on mRNA expression levels of lncRNA-ATB and fibrosis factors in pulmonary fibroblasts
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摘要:
目的 研究转化生长因子（TGF）-β1刺激肺成纤维细胞MRC-5后被TGF-β活化的长链非编码RNA（lncRNA-ATB）和Ⅰ型胶原蛋白（Col I）、Ⅲ型胶原蛋白（Col Ⅲ）以及纤连蛋白（FN）等纤维化因子基因mRNA水平的变化及其相关性。
方法 将成纤维细胞株MRC-5以5×103个/孔的密度种于96孔板中，分别经浓度为0、2.5、5.0、10.0 ng/mL的TGF-β1刺激24 h后，通过MTT法测定细胞增殖活性，采用实时荧光定量PCR法检测MRC-5细胞中lncRNA-ATB、Col I、Col Ⅲ、FN的mRNA相对水平。采用Spearman秩相关检验分析lncRNA-ATB与Col I、Col Ⅲ、FN 3个基因mRNA相对水平之间的相关性。
结果 随着TGF-β1刺激浓度的增加，MRC-5细胞增殖活性增高，5.0、10.0 ng/mL组的细胞增殖活性分别是0 ng/mL组的1.07倍和1.12倍（P < 0.05）。随TGF-β1浓度的增加，2.5、5.0、10.0 ng/mL组中FN的mRNA水平分别是0 ng/mL组的2.11倍（P=0.018）、3.58倍（P=0.03）和6.51倍（P < 0.01）；2.5、5.0、10.0 ng/mL组中Col I的mRNA水平分别是0 ng/mL组的1.28倍（P=0.324）、4.64倍（P < 0.01）和3.97倍（P < 0.01）；2.5、5.0、10.0 ng/mL组中Col Ⅲ的mRNA水平分别是0 ng/mL组的1.21倍（P=0.400）、1.42倍（P=0.120）和4.23倍（P < 0.01）。2.5、5.0、10.0 ng/mL组中lncRNA-ATB的mRNA水平分别是0 ng/mL组的1.80倍（P=0.073）、3.57倍（P < 0.01）和4.55倍（P < 0.01）。此外，相关性分析结果显示，lncRNA-ATB与FN（r=0.86）、Col I（r=0.85）和Col Ⅲ（r=0.82）的mRNA相对水平呈正相关关系（均P < 0.05）。
结论 TGF-β1可刺激成纤维细胞增殖，升高lncRNA-ATB、Col I、Col Ⅲ、FN的mRNA相对水平。lncRNA-ATB与纤维化因子的表达呈正相关，提示其可能与肺纤维化的发生有关。
Abstract:Objective To study the changes of mRNA expression levels of long non-coding RNA activated by transforming growth factor β (lncRNA-ATB), collagen type Ⅰ (Col I), collagen type Ⅲ (Col Ⅲ) and fibronectin (FN) in lung fibroblasts MRC-5 stimulated by transforming growth factor (TGF)-β1.
Methods The fibroblast cell line MRC-5 was seeded in a 96-well plate at a density of 5×103 cells/well, and stimulated by TGF-β1 at concentrations of 0, 2.5, 5.0, and 10.0 ng/mL for 24 h. The proliferation activity was measured by MTT method, and the relative mRNA levels of lncRNA-ATB, Col I, Col Ⅲ, and FN in stimulated MRC-5 cells were detected by real-time quantitative PCR. Spearman rank correlation test was used to assess the correlations of lncRNA-ATB mRNA levels with Col I, Col Ⅲ, and FN mRNA levels.
Results With the increasing of TGF-β1 dose, the proliferation activity of MRC-5 cells increased, and the cell proliferation activities of the 5.0 and 10.0 ng/mL groups were 1.07 and 1.12 times of the 0 ng/mL group (P < 0.05). With the increasing of TGF-β1 dose, the mRNA levels of FN in the 2.5, 5.0, and 10.0ng/mL groups were 2.11 (P=0.018), 3.58 (P=0.03), and 6.51 (P < 0.01) times of the 0ng/mL group, respectively; the mRNA levels of Col I in the 2.5, 5.0, and 10.0 ng/mL groups were 1.28 (P=0.324), 4.64 (P < 0.01), and 3.97 (P < 0.01) times of the 0 ng/mL group, respectively; the mRNA levels of Col Ⅲ in the 2.5, 5.0, and 10.0 ng/mL groups were 1.21 (P=0.400), 1.42 (P=0.120), and 4.23 (P < 0.01) times of the 0 ng/mL group, respectively; the mRNA levels of lncRNA-ATB in the 2.5, 5.0, 10.0 ng/mL groups were 1.80 (P=0.073), 3.57 (P < 0.01), and 4.55 (P < 0.01) times of the 0 ng/mL group, respectively. The results of correlation analysis showed that the mRNA level of lncRNA-ATB was positively correlated with the mRNA levels of FN (r=0.86), Col I (r=0.85) and Col Ⅲ (r=0.82) (Ps < 0.05).
Conclusion TGF-β1 could stimulate cell proliferation of fibroblasts, and increase the relative mRNA levels of lncRNA-ATB, Col I, Col Ⅲ, and FN. LncRNA-ATB is positively correlated with Col I, Col Ⅲ, and FN, suggesting that it may be related to the occurrence of pulmonary fibrosis.
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