Mechanism of leflunomide in regulating pulmonary fibrosis by regulating miR-449a

LIU Dong; LAI Weinan

doi:10.12206/j.issn.1006-0111.201910073

Volume 38 Issue 4

Jul. 2020

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LIU Dong, LAI Weinan. Mechanism of leflunomide in regulating pulmonary fibrosis by regulating miR-449a[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(4): 296-300, 306. doi: 10.12206/j.issn.1006-0111.201910073

Citation:

LIU Dong, LAI Weinan. Mechanism of leflunomide in regulating pulmonary fibrosis by regulating miR-449a[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(4): 296-300, 306. doi: 10.12206/j.issn.1006-0111.201910073

Mechanism of leflunomide in regulating pulmonary fibrosis by regulating miR-449a

doi: 10.12206/j.issn.1006-0111.201910073

LIU Dong^1
,,
LAI Weinan²

1.
Department of Pharmacy, Xinqiao Hospital, Army Military Medical University, Chongqing 400037, China
2.
Department of Rheumatology and Immunology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

Received Date: 2019-10-22
Rev Recd Date: 2020-03-09

Available Online: 2020-07-27

Publish Date: 2020-07-25

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.
- leflunomide,
- pulmonary fibrosis,
- fibroblasts,
- alpha smooth muscle actin,
- microRNA-449a,
- c-Jun N-terminal kinase

References

[1]	JOSHI S, SINGH A R, WONG S S, et al. Rac2 is required for alternative macrophage activation and bleomycin induced pulmonary fibrosis; a macrophage autonomous phenotype[J]. PLoS One,2017,12(8):e0182851-e0182856. doi: 10.1371/journal.pone.0182851
[2]	REN L M, LI R, CHEN L N, et al. Efficacy and safety of weekly leflunomide for the treatment of early rheumatoid arthritis: a randomized, multi-center study[J]. Int J Rheum Dis,2016,19(7):651-657. doi: 10.1111/1756-185X.12677
[3]	BOCKHORN J, DALTON R, NWACHUKWU C, et al. MicroRNA-30c inhibits human breast tumour chemotherapy resistance by regulating TWF1 and IL-11[J]. Nat Commun,2013,4: 4(1):1393-1398.
[4]	LI J, LU M J, JIN J, et al. MiR-449a suppresses tamoxifen resistance in human breast cancer cells by targeting ADAM22[J]. Cell Physiol Biochem,2018,50(1):136-149. doi: 10.1159/000493964
[5]	HAN R H, JI X M, RONG R, et al. MiR-449a regulates autophagy to inhibit silica-induced pulmonary fibrosis through targeting Bcl2[J]. J Mol Med,2016,94(11):1267-1279. doi: 10.1007/s00109-016-1441-0
[6]	TANG Y N, GENG Q, CHEN D, et al. Germline proliferation is regulated by somatic endocytic genes via JNK and BMP signaling in Drosophila[J]. Genetics,2017,206(1):189-197. doi: 10.1534/genetics.116.196535
[7]	HUANG Y, MA S F, ESPINDOLA M S, et al. Microbes are associated with host innate immune response in idiopathic pulmonary fibrosis[J]. Am J Respir Crit Care Med,2017,196(2):208-219. doi: 10.1164/rccm.201607-1525OC
[8]	DENG F, ZHANG P, FENG J, et al. Effect of leflunomideon inflammatory factors and immune function in rats with chronic glomerulonephritis[J]. J Sichuan Univ Med Sci Ed,2016,47(2):217-221.
[9]	PATEL A, ZHANG S J, PARAMAHAMSA M, et al. Leflunomide induces pulmonary and hepatic CYP1A enzymes via aryl hydrocarbon receptor[J]. Drug Metab Dispos,2015,43(12):1966-1970. doi: 10.1124/dmd.115.066084
[10]	SCOTT D L. Interstitial lung disease and disease modifying anti-rheumatic drugs[J]. Lancet,2004,363(9416):1239-1240.
[11]	SAKAI F, NOMA S, KURIHARA Y, et al. Leflunomide-related lung injury in patients with rheumatoid arthritis: imaging features[J]. Mod Rheumatol,2005,15(3):173-179. doi: 10.3109/s10165-005-0387-9
[12]	RYU C, SUN H X, GULATI M, et al. Extracellular mitochondrial DNA is generated by fibroblasts and predicts death in idiopathic pulmonary fibrosis[J]. Am J Respir Crit Care Med,2017,196(12):1571-1581. doi: 10.1164/rccm.201612-2480OC
[13]	ISHIDA Y, KIMURA A, NOSAKA M, et al. Essential involvement of the CX3CL1-CX3CR1 axis in bleomycin-induced pulmonary fibrosis via regulation of fibrocyte and M2 macrophage migration[J]. Sci Rep,2017,7(1):16833-16837. doi: 10.1038/s41598-017-17007-8
[14]	LIANG L, HUIJUAN L, LIANJIANG D, et al. MiR-449a suppresses LDHA-mediated glycolysis to enhance the sensitivity of non-small cell lung cancer cells to ionizing radiation[J]. Oncol Res Feat Preclin Clin Cancer Therap,2017,26(4):547-556.
[15]	WU D D, LIU J, CHEN J L, et al. MiR-449a suppresses tumor growth, migration, and invasion in non-small cell lung cancer by targeting a HMGB1-mediated NF-κB signaling pathway[J]. Oncol Res,2019,27(2):227-235. doi: 10.3727/096504018X15213089759999
[16]	ZHANG J, GAO F D, NI T J, et al. Linc-POU3F3 is overexpressed in in-stent restenosis patients and induces VSMC phenotypic transformation via POU3F3/miR-449a/KLF4 signaling pathway[J]. Am J Transl Res,2019,11(7):4481-4490.
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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。

