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成纤维细胞的增殖和活化引起的细胞外基质(extracellular matrix,ECM)的累积是肺纤维化的主要病理基础[1]。肺纤维化治疗难度大,且发展到晚期纤维化过程不可逆转,而一些药物的长期使用会提高肺纤维化的发生风险,对于这临床的一类并发症应格外重视。来氟米特(leflunomide,LEF)是治疗类风湿性关节炎的常用药物,但是有临床报道称,长期服用LEF可能提高肺纤维化的发生风险,但是也有研究认为LEF对肺纤维化影响不大[2]。微小RNA(microRNA,miRNA)长度约为18~22个核苷酸,虽然不具备编码功能,但是可通过识别和碱基配对的方式与靶基因信使RNA(message RNA,mRNA)的3'非翻译区(3'UTR)结合,从而参与基因表达的调控[3]。miR-449a具有抑制肿瘤细胞的增殖并诱导凋亡的作用[4],并且最新研究发现miR-449a可能与肺纤维化有关,在二氧化硅诱导的肺纤维化模型中,miR-449a可通过调节自噬缓解纤维化[5]。c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)是调节细胞增殖、凋亡和分化的重要蛋白,其磷酸化后可通过信号转导调控细胞生物学行为[6]。本文发现了miR-449a的过表达会显著缓解由LEF引起的肺成纤维细胞的增殖,而沉默miR-449a对细胞的影响相反,这可能是LEF引起肺纤维化的机制之一,报道如下。
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使用qPCR检测各组细胞中miR-449a表达水平。结果显示mimic组miR-449a水平显著高于对照组,inhibitor组的miR-449a表达水平显著低于对照组(P<0.05),说明转染实验成功。LEF组的miR-449a水平显著低于对照组(P<0.05),并且LEF+mimic组的miR-449a的表达水平显著高于LEF组,LEF+inhibitor组的miR-449a显著低于LEF组(P<0.05)。表明LEF可抑制人成纤维细胞中miR-449a的表达,见表1。
表 1 各组miR-449a表达水平比较
组别 miR-449a 对照组 1.16±0.08 LEF组 0.58±0.05* LEF+mimic组 2.04±0.16# mimic组 6.32±0.63* LEF+inhibitor组 0.41±0.06# inhibitor组 0.77±0.07* *P<0.05,与对照组比较;#P<0.05,与LEF组比较 -
使用CCK-8法检测各组细胞的相对细胞活力。结果显示在第48小时和第72小时,LEF组和inhibitor组的细胞活力显著高于对照组(P<0.05),而mimic组的细胞活力显著低于对照组(P<0.05)。此外,LEF+mimic组的细胞活力显著低于LEF组,LEF+inhibitor组的细胞活力显著高于LEF组(P<0.05),过表达miR-449a可部分逆转LEF对促进人成纤维细胞的细胞活力的作用,而降低miR-449a的水平会进一步促进细胞活力,见表2。
表 2 各组细胞的相对细胞活力比较(%)
组别 24 h 48 h 72 h 对照组 100.07±1.83 100.76±2.07 100.16±1.96 LEF组 103.67±2.06 110.83±2.15* 121.17±2.65* LEF+mimic组 99.98±2.14 98.57±2.11# 97.37±2.01# mimic组 97.54±1.97 91.79±2.35* 81.77±1.78* LEF+inhibitor组 107.68±2.08 118.67±3.07# 132.84±2.07# inhibitor组 104.31±1.79 111.38±2.67* 119.35±2.18* *P<0.05,与对照组比较;#P<0.05,与LEF组比较 -
LEF组和inhibitor组的克隆形成数目显著高于对照组而细胞凋亡率低于对照组(P<0.05),mimic组的克隆形成数目显著低于对照组而细胞凋亡率显著高于对照组(P<0.05)。此外,LEF+mimic组的克隆形成数目显著低于LEF组而细胞凋亡率显著高于LEF组(P<0.05),LEF+inhibitor组的克隆形成数目在LEF的基础上进一步升高而细胞凋亡率进一步降低(P<0.05)。过表达miR-449a可逆转LEF促进肺成纤维细胞增殖和抑制凋亡的作用,而低表达miR-449a会加剧LEF的作用,见表3。
表 3 各组细胞增殖和凋亡情况比较
组别 克隆形成数目(个) 细胞凋亡率(%) 对照组 54.32±4.36 5.53±0.94 LEF组 87.66±7.24* 3.11±0.76* LEF+mimic组 60.82±6.06# 6.73±1.26# mimic组 31.12±3.78* 17.32±3.28* LEF+inhibitor组 119.35±5.08# 2.14±0.62# inhibitor组 92.71±7.89* 3.45±0.83* *P<0.05,与对照组比较;#P<0.05,与LEF组比较 -
本次研究通过免疫荧光技术检测了各组α-SMA的水平来分析细胞向肌细胞转化情况,检测Col I的水平来分析ECM水平。其中蓝色荧光为细胞核,红色荧光为α-SMA或Col I蛋白。LEF组和inhibitor组的荧光强度显著高于对照组(P<0.05),而mimic组的相对荧光强度低于对照组(P<0.05)。此外,LEF+mimic组的相对荧光强度显著低于LEF组(P<0.05),LEF+inhibitor组的相对荧光强度显著高于LEF组(P<0.05)。过表达miR-449a可部分逆转LEF对促进人成纤维细胞α-SMA和Col I表达的促进作用,见图1、图2和表4。
