LncRNA NEAT1/microRNA‑124 regulates cell viability, inflammation and fibrosis in high‑glucose‑treated mesangial cells

Zhao,Na; Du,Likun; Ma,Yingli; Wang,Yang; Ma,Jian; Fang,Zhaohui

doi:10.3892/etm.2022.11434

LncRNA NEAT1/microRNA‑124 regulates cell viability, inflammation and fibrosis in high‑glucose‑treated mesangial cells

Authors:
- Na Zhao
- Likun Du
- Yingli Ma
- Yang Wang
- Jian Ma
- Zhaohui Fang
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Affiliations: Department of Endocrinology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, P.R. China, School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, P.R. China, Department of Endocrinology, First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui 230031, P.R. China
Published online on: June 9, 2022 https://doi.org/10.3892/etm.2022.11434
Article Number: 507
Copyright: © Zhao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Long non‑coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) has been frequently found to be dysregulated, which contributes to diabetes‑related complications. The present study aimed to explore the effect of knockdown on mouse mesangial cell (MMC) viability, apoptosis, inflammation and fibrosis in an in vitro model of diabetic nephropathy (DN). The SV40 MES13 MMC cell line was first cultured with high glucose to establish an in vitro MMC DN cell model. Lnc‑NEAT1 shRNA or the negative control shRNA were transfected into MMC DN cells, followed by the measurement of cell viability, apoptosis, inflammation, fibrosis and microRNA (miR)‑124 expression, a known target of lnc‑NEAT1, using Cell Counting Kit‑8, flow cytometry, ELISA, western blotting [Capain1 (capn1), β‑catenin (CTNNB1), cleaved caspase 3, cleaved poly‑(ADP ribose) polymerase, fibronectin and Collagen] and reverse transcription‑quantitative PCR (Capn1, CTNNB1, lnc‑NEAT1, fibronectin, collagen and miR‑124), respectively. In rescue experiments, the miR‑124 and negative control inhibitor were co‑transfected into lnc‑NEAT1‑downregulated cells, following which cell viability, apoptosis, inflammation, fibrosis, capn1 and CTNNB1 expression were measured. Lnc‑NEAT1 expression was increased in high glucose‑treated cells compared with that in normal glucose‑treated cells and osmotic control cells, suggesting that lnc‑NEAT1 is overexpressed in the MMC DN cell model. In the MMC DN cell model, lncRNA‑NEAT1 knockdown enhanced cell apoptosis but reduced cell viability and the secretion of inflammatory cytokines in the supernatant (IL‑1β, IL‑8, monocyte chemotactic protein 1 and TNF‑α), in addition to reducing the expression of fibrosis markers fibronectin and collagen I in the lysates. Lnc‑NEAT1 knockdown increased miR‑124 expression. Furthermore, transfection with the miR‑124 inhibitor reduced cell apoptosis but increased cell viability, inflammation and fibrosis in lnc‑NEAT1-downregulated MMC DN cells. miR‑124 inhibitor transfection also increased the expression levels of Capn1 and CTNNB1. Taken together, the findings of the present study demonstrated that lnc‑NEAT1 knockdown was able to attenuate MMC viability, inflammation and fibrosis by regulating miR‑124 expression and the Capn1/β‑catenin signaling pathway downstream. Therefore, Lnc‑NEAT1 may serve as a potential therapeutic target for DN.