1. 材料与方法

1.1. 试剂与仪器

人肺成纤维细胞MRC-5购自美国ATCC；LEF（苏州长征-欣凯制药有限公司，国药准字H20000550）；RPMI-1640培养基以及血清购自美国Gibco公司；miR-449a mimic和inhibitor质粒由Genepharma公司构建；

Lipofectamine^TM 2000（美国Invitrogen公司）；荧光显微镜（Olympus BX51）；Model 680酶标仪（Bio-Rad，美国）；流式细胞仪（BD FACScanto II，Becton Dickinson，美国）。CCK-8试剂盒（武汉华美公司）；凋亡试剂盒（美国Thermo Fisher）；PVDF膜（美国Bio-Rad公司）；抗体购自美国Abcam公司，逆转录试剂盒TaKaRa和SYBR Prellix Ex TaqTM实时PCR试剂盒购自TaKaRa（日本）。

1.2. 细胞分组和转染

MRC-5细胞在RPMI-1640培养基中培养，温度为37 ℃，CO₂浓度为5%。细胞被分为6组，即对照组、LEF组、LEF+mimic组、mimic组、LEF+inhibitor组和inhibitor组。其中LEF+mimic组和mimic组通过转染miR-449a mimic质粒过表达miR-449a的水平，LEF+inhibitor组和inhibitor组通过转染miR-449a inhibitor质粒使miR-449a的水平降低。对照组转染空载质粒。LEF组、LEF+mimic组和LEF+inhibitor组分别在5 mg/L LEF的条件下培养48 h。

1.3. 检测指标和方法

1.3.1. qPCR检测miR-449a

将细胞裂解后收集总RNA并检测纯度，通过逆转录试剂盒合成cDNA，然后进行PCR反应，步骤如下：95 ℃下2 min，95 ℃下15 s，60 ℃下25 s和72 ℃下60 s，共进行40个循环。以U6作为内参，使用2^-ΔΔCT法分析miR-449a水平。引物序列如下（5′-3′），miR-449a上游引物：TGCGGTGGCAGTGTATTGTTAGC，下游引物：CCAGTGCAGGGTCCGAGGT；U6上游引物：GGGCAGGAAGAGGGCCTAT，下游引物：TATGGCTAGCATGACTGGT。

1.3.2. CCK-8法检测细胞活力

将细胞调节至2×10⁴个细胞/ml的密度，接种于96孔板中，100 μl/孔。再培养24、48和72 h后将10 μl的CCK-8试剂加入至每孔中，37 ℃下培养2 h。在酶标仪上测量450 nm处的吸光度（A），计算相对细胞活力。

1.3.3. 克隆形成检测细胞增殖能力

分别将各组细胞200个细胞在6孔板中培养，每3天补充一次培养基，培养2周。用PBS洗涤细胞并加入甲醇固定15 min，加入使用结晶紫染色30 min，在显微镜下观察克隆形成的数目，≥50个细胞的集落为一个克隆形成。