表 4 各组细胞α-SMA相对荧光强度比较
组别 α-SMA Col I 对照组 1.02±0.11 1.24±0.14 LEF组 2.36±0.47* 2.57±0.38* LEF+mimic组 1.53±0.34# 1.89±0.25# mimic组 0.47±0.05* 0.45±0.06* LEF+inhibitor组 3.25±0.18# 4.13±0.54# inhibitor组 2.48±0.15* 3.11±0.39* *P<0.05,与对照组比较;#P<0.05,与LEF组比较 -
LEF组和inhibitor组的p-JNK/JNK水平高于对照组,mimic组的p-JNK/JNK水平显著低于对照组(P<0.05),并且LEF+mimic组中p-JNK/JNK水平显著低于LEF组(P<0.05),LEF+inhibitor组中p-JNK/JNK水平显著高于LEF组(P<0.05)。过表达miR-449a可逆转LEF促进JNK蛋白磷酸化的作用,见表5。
表 5 各组p-JNK/JNK相对水平比较
组别 p-JNK JNK p-JNK/JNK 对照组 2.04±0.18 2.16±0.16 0.94±0.09 LEF组 2.87±0.31 1.05±0.10 2.73±0.18* LEF+mimic组 1.67±0.19 2.24±0.21 0.75±0.07# mimic组 0.96±0.11 3.11±0.28 0.31±0.04* LEF+inhibitor组 3.04±0.24 1.10±0.10 2.76±0.25# inhibitor组 3.78±0.34 1.02±0.09 3.71±0.31* *P<0.05,与对照组比较;#P<0.05,与LEF组比较
Mechanism of leflunomide in regulating pulmonary fibrosis by regulating miR-449a
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摘要:
目的 探究来氟米特(leflunomide,LEF)通过调节微小RNA(microRNA,miR)-449a在肺纤维化中的机制研究。 方法 将人肺成纤维细胞MRC-5分为6组,即对照组、LEF组、LEF+mimic组、mimic组、LEF+inhibitor组和inhibitor组。通过质粒转染miR-449a mimic或inhibitor来过表达或沉默miR-449a,在5 mg/L LEF的条件下培养48 h。分别通过CCK-8法、克隆形成实验和流式细胞术检测各组细胞活力、细胞增殖能力和凋亡率。使用免疫荧光染色检测α平滑肌肌动蛋白(α smooth muscle actin,α-SMA)胶质蛋白I(collagen I,col I)。分别使用qPCR和Western blot检测miRNA和蛋白的水平。 结果 mimic组miR-449a水平显著高于对照组(P<0.05)。LEF组和inhibitor组的miR-449a水平显著低于对照组(P<0.05)。LEF+mimic组的miR-449a的表达水平显著高于LEF组,LEF+inhibitor组的miR-449a水平显著低于LEF组(P<0.05)。LEF组和inhibitor组的细胞活力和细胞增殖能力显著高于对照组(P<0.05)。mimic组的细胞活力和细胞增殖能力显著低于对照组(P<0.05)。LEF+mimic组的细胞活力和细胞增殖能力显著低于LEF组而LEF+inhibitor组的细胞活力显著高于LEF组(P<0.05)。LEF组和inhibitor组的细胞凋亡率低于对照组(P<0.05),mimic组的细胞凋亡率显著高于对照组(P<0.05)。LEF+mimic组的细胞凋亡率显著高于LEF组而LEF+inhibitor组的凋亡率显著低于LEF组(P<0.05)。LEF组和inhibitor组的α-SMA和Col I蛋白的荧光强度显著高于对照组(P<0.05),mimic组的相对荧光强度低于对照组(P<0.05)。LEF+mimic组的α-SMA和Col I蛋白相对荧光强度显著低于LEF组,LEF+inhibitor组的α-SMA和Col I蛋白相对荧光强度显著高于LEF组(P<0.05)。LEF组和inhibitor组的p-JNK/JNK水平高于对照组(P<0.05),mimic组的p-JNK/JNK水平显著低于对照组(P<0.05),LEF+mimic组中p-JNK/JNK水平显著低于LEF组而LEF+inhibitor组的p-JNK/JNK水平显著高于LEF组(P<0.05)。 结论 LEF可能通过抑制肺成纤维细胞中miR-449a的表达激活JNK途径,从而诱导成纤维细胞的活化和增殖,抑制其凋亡,从而引起肺纤维化。 -
关键词:
- 来氟米特 /
- 肺纤维化 /
- 成纤维细胞 /
- α平滑肌肌动蛋白 /
- 微小RNA-449a /
- c-Jun氨基末端激酶
