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Long non-coding RNA Neat1 triggers renal tubular epithelial cell apoptosis via activating BH3-only protein in membranous nephropathy
Autoimmunity ( IF 3.5 ) Pub Date : 2021-09-03 , DOI: 10.1080/08916934.2021.1972289
Pei Pi 1 , Qingqiao Yin 1 , Ling Xiao 1 , Dan Luo 1
Affiliation  

Abstract

Background and Objective

Membranous nephropathy (MN) is an autoimmune disease. The up-regulation of the long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (Neat1) has been found in MN but the mechanism is still unclear. Here, we explored the effect and the underlying mechanism of lncRNA Neat1 on the apoptosis of renal tubular epithelial cells in MN.

Methods

Albumin-stimulated E11 podocytes and proximal tubular epithelial cells in vitro and the cationic-bovine serum albumin-induced MN mouse model in vivo were established. The expression of Neat1 in E11 podocytes, renal tubular epithelial cells, and renal tubules and the mRNA expression of BH3-only (the Bcl-2 homology 3-only) proteins were determined by quantitative reverse transcription-polymerase chain reaction. Levels of Cleaved Caspase 3, 6, 7, and Noxa were examined by western blotting. The number of apoptotic cells was detected by flow cytometry. Cellular proliferation was determined by 5-Ethynyl-2’-deoxyuridine and Cell Counting Kit-8 assay. Interactions between BH3-only protein Noxa and Bcl-2 as well as Bcl-xL were evaluated with co-immunoprecipitation.

Results

The expression of lncRNA Neat1 was unchanged in albumin-stimulated E11 podocytes, but it was up-regulated in albumin-stimulated renal tubular epithelial cells and MN renal tubule tissues and there was a time-dependent increase in vivo. In the albumin-stimulated proximal tubular epithelial cells, overexpression of Neat1 could increase apoptosis and decrease proliferation. In turn, interference with Neat1 reduced apoptosis and increased proliferation accordingly. The mRNA expression levels of BH3-only proteins (Bad, Bim, Bid, Puma, Noxa) were detected with qRT-PCR, the results indicated that after overexpression of Neat1, mRNA and protein levels of Noxa were significantly increased, and the interference with BH3-only protein Noxa alleviated apoptosis of renal tubular epithelial cells in vitro.

Conclusion

In our study, we proved that lncRNA Neat1 promoted the development of MN by inducing apoptosis and this effect may be exerted by inhibiting the anti-apoptotic protein activity mediated by Noxa.



中文翻译:

长链非编码 RNA Neat1 通过激活膜性肾病中的 BH3-only 蛋白触发肾小管上皮细胞凋亡

摘要

背景与目的

膜性肾病(MN)是一种自身免疫性疾病。已在 MN 中发现长非编码 RNA (lncRNA) 核副啄木鸟组装转录本 1 (Neat1) 的上调,但其机制仍不清楚。在这里,我们探讨了 lncRNA Neat1 对 MN 肾小管上皮细胞凋亡的影响及其潜在机制。

方法

建立体外白蛋白刺激的E11足细胞和近端肾小管上皮细胞和体内阳离子牛血清白蛋白诱导的MN小鼠模型。通过定量逆转录聚合酶链反应测定 E11 足细胞、肾小管上皮细胞和肾小管中 Neat1 的表达以及 BH3-only(Bcl-2 同源性 3-only)蛋白的 mRNA 表达。通过蛋白质印迹检查 Cleaved Caspase 3、6、7 和 Noxa 的水平。流式细胞仪检测凋亡细胞数。通过 5-Ethynyl-2'-deoxyuridine 和 Cell Counting Kit-8 测定法测定细胞增殖。BH3-only 蛋白 Noxa 和 Bcl-2 以及 Bcl-xL 之间的相互作用通过免疫共沉淀进行了评估。

结果

lncRNA Neat1 在白蛋白刺激的 E11 足细胞中的表达没有变化,但在白蛋白刺激的肾小管上皮细胞和 MN 肾小管组织中上调,并且在体内呈时间依赖性增加。在白蛋白刺激的近端肾小管上皮细胞中,Neat1 的过表达可增加细胞凋亡并减少增殖。反过来,对 Neat1 的干扰减少了细胞凋亡并相应地增加了增殖。qRT-PCR检测BH3-only蛋白(Bad、Bim、Bid、Puma、Noxa)的mRNA表达水平,结果表明过表达Neat1后,Noxa的mRNA和蛋白水平显着升高,干扰BH3-only蛋白Noxa在体外减轻肾小管上皮细胞的凋亡.

结论

在我们的研究中,我们证明了lncRNA Neat1通过诱导细胞凋亡来促进MN的发展,这种作用可能是通过抑制Noxa介导的抗凋亡蛋白活性来发挥的。

更新日期:2021-09-03
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