LncRNA TCF7 contributes to high glucose-induced damage in human podocytes by up-regulating SEMA3A via sponging miR-16-5p

Abstract

Aims/introduction: Long non-coding RNAs (lncRNAs) exert essential functions in the pathogenesis of diabetic nephropathy (DN). LncRNA T-cell factor 7 (TCF7) and semaphorin-3A (SEMA3A) have been found to be involved in the progression of diabetic nephropathy. However, whether the effect of TCF7 on the pathogenesis of diabetic nephropathy is mediated by SEMA3A remains unclear.

Materials and methods: TCF7, miR-16-5p, and SEMA3A were quantified by a qRT-PCR or immunoblotting method. A CCK-8 assay gauged the cell viability. Measurement of cell apoptosis was done using flow cytometry. RNA immunoprecipitation (RIP), dual-luciferase reporter, and RNA pull-down assays were utilized to assay the targeted interactions among the variables.

Results: The TCF7 and SEMA3A levels were elevated in serum from patients with diabetic nephropathy. TCF7 silencing or SEMA3A depletion ameliorated high glucose (HG)-induced podocyte injury. Moreover, TCF7 silencing protected against HG-induced podocyte injury by down-regulating SEMA3A. TCF7 targeted miR-16-5p, and miR-16-5p targeted SEMA3A. Furthermore, TCF7 affected the expression of SEMA3A by competing specifically for shared miR-16-5p.

Conclusions: These findings suggested that TCF7 silencing attenuated high glucose-induced podocyte damage partially through the miR-16-5p/SEMA3A regulation cascade.

Keywords: Diabetic nephropathy; Podocyte injury; TCF7.

Figure 1

TCF7 and SEMA3A levels were elevated in serum of patients with diabetic nephropathy. TCF7 expression (a) and SEMA3A mRNA level (b) in serum samples of 30 patients with diabetic nephropathy, 30 non‐diabetic patients with CKD, and 30 healthy volunteers. (c) SEMA3A protein level by immunoblotting in serum samples of 3 patients with diabetic nephropathy and 3 healthy volunteers. (d) Correlation between TCF7 and SEMA3A levels in serum from patients with diabetic nephropathy using the Spearman test. *P < 0.05.

Figure 2

TCF7 depletion protected podocytes from cytotoxicity induced by high glucose. TCF7 expression in podocytes after high glucose or normal glucose treatment (a), after transfection by si‐TCF7 or si‐NC (b). Podocytes were introduced with si‐TCF7 or siRNA mimic prior to high glucose treatment and checked for cell viability by CCK‐8 assay (d), cell apoptosis by flow cytometry (e, f), Bax and Bcl‐2 expression by immunoblotting (g, h), ROS production, MDA consent, SOD activity, and CAT activity using assay kits (i–l), IL‐1β, TNF‐α and IL‐6 secretion by ELISA (m–o). *P < 0.05.

Figure 3

SEMA3A deficiency attenuated high glucose‐induced podocyte damage. (a, b) SEMA3A mRNA expression and SEMA3A protein in podocytes treated with high glucose. (c, d) SEMA3A expression in podocytes transfected as indicated. Podocytes were introduced with si‐SEMA3A or siRNA mimic prior to high glucose simulation and assayed by cell viability by CCK‐8 assay (e), cell apoptosis by flow cytometry (f), Bax and Bcl‐2 expression by immunoblotting (g, h), ROS production, MDA consent, SOD activity and CAT activity using assay kits (i–l), IL‐1β, IL‐6 and TNF‐α secretion by ELISA (m–o). *P < 0.05.

Figure 4

TCF7 knockdown protected against high glucose‐induced podocyte damage through down‐regulating SEMA3A. (a, b) SEMA3A mRNA expression and protein level in podocytes transfected as indicated. Podocytes after introduction by si‐NC, si‐TCF7, si‐TCF7 + pcDNA or si‐TCF7 + pcDNA‐SEMA3A were subjected to high glucose treatment and checked for cell viability by CCK‐8 assay (c), cell apoptosis by flow cytometry (d), Bax and Bcl‐2 expression by immunoblotting (e, f), ROS production, MDA consent, SOD activity, and CAT activity using assay kits (g–j), IL‐1β, IL‐6 and TNF‐α secretion by ELISA (k–m). *P < 0.05.

Figure 5

MiR‐16‐5p directly interacted with TCF7 and SEMA3A 3′‐UTR. (a) The miR‐16‐5p pairing sequence in TCF7 and the mutant in seed region. (b) Luciferase activity in podocytes after introduction by TCF7‐WT or TCF7‐MUT. (c) TCF7 and miR‐16‐5p enrichment in the RISC of podocytes using an anti‐Ago2 antibody. (d) TCF7 enrichment in cell lysates of podocytes using Bio‐NC or Bio‐miR‐16‐5p. (e) MiR‐16‐5p expression in podocytes after introduction by si‐TCF7 or siRNA mock. (f) Relative miR‐16‐5p expression in serum samples of 30 patients with diabetic nephropathy, 30 non‐diabetic patients with CKD, and 30 healthy volunteers. (g) Schematic model of SEMA3A 3′‐UTR illustrating the miR‐16‐5p‐pairing sequence and mutated seed region. (h) Luciferase activity in podocytes after transfection by SEMA3A 3′‐UTR‐WT or SEMA3A 3′‐UTR‐MUT. (i) The enrichment of miR‐16‐5p and SEMA3A in the RISC of podocytes using the anti‐Ago2 antibody. (j) MiR‐16‐5p expression in podocytes after introduction by anti‐miR‐16‐5p or anti‐miR‐NC. (k–m) SEMA3A level in podocytes transfected as indicated. *P < 0.05.

Figure 6

TCF7 positively modulated SEMA3A via miR‐16‐5p. SEMA3A mRNA expression (a) and SEMA3A protein level (b) in podocytes transfected as indicated. *P < 0.05.