Abstract:Objective To investigate the mechanism of leflunomide (LEF) in regulating pulmonary fibrosis by regulating microRNA (miR)-449a. Methods Human lung fibroblasts MRC-5 were divided into 6 groups: control group, LEF group, LEF+mimic group, mimic group, LEF+inhibitor group and inhibitor group. MiR-449a was overexpressed or silenced by plasmid transfection with miR-449a mimic or inhibitor and ncubate for 48 h at 5 mg / L LEF. The cell viability, cell proliferation ability and apoptotic rate of each group were measured by CCK-8 method, clone formation experiment and flow cytometry. Immunofluorescent staining was used to detect α smooth muscle actin (α-SMA) and collagen I (col I). The levels of miRNA and protein were detected using qPCR and Western blot, respectively. Results The miR-449a level in the mimic group was significantly higher than that in the control group (P<0.05). The level of miR-449a in LEF group and inhibitor group was significantly lower than that in control group (P<0.05). The expression level of miR-449a in LEF+mimic group was significantly higher than that in LEF group, and the level of miR-449a in LEF+inhibitor group was significantly lower than that in LEF group (P<0.05). The cell viability and cell proliferation ability of the LEF group and inhibitor group were significantly higher than those of the control group (P<0.05). The cell viability and cell proliferation ability of the mimic group were significantly lower than those of the control group (P<0.05). The cell viability and cell proliferation ability of the LEF+mimic group were significantly lower than those of the LEF group, while the cell viability of the LEF+inhibitor group was significantly higher than that of the LEF group (P<0.05). The apoptosis rate of LEF group and inhibitor group was lower than that of control group (P<0.05). The apoptosis rate of mimic group was significantly higher than that of control group (P<0.05). The apoptosis rate of LEF+mimic group was significantly higher than that of LEF group, while the apoptosis rate of LEF+inhibitor group was significantly lower than that of LEF group (P<0.05). The fluorescence intensity of α-SMA and Col I proteins in LEF group and inhibitor group were significantly higher than those in control group (P<0.05). The relative fluorescence intensity of mimic group was lower than that of control group (P<0.05). The relative fluorescence intensities of α-SMA and Col I proteins in LEF+mimic group were significantly lower than those in LEF