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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-kappa B 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-kappa B pathway

机译：通过NF-κB途径抑制MDR1 / P-GP表达，抑制miR-16通过抑制MDR1 / P-GP表达来增强成纤维细胞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-kappa B 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-kappa B 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-kappa B pathway.
机译：对MicroRNA（miRNA）进行了证明有助于在许多疾病中调节耐药性。尽管如此，关于miR-16在类风湿性关节炎（Ra）甲氨蝶呤抗性中的作用和潜在机制很少。在这项研究中，我们首先检查了来自对甲氨蝶呤单药治疗（UR-MTX患者）和反应性RA患者对甲氨蝶呤的RA患者的血清和滑膜液中的miR-16表达，并响应RA患者（R-MTX患者）。其次，在成纤维细胞状滑膜细胞（FLS）和甲氨蝶呤抗性RA-FL-FLS细胞（FLS-MTX）中测量miR-16表达。本研究中使用的FLS细胞与从患有总关节置换的RA患者获得的滑膜组织标本中分离出来的。通过逐渐增加培养基中甲氨蝶呤的浓度进行FLS-MTX细胞。通过3-（4,5） - 二甲基噻唑（-Z-Y1）-3,5-二苯基 - 甲腈（MTT）测定证实了FLS-MTX细胞的构建。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抑制剂引入到FLS-MTX细胞中以敲击MIR-16的表达，使用荧光定量PCR检测抑制效率。用MTT和流式细胞术分析监测MIR-16抑制对细胞活力，细胞周期停用和细胞凋亡的影响。使用QRT-PCR，Western印迹和免疫荧光染色进行P-糖蛋白（P-GP）的miR-16的调节。第四，吡咯烷二氨基氨基甲酸铵（PDTC），NF-Kappa途径抑制剂，用于验证miR-16所涉及调节p-gp表达的机制，从而有助于甲氨蝶呤在甲基氨基甲酸溶液中的抗性。 MiR-16在来自UR-MTX患者的血清和滑膜液中均上调，以及在FLS-MTX细胞中。 MiR-16对MiR-16的抑制通过抑制细胞活力，细胞促进G0 / G1相的循环滞留和增强细胞凋亡，将FLS-MTX细胞重新敏感到甲氨蝶呤中。 miR-16的敲低显着降低了FLS-MTX细胞中的MDR1 mRNA表达和P-GP蛋白表达。此外，PDTC对NF-κB途径的抑制增强了MIR-16敲低对P-GP表达，细胞活力，细胞周期停滞和细胞凋亡的影响。总之，我们的研究表明，通过抑制NF-κB途径的灭活，通过抑制MDR1 / P-GP表达，抑制miR-16在氟甲酸甲醇溶液中抑制甲氨蝶呤抗性。

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    《RSC Advances》 |2019年第46期|共9页
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  • 正文语种 eng
  • 中图分类 化学;
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