长链非编码RNA-GAS5靶向miR-29下调铜转运蛋白CTR1抑制肾脏纤维化的机制研究

Long non-coding RNA-GAS5 downregulates copper transporter protein CTR1 to inhibit renal fibrosis through targeting miR-29

目的研究长链非编码RNA-生长阻滞特异转录物5(GAS5)对肾脏纤维化的作用及机制。方法收集临床肾穿刺患者的组织标本,用原位免疫荧光方法检测GAS5在组织上的定位和表达情况;构建小鼠单侧输尿管梗阻肾脏纤维化模型,用原位免疫荧光方法检测GAS5在肾组织上的定位以及表达情况;用转化生长因子-β(TGF-β)刺激肾小管上皮细胞后,用Real-time PCR方法检测GAS5的表达水平;在肾小管上皮细胞中转染GAS5过表达质粒后,用Western印迹法检测细胞外基质蛋白胶原蛋白3(Col3)、纤连蛋白1(FN1)、铜转运蛋白1(CTR1)和铜离子转运ATP酶α肽(ATP7a)的表达水平,用Real-time PCR方法检测miR-21、miR-29、CTR1和ATP7a的表达。在肾小管上皮细胞中过表达miR-29后,用Western印迹法检测CTR1的表达;在肾小管上皮细胞中过表达miR-21后,用Western印迹法检测ATP7a的表达。结果在不同肾脏纤维化模型中,GAS5均较对照组表达下调。在肾小管上皮细胞中,过表达GAS5抑制Col3和FN1的合成(P<0.05);过表达GAS5可上调miR-29的表达,并下调miR-21的表达(P<0.05)。miR-29在转录后水平抑制CTR1的蛋白表达(P<0.05),且过表达GAS5能够降低CTR1的蛋白表达(P<0.05);miR-21在转录后水平抑制ATP7a的蛋白表达(P<0.05),但过表达GAS5不影响ATP7a的表达。结论长链非编码RNA-GAS5通过上调miR-29,在转录后水平抑制铜转运蛋白CTR1,进而抑制肾脏纤维化。

Objective To study the effect of long non-coding RNA-GAS5 on renal fibrosis and the involving mechanisms.Methods Renal tissue specimens were collected from renal puncture patients and mouse kidney fibrosis model,the localization and expression of GAS5 in human and mouse renal tissues were detected by in situ immunofluorescence.Rat renal tubular epithelial NRK-52E cells were treated with transforming growth factor-β(TGF-β),the expression level of GAS5 was detected by Real-time PCR.The NRK-52E cells were transfection with GAS5 overexpression plasmid,the expression levels of extracellular matrix proteins collagen 3(Col3),fibronectin 1(FN1),copper transporter protein 1(CTR1)and copper-transporting ATPase alpha peptide(ATP7a)were detected by Western blotting,and the expression level of miR-21,miR-29,CTR1 and ATP7a was detected by Real-time PCR.After overexpression of miR-29 in NRK-52E cells,the expression of CTR1 and ATP7a was detected by Western blotting.Results In situ immunofluorescence showed that in renal fibrosis models,the expression of GAS5 was down-regulated compared with the control group.GAS5 overexpression inhibited the synthesis of Col3 and FN1 in NRK-52E cells(P<0.05).Overexpression of GAS5 up-regulated the expression of miR-29 and down-regulated the expression of miR-21(P<0.05).miR-29 inhibited the expression of CTR1 at the post-transcriptional level(P<0.05),and overexpression of GAS5 reduced the expression of CTR1(P<0.05).miR-21 inhibited ATP7a protein expression at the post-transcriptional level(P<0.05),but GAS5 overexpression did not affect ATP7a expression.Conclusion Long non-coding RNA-GAS5 inhibited the copper transporter CTR1 at the post-transcriptional level by upregulating the expression of miR-29,thereby attenuating renal fibrosis.