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

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.

Keywords: calpain 1; diabetic nephropathy; lncRNA nuclear enriched abundant transcript 1; mesangial cell; microRNA-124; β-catenin.

Figure 1

Lnc-NEAT1 expression, cell viability and apoptosis in lnc-NEAT1-downregulated MMC DN cells. (A-F) MMC cells were treated with either HG or NG. (A) Cell viability was measured using CCK-8 assay. (B) Capn1 and (C) CTNNB1 mRNA expression was measured by RT-qPCR. (D) Capn1 and CTNNB1 protein expression was measured by western blotting, (E) which was quantified. (F) Lnc-NEAT1 expression was measured in HG cells by RT-qPCR. (G-L) MMC cells were transfected with either lnc-KD or NC-KD plasmids after HG treatment. (G) Lnc-NEAT1 expression was measured by RT-qPCR after Lnc-NEAT1 knockdown. (H) Cell viability was measured using CCK-8 assay. (I) Cell apoptosis was measured using flow cytometry, (J) which was quantified. (K) Protein expression levels of cleaved caspase 3 and cleaved PARP in the lnc-KD and NC-KD groups were measured by western blotting, (L) which was quantified. Lnc-NEAT1, long non-coding RNA nuclear enriched abundant transcript 1; NG cells, normal glucose-treated cells; HG cells, high glucose-treated cells; lnc-KD, lnc-NEAT1 knockdown; NC-KD, negative control knockdown; MMC, mouse mesangial cells; DN, diabetic nephropathy; RT-qPCR, reverse transcription-quantitative PCR; CCK-8, Cell Counting Kit-8; OD, optical density; OC, osmotic control; capn1, calpain 1; CTNNB1, β-catenin; PARP, poly-ADP ribose polymerase.

Figure 2

Secretion levels of inflammatory cytokines and expression of fibrosis markers in lnc-NEAT1-downregulated MMC DN cells. (A) IL-1β, (B) IL-8, (C) MCP-1, (D) TNF-α secretion into the cell supernatant of lnc-KD and NC-KD groups were measured by ELISA. (E) Fibronectin and (F) collagen I mRNA expression in the lnc-KD and NC-KD groups were measured by reverse transcription-quantitative PCR. (G) Fibronectin and collagen I protein expression was measured by western blotting, (H) which was quantified. lnc-NEAT1, long non-coding RNA nuclear enriched abundant transcript 1; lnc-KD, lnc-NEAT1 knockdown; NC-KD, negative control knockdown; MMC, mouse mesangial cell; DN, diabetic nephropathy; MCP-1, monocyte chemotactic protein 1.

Figure 3

miR-124 expression and effect of miR-124 inhibition on cell viability and apoptosis in a lnc-NEAT1-downregulated MMC DN cell model. (A) miR-124 expression in the lnc-KD and NC-KD groups was measured by RT-qPCR. (B-H) The MMC DN cell model were transfected with the Lnc-KD or NC-KD plasmid and miR-124 inhibitor or NC-inhibitor. (B) Lnc-NEAT1 and (C) miR-124 expression was measured by RT-qPCR. (D) Cell viability was measured using CCK-8 assay. (E) Cell apoptosis was measured using flow cytometry, (F) which was quantified. (G) Cleaved caspase 3 and cleaved PARP protein expression was measured by western blotting, (H) which was quantified. miR, microRNA; lnc-NEAT1, long non-coding RNA nuclear enriched abundant transcript 1; MMC, mouse mesangial cell; DN, diabetic nephropathy; lnc-KD, lnc-NEAT1 knockdown; NC-KD, negative control knockdown; RT-qPCR, reverse transcription-quantitative PCR; PARP, poly-ADP ribose polymerase; CCK-8, Cell Counting Kit-8; OD, optical density.

Figure 4

Effect of miR-124 inhibition on the secretion of inflammatory cytokines and expression of fibrosis markers in a lnc-NEAT1-downregulated MMC DN cell model. Secretion of (A) IL-1β, (B) IL-8, (C) MCP-1 and (D) TNF-α into the supernatant of cells from lnc-KD + miR-124-inhibitor and lnc-KD + NC-inhibitor groups were measured using ELISA. (E) Fibronectin and (F) collagen I mRNA expression was measured using reverse transcription-quantitative PCR. (G) Fibronectin and collagen I protein expression in the lnc-KD + miR-124-inhibitor and lnc-KD + NC-inhibitor groups was measured by western blotting, (H) which was quantified. miR, microRNA; lnc-NEAT1, long non-coding RNA nuclear enriched abundant transcript 1; MMC, mouse mesangial cell; DN, diabetic nephropathy; lnc-KD, lnc-NEAT1 knockdown; NC-KD, negative control knockdown; MCP-1, monocyte chemotactic protein 1; TNF-α, tumor necrosis factor α; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

miR-124 inhibition increases Capn1 and CTNNB1 expression the lnc-NEAT1-downregulated MMC DN cell model. The MMC DN cell model was transfected with either lnc-KD + miR-124-inhibitor or lnc-KD + NC-inhibitor. (A) Capn1 and (B) CTNNB1 mRNA expression was measured by reverse transcription-quantitative PCR. (C) Capn1 and CTNNB1 protein expression was measured by western blotting, (D) which was quantified. miR, microRNA; Capn1, calpain 1; CTNNB1, catenin β1; lnc-NEAT1, long non-coding RNA nuclear enriched abundant transcript 1; MMC, mouse mesangial cell; DN, diabetic nephropathy; lnc-KD, lnc-NEAT1 knockdown; NC-KD, negative control knockdown.