1.3.4. 流式细胞术检测细胞凋亡

将细胞洗涤后重悬于结合缓冲液中，使细胞浓度为2.5×10⁵个/ml。根据试剂盒说明书将试剂加入细胞中，并通过流式细胞术分析细胞凋亡情况。

1.3.5. 免疫荧光染色

通过免疫荧光染色检测α平滑肌肌动蛋白（α smooth muscle actin，α-SMA）和胶质蛋白I（collagen I，Col I）的表达情况分析细胞的表型和细胞外基质。然后将细胞在4 ℃下使用α-SMA的抗体染色过夜，用异硫氰酸四甲基罗丹明的山羊抗兔抗体染色30 min。然后再于避光条件下利用4',6-二脒基-2-苯基吲哚（4',6-diamidino-2-phenylindole，DAPI）对细胞核染色10 min，通过荧光显微镜观察。

1.3.6. Western blot检测蛋白表达

通过Western blot检测JNK和磷酸化JNK（p-JNK）蛋白的水平。将细胞裂解、离心收集总蛋白并检测蛋白浓度。使用10%的SDS-PAGE凝胶用于电泳分离蛋白，电泳后使用PVDF膜转膜并在室温下用5%无脂牛奶封闭2 h。分别加入一抗（稀释1:1 000）室温震荡2 h，后在4 ℃孵育过夜，加入二抗（稀释1:5 000），孵育3 h。通过Quantity One软件分析条带的灰度值并以GAPDH为参照计算目标蛋白质的表达量。

1.4. 统计学处理

实验数据采用SPSS 19软件进行处理，实验结果以平均值±标准偏差（SD）表示，组间比较采用单因素方差分析和t检验，统计学显著性表示为P<0.05。

3. 讨论

肺纤维化是一种慢性进行性肺部疾病，但是临床上尚无治疗肺纤维化的特效方法和药物，目前用于进行性肺纤维化的唯一有效治疗方法是肺移植^[7]，若患者未接受肺移植，通常在诊断后的3至5年内出现肺功能丧失导致呼吸衰竭和死亡。肺纤维化的病理特征包括纤维增生和ECM沉积过多，但是这个过程较为漫长，并且在早期症状不明显，也缺乏相应的诊断手段，在患者确诊为肺纤维化时再采取治疗效果有限。因此虽然LEF是否会引起肺纤维化尚无定论，但是由于肺纤维化的恶性预后和致死率，LEF治疗过程中的肺纤维化风险仍是临床重点关注的问题。研究LEF促进肺纤维化的机制是寻找诊断和治疗肺纤维化新方法的重要途径。

LEF是一种调节免疫的药物，其作用机制通过抑制二氢乳清酸脱氢酶来抑制T淋巴细胞和其他类型细胞的细胞周期进程^[8]。LEF引起的肺纤维化并导致患者死亡的病例随着LEF使用时间的增加而升高^[9]。一项长期的调查随访报告指出，在5911例使用LEF治疗的患者中，共出现了80例间质性肺病，其中有27例患者死亡，并且结果判定其中有18例患者的死亡是由于LEF直接导致^[10-11]。在肺纤维化的过程中，成纤维细胞向成肌纤维细胞转化和ECM的累积是两大特点^[12]。因此本文主要分析了LEF对人肺成纤维细胞的影响，结果显示LEF可显著促进成纤维细胞的细胞活力和增殖，抑制其凋亡，并诱导细胞表达大量的α-SMA蛋白和ECM累积。α-SMA是上皮细胞向间质细胞转化的检测指标之一，也是体外研究肺纤维化的最常用指标，而Col I是ECM的主要成分^[13]。作者提示了LEF可通过活化成纤维细胞和促进其增殖参与肺纤维化。

为进一步分析LEF调节肺成纤维细胞增殖和表达α-SMA的机制，我们检测了miR-449a在其中的作用。miR-449a是近年来新发现的一种miRNA，研究已经证实了其可通过靶向并诱导靶基因mRNA降解，抑制肺癌细胞的增殖、上皮间充质转化^[14-15]。本次研究结果显示LEF可抑制miR-449a的表达水平，并且过表达miR-449a可抑制成纤维细胞的细胞活力、细胞增殖能力，抑制α-SMA和Col I蛋白的表达，并促进其凋亡。过表达miR-449a会逆转由LEF引起的细胞活化、增殖以及α-SMA和Col I蛋白的表达，而抑制miR-449a的水平会进一步加剧LEF的促纤维化作用。Zhang等^[16]的研究结果也显示miR-449a具有调节α-SMA蛋白表达的作用。通过进一步的研究我们还发现LEF可促进JNK的磷酸化，过表达miR-449a会抑制JNK磷酸化水平并显著逆转LEF促进JNK磷酸化的作用。JNK的活化在促进肺癌发生和发展中的作用已经被广泛证实，此外，JNK可活化可能通过活化肝星状细胞引起肝纤维化^[17]。Yang等^[18]的研究结果显示阻断JNK途径可抑制成纤维细胞样滑膜细胞的活性，并抑制迁移和侵袭。Shingyochi等^[19]的研究结果也显示了激活JNK通路会促进成纤维细胞的增殖和迁移。这提示LEF可能通过miR-499a促进JNK蛋白的磷酸化，从而促进肺成纤维细胞的活化和增殖，并促进细胞向肌纤维细胞转化和ECM的累积，进而引起肺纤维化。