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1	Han Q, Zhu H, Chen X and Liu Z: Non-genetic mechanisms of diabetic nephropathy. Front Med. 11:319–332. 2017.PubMed/NCBI View Article : Google Scholar
2	Szrejder M and Piwkowska A: AMPK signalling: Implications for podocyte biology in diabetic nephropathy. Biol Cell. 111:109–120. 2019.PubMed/NCBI View Article : Google Scholar
3	Li Y, Xu K, Xu K, Chen S, Cao Y and Zhan H: Roles of identified long noncoding RNA in diabetic nephropathy. J Diabetes Res. 2019(5383010)2019.PubMed/NCBI View Article : Google Scholar
4	Srivastava SP, Goodwin JE, Tripathi P, Kanasaki K and Koya D: Interactions among long non-coding RNAs and microRNAs influence disease phenotype in diabetes and diabetic kidney disease. Int J Mol Sci. 22(6027)2021.PubMed/NCBI View Article : Google Scholar
5	Coellar JD, Long J and Danesh FR: Long noncoding RNAs and their therapeutic promise in diabetic nephropathy. Nephron. 145:404–414. 2021.PubMed/NCBI View Article : Google Scholar
6	Chen Y, Qiu J, Chen B, Lin Y, Chen Y, Xie G, Qiu J, Huasheng H and Jiang D: Long non-coding RNA NEAT1 plays an important role in sepsis-induced acute kidney injury by targeting miR-204 and modulating the NF-κB pathway. Int Immunopharmacol. 59:252–260. 2018.PubMed/NCBI View Article : Google Scholar
7	Ma M, Hui J, Zhang QY, Zhu Y, He Y and Liu XJ: Long non-coding RNA nuclear-enriched abundant transcript 1 inhibition blunts myocardial ischemia reperfusion injury via autophagic flux arrest and apoptosis in streptozotocin-induced diabetic rats. Atherosclerosis. 277:113–122. 2018.PubMed/NCBI View Article : Google Scholar
8	Zhou Y, Deng J, Chu X, Zhao Y and Guo Y: Role of post-transcriptional control of calpain by miR-124-3p in the development of Alzheimer's disease. J Alzheimers Dis. 67:571–581. 2019.PubMed/NCBI View Article : Google Scholar
9	Azoulay-Alfaguter I, Yaffe Y, Licht-Murava A, Urbanska M, Jaworski J, Pietrokovski S, Hirschberg K and Eldar-Finkelman H: Distinct molecular regulation of glycogen synthase kinase-3alpha isozyme controlled by its N-terminal region: Functional role in calcium/calpain signaling. J Biol Chem. 286:13470–13480. 2011.PubMed/NCBI View Article : Google Scholar
10	Lade A, Ranganathan S, Luo J and Monga SP: Calpain induces N-terminal truncation of β-catenin in normal murine liver development: Diagnostic implications in hepatoblastomas. J Biol Chem. 287:22789–22798. 2012.PubMed/NCBI View Article : Google Scholar
11	Potz BA, Sabe AA, Elmadhun NY, Clements RT, Abid MR, Sodha NR and Sellke FW: Calpain inhibition modulates glycogen synthase kinase 3β pathways in ischemic myocardium: A proteomic and mechanistic analysis. J Thorac Cardiovasc Surg. 153:342–357. 2017.PubMed/NCBI View Article : Google Scholar
12	Liu Y, Liu B, Zhang GQ, Zou JF, Zou ML and Cheng ZS: Calpain inhibition attenuates bleomycin-induced pulmonary fibrosis via switching the development of epithelial-mesenchymal transition. Naunyn Schmiedebergs Arch Pharmacol. 391:695–704. 2018.PubMed/NCBI View Article : Google Scholar
13	Muniappan L, Javidan A, Jiang W, Mohammadmoradi S, Moorleghen JJ, Katz WS, Balakrishnan A, Howatt DA and Subramanian V: Calpain inhibition attenuates adipose tissue inflammation and fibrosis in diet-induced obese mice. Sci Rep. 7(14398)2017.PubMed/NCBI View Article : Google Scholar
14	Zhao YL, Li JB, Li YJ, Li SJ, Zhou SH and Xia H: Capn4 promotes esophageal squamous cell carcinoma metastasis by regulating ZEB1 through the Wnt/β-catenin signaling pathway. Thorac Cancer. 10:24–32. 2019.PubMed/NCBI View Article : Google Scholar