group, while the relative fluorescence intensities of α-SMA and Col I protein in LEF+inhibitor group were significantly higher than those in LEF group (P<0.05). The levels of p-JNK / JNK in LEF group and inhibitor group were higher than those in control group (P<0.05). The p-JNK / JNK level in the mimic group was significantly lower than that in the control group (P<0.05). The level of p-JNK / JNK in LEF+mimic group was significantly lower than that in LEF group, while the level of p-JNK / JNK in LEF+inhibitor group was significantly higher than that in LEF group (P<0.05). Conclusion LEF may activate the JNK pathway by inhibiting the expression of miR-449a in lung fibroblasts, thereby inducing fibroblast activation and proliferation, inhibiting apoptosis, and causing pulmonary fibrosis. -
Key words:
- leflunomide /
- pulmonary fibrosis /
- fibroblasts /
- alpha smooth muscle actin /
- microRNA-449a /
- c-Jun N-terminal kinase
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表 1 各组miR-449a表达水平比较
组别 miR-449a 对照组 1.16±0.08 LEF组 0.58±0.05* LEF+mimic组 2.04±0.16# mimic组 6.32±0.63* LEF+inhibitor组 0.41±0.06# inhibitor组 0.77±0.07* *P<0.05,与对照组比较;#P<0.05,与LEF组比较 表 2 各组细胞的相对细胞活力比较(%)
组别 24 h 48 h 72 h 对照组 100.07±1.83 100.76±2.07 100.16±1.96 LEF组 103.67±2.06 110.83±2.15* 121.17±2.65* LEF+mimic组 99.98±2.14 98.57±2.11# 97.37±2.01# mimic组 97.54±1.97 91.79±2.35* 81.77±1.78* LEF+inhibitor组 107.68±2.08 118.67±3.07# 132.84±2.07# inhibitor组 104.31±1.79 111.38±2.67* 119.35±2.18* *P<0.05,与对照组比较;#P<0.05,与LEF组比较 表 3 各组细胞增殖和凋亡情况比较
组别 克隆形成数目(个) 细胞凋亡率(%) 对照组 54.32±4.36 5.53±0.94 LEF组 87.66±7.24* 3.11±0.76* LEF+mimic组 60.82±6.06# 6.73±1.26# mimic组 31.12±3.78* 17.32±3.28* LEF+inhibitor组 119.35±5.08# 2.14±0.62# inhibitor组 92.71±7.89* 3.45±0.83* *P<0.05,与对照组比较;#P<0.05,与LEF组比较 表 4 各组细胞α-SMA相对荧光强度比较
组别 α-SMA Col I 对照组 1.02±0.11 1.24±0.14 LEF组 2.36±0.47* 2.57±0.38* LEF+mimic组 1.53±0.34# 1.89±0.25# mimic组 0.47±0.05* 0.45±0.06* LEF+inhibitor组 3.25±0.18# 4.13±0.54# inhibitor组 2.48±0.15* 3.11±0.39* *P<0.05,与对照组比较;#P<0.05,与LEF组比较 表 5 各组p-JNK/JNK相对水平比较
组别 p-JNK JNK p-JNK/JNK 对照组 2.04±0.18 2.16±0.16 0.94±0.09 LEF组 2.87±0.31 1.05±0.10 2.73±0.18* LEF+mimic组 1.67±0.19 2.24±0.21 0.75±0.07# mimic组 0.96±0.11 3.11±0.28 0.31±0.04* LEF+inhibitor组 3.04±0.24 1.10±0.10 2.76±0.25# inhibitor组 3.78±0.34 1.02±0.09 3.71±0.31* *P<0.05,与对照组比较;#P<0.05,与LEF组比较 -
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