综上所述，LEF可能通过抑制肺成纤维细胞中miR-449a的表达激活JNK途径，促进α-SMA的表达和ECM的累积，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。但是，关于LEF调节miR-449a的机制和miR-449a在JNK途径中的作用仍需要进一步的研究。

Reference (19)

[1]	JOSHI S, SINGH A R, WONG S S, et al. Rac2 is required for alternative macrophage activation and bleomycin induced pulmonary fibrosis; a macrophage autonomous phenotype[J]. PLoS One,2017,12(8):e0182851-e0182856.
[2]	REN L M, LI R, CHEN L N, et al. Efficacy and safety of weekly leflunomide for the treatment of early rheumatoid arthritis: a randomized, multi-center study[J]. Int J Rheum Dis,2016,19(7):651-657.
[3]	BOCKHORN J, DALTON R, NWACHUKWU C, et al. MicroRNA-30c inhibits human breast tumour chemotherapy resistance by regulating TWF1 and IL-11[J]. Nat Commun,2013,4: 4(1):1393-1398.
[4]	LI J, LU M J, JIN J, et al. MiR-449a suppresses tamoxifen resistance in human breast cancer cells by targeting ADAM22[J]. Cell Physiol Biochem,2018,50(1):136-149.
[5]	HAN R H, JI X M, RONG R, et al. MiR-449a regulates autophagy to inhibit silica-induced pulmonary fibrosis through targeting Bcl2[J]. J Mol Med,2016,94(11):1267-1279.
[6]	TANG Y N, GENG Q, CHEN D, et al. Germline proliferation is regulated by somatic endocytic genes via JNK and BMP signaling in Drosophila[J]. Genetics,2017,206(1):189-197.
[7]	HUANG Y, MA S F, ESPINDOLA M S, et al. Microbes are associated with host innate immune response in idiopathic pulmonary fibrosis[J]. Am J Respir Crit Care Med,2017,196(2):208-219.
[8]	DENG F, ZHANG P, FENG J, et al. Effect of leflunomideon inflammatory factors and immune function in rats with chronic glomerulonephritis[J]. J Sichuan Univ Med Sci Ed,2016,47(2):217-221.
[9]	PATEL A, ZHANG S J, PARAMAHAMSA M, et al. Leflunomide induces pulmonary and hepatic CYP1A enzymes via aryl hydrocarbon receptor[J]. Drug Metab Dispos,2015,43(12):1966-1970.
[10]	SCOTT D L. Interstitial lung disease and disease modifying anti-rheumatic drugs[J]. Lancet,2004,363(9416):1239-1240.
[11]	SAKAI F, NOMA S, KURIHARA Y, et al. Leflunomide-related lung injury in patients with rheumatoid arthritis: imaging features[J]. Mod Rheumatol,2005,15(3):173-179.
[12]	RYU C, SUN H X, GULATI M, et al. Extracellular mitochondrial DNA is generated by fibroblasts and predicts death in idiopathic pulmonary fibrosis[J]. Am J Respir Crit Care Med,2017,196(12):1571-1581.
[13]	ISHIDA Y, KIMURA A, NOSAKA M, et al. Essential involvement of the CX3CL1-CX3CR1 axis in bleomycin-induced pulmonary fibrosis via regulation of fibrocyte and M2 macrophage migration[J]. Sci Rep,2017,7(1):16833-16837.
[14]	LIANG L, HUIJUAN L, LIANJIANG D, et al. MiR-449a suppresses LDHA-mediated glycolysis to enhance the sensitivity of non-small cell lung cancer cells to ionizing radiation[J]. Oncol Res Feat Preclin Clin Cancer Therap,2017,26(4):547-556.
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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

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

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

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

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