15	Hu W, Han Q, Zhao L and Wang L: Circular RNA circRNA_15698 aggravates the extracellular matrix of diabetic nephropathy mesangial cells via miR-185/TGF-β1. J Cell Physiol. 234:1469–1476. 2019.PubMed/NCBI View Article : Google Scholar
16	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar
17	Gao J, Wang W, Wang F and Guo C: LncRNA-NR_033515 promotes proliferation, fibrogenesis and epithelial-to-mesenchymal transition by targeting miR-743b-5p in diabetic nephropathy. Biomed Pharmacother. 106:543–552. 2018.PubMed/NCBI View Article : Google Scholar
18	Hu M, Wang R, Li X, Fan M, Lin J, Zhen J, Chen L and Lv Z: LncRNA MALAT1 is dysregulated in diabetic nephropathy and involved in high glucose-induced podocyte injury via its interplay with β-catenin. J Cell Mol Med. 21:2732–2747. 2017.PubMed/NCBI View Article : Google Scholar
19	Long J and Danesh FR: Values and limitations of targeting lncRNAs in diabetic nephropathy. Diabetes. 67:552–553. 2018.PubMed/NCBI View Article : Google Scholar
20	Alvarez ML, Khosroheidari M, Eddy E and Kiefer J: Role of microRNA 1207-5P and its host gene, the long non-coding RNA Pvt1, as mediators of extracellular matrix accumulation in the kidney: Implications for diabetic nephropathy. PLoS One. 8(e77468)2013.PubMed/NCBI View Article : Google Scholar
21	Zou G, Zhong W, Wu F, Wang X and Liu L: Catalpol attenuates cardiomyocyte apoptosis in diabetic cardiomyopathy via Neat1/miR-140-5p/HDAC4 axis. Biochimie. 165:90–99. 2019.PubMed/NCBI View Article : Google Scholar
22	Huang S, Xu Y, Ge X, Xu B, Peng W, Jiang X, Shen L and Xia L: Long noncoding RNA NEAT1 accelerates the proliferation and fibrosis in diabetic nephropathy through activating Akt/mTOR signaling pathway. J Cell Physiol. 234:11200–11207. 2019.PubMed/NCBI View Article : Google Scholar
23	Yu F, Jiang Z, Chen B, Dong P and Zheng J: NEAT1 accelerates the progression of liver fibrosis via regulation of microRNA-122 and Kruppel-like factor 6. J Mol Med (Berl). 95:1191–1202. 2017.PubMed/NCBI View Article : Google Scholar
24	Chen J, Cui L, Yuan J, Zhang Y and Sang H: Circular RNA WDR77 target FGF-2 to regulate vascular smooth muscle cells proliferation and migration by sponging miR-124. Biochem Biophys Res Commun. 494:126–132. 2017.PubMed/NCBI View Article : Google Scholar
25	Gong Q, Xie J, Liu Y, Li Y and Su G: Differentially expressed microRNAs in the development of early diabetic retinopathy. J Diabetes Res. 2017(4727942)2017.PubMed/NCBI View Article : Google Scholar
26	Li XY, Zhang YQ, Xu G, Li SH and Li H: miR-124/MCP-1 signaling pathway modulates the protective effect of itraconazole on acute kidney injury in a mouse model of disseminated candidiasis. Int J Mol Med. 41:3468–3476. 2018.PubMed/NCBI View Article : Google Scholar
27	Zhao MY, Wang GQ, Wang NN, Yu QY, Liu RL and Shi WQ: The long-non-coding RNA NEAT1 is a novel target for Alzheimer's disease progression via miR-124/BACE1 axis. Neurol Res. 41:489–497. 2019.PubMed/NCBI View Article : Google Scholar
28	He F, Zhang C and Huang Q: Long noncoding RNA nuclear enriched abundant transcript 1/miRNA-124 axis correlates with increased disease risk, elevated inflammation, deteriorative disease condition, and predicts decreased survival of sepsis. Medicine (Baltimore). 98(e16470)2019.PubMed/NCBI View Article : Google Scholar
29	Ren X, Zhu R, Liu G, Xue F, Wang Y, Xu J, Zhang W, Yu W and Li R: Effect of sitagliptin on tubulointerstitial Wnt/β-catenin signalling in diabetic nephropathy. Nephrology (Carlton). 24:1189–1197. 2019.PubMed/NCBI View Article : Google Scholar
30	Wang Y, Xiang K, Zheng T, Jia W, Shen K and Li J: The UCSNP44 variation of calpain 10 gene on NIDDM1 locus and its impact on plasma glucose levels in type 2 diabetic patients. Zhonghua Yi Xue Za Zhi. 82:613–616. 2002.PubMed/NCBI(In Chinese).