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首页> 外文期刊>RSC Advances >Inhibition of miR-16 enhances the sensitivity of fibroblast-like synovial cells to methotrexate by restraining MDR1/P-gp expression via NF-κB pathway
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Inhibition of miR-16 enhances the sensitivity of fibroblast-like synovial cells to methotrexate by restraining MDR1/P-gp expression via NF-κB pathway

机译：通过抑制NF-κB途径抑制MDR1 / P-gp的表达，抑制miR-16可增强成纤维样滑膜细胞对甲氨蝶呤的敏感性。

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摘要

MicroRNAs (miRNAs) are demonstrated to contribute to the regulation of drug resistance in a number of diseases. Nevertheless, little is known about the role and the underlying mechanism of miR-16 in rheumatoid arthritis (RA) methotrexate resistance. In this study, we firstly examined the miR-16 expression in the serum and synovial fluid from RA patients who were unresponsive to methotrexate monotherapy (UR-MTX patients) and responsive RA patients (R-MTX patients). Secondly, the miR-16 expression was measured in both fibroblast-like synovial cells (FLS) and methotrexate resistance RA-FLS cells (FLS-MTX). FLS cells used in this study were isolated from synovial tissue specimens obtained from patients with RA who underwent total joint replacement. FLS-MTX cells were conducted by gradually increasing the concentration of methotrexate in the medium. The construction of FLS-MTX cells was confirmed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay. Thirdly, in order to further investigate the role of miR-16 in FLS-MTX cells, we introduced miR-16 inhibitor into FLS-MTX cells to knockdown the expression of miR-16, used fluorescence quantitative PCR to detect the inhibition efficiency. The effects of miR-16 inhibition on cell viability, cell cycle arrest and apoptosis in FLS-MTX cells were monitored with MTT and flow cytometry analysis, respectively. And the regulation of miR-16 on P-glycoprotein (P-gp) was performed using qRT-PCR, western blotting, and immunofluorescence staining. Fourthly, ammonium pyrrolidinedithiocarbamate (PDTC), a NF-κB pathway inhibitor, was applied to verify the mechanism by which miR-16 involved in to regulate the P-gp expression, and thus contributing to the methotrexate resistance in FLS-MTX cells. MiR-16 was upregulated in the in serum and synovial fluid from UR-MTX patients as well as in FLS-MTX cells. Inhibition of miR-16 re-sensitized the FLS-MTX cells to methotrexate by suppressing the cell viability, cell promoting cycle arrest at G0/G1 phase and enhancing apoptosis. Knockdown of miR-16 significantly reduced MDR1 mRNA expression and P-gp protein expression in FLS-MTX cells. Furthermore, inhibition of NF-κB pathway by PDTC reinforced the effect of miR-16 knockdown on P-gp expression, cell viability, cell cycle arrest and apoptosis. In conclusion, our study illustrated that inhibition of miR-16 in FLS-MTX cells alleviated methotrexate resistance by inhibiting MDR1/P-gp expression through inactivation of the NF-κB pathway.
机译：MicroRNA（miRNA）被证明有助于调节多种疾病中的耐药性。然而，关于miR-16在类风湿关节炎（RA）甲氨蝶呤耐药性中的作用及其潜在机制知之甚少。在这项研究中，我们首先检查了对甲氨蝶呤单一疗法（UR-MTX患者）无反应的RA患者和反应性RA患者（R-MTX患者）的血清和滑液中miR-16的表达。其次，在成纤维样滑膜细胞（FLS）和甲氨蝶呤抗性RA-FLS细胞（FLS-MTX）中均检测到miR-16表达。本研究中使用的FLS细胞是从滑膜组织标本中分离的，该标本取自接受全关节置换的RA患者。通过逐渐增加培养基中甲氨蝶呤的浓度进行FLS-MTX细胞。 FLS-MTX细胞的构建通过3-（4,5）-二甲基噻唑并（-z-y1）-3,5-di-phenytetrazoliumromide（MTT）分析得以证实。第三，为了进一步研究miR-16在FLS-MTX细胞中的作用，我们将miR-16抑制剂引入FLS-MTX细胞中以敲低miR-16的表达，采用荧光定量PCR检测其抑制效率。分别用MTT和流式细胞术分析了miR-16抑制对FLS-MTX细胞中细胞活力，细胞周期停滞和凋亡的影响。使用qRT-PCR，western印迹和免疫荧光染色进行miR-16对P-糖蛋白（P-gp）的调节。第四，应用NF-κB途径抑制剂吡咯烷二硫代氨基甲酸铵（PDTC）来验证miR-16参与调节P-gp表达的机制，从而促进FLS-MTX细胞对甲氨蝶呤的耐药性。 UR-MTX患者的血清和滑液以及FLS-MTX细胞中的MiR-16上调。抑制miR-16可通过抑制细胞活力，促进细胞在G0 / G1期停滞并增强凋亡来使FLS-MTX细胞对甲氨蝶呤重新敏感。敲除miR-16可显着降低FLS-MTX细胞中MDR1 mRNA表达和P-gp蛋白表达。此外，PDTC对NF-κB通路的抑制作用增强了miR-16敲低对P-gp表达，细胞活力，细胞周期阻滞和凋亡的影响。总之，我们的研究表明，通过抑制NF-κB通路抑制MDR1 / P-gp表达，抑制FLS-MTX细胞中的miR-16可以减轻甲氨蝶呤的耐药性。

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