A novel identified circular RNA, circ_0000491, aggravates the extracellular matrix of diabetic nephropathy glomerular mesangial cells through suppressing miR‑101b by targeting TGFβRI

Abstract

Circular RNAs (circRNAs) have crucial roles in various diseases; however, the mechanisms of action underlying circRNAs in the occurrence and development of diabetic nephropathy (DN) remains largely unknown. The present study investigated the differentially expressed circRNAs in the DN mice kidney cortex using circRNA sequencing and elucidated the role of circRNAs in mesangial cells. It was revealed that 40 circRNAs were unconventionally expressed, including 18 upregulated circRNAs and 22 downregulated circRNAs. Furthermore, circ_0000491 levels were significantly augmented in both DN mice and high glucose (HG, 30 mM)‑induced mouse mesangial cells (MES13 cells). Knockdown of circ_0000491 significantly suppressed the increase of vimentin, fibronectin and α‑smooth muscle actin, as well as collagen type I, III and IV, whilst reversing the decrease of E‑cadherin in HG‑induced MES13 cells. It was further revealed that circRNA_0000491 sponged miR‑101b and that miR‑101b directly targets TGFβRI. In addition, the expression levels of miR‑101b were negatively associated with the transcriptional level of circRNA_0000491 and miR‑101b inhibitors reversed the suppression of extracellular matrix (ECM)‑associated protein synthesis mediated by knocking‑down circRNA_0000491. In conclusion, the present study investigated the circRNA_0000491/miR‑101b/TGFβRI axis in ECM accumulation and fibrosis‑associated protein expression levels of mesangial cells, which suggested that circRNA_0000491 may be beneficial for the development of an effective therapeutic target for DN.

Figure 1.

circRNA microarray analysis and circRNA_0000491 are upregulated in db/db diabetic nephropathy mice kidney tissues and MES13 cells treated with high glucose. (A) Heat maps and (B) volcano plots displaying the differentially expressed circRNAs. (C) A schematic diagram of the genomic location and structure of circ_0000491. circRNA, circular RNA.

Figure 2.

Expression of circRNA_0000491 in the kidney tissue of db/db mice and high glucose-induced mouse MES13 cells. (A) Reverse transcription-quantitative PCR estimated the expression levels of circ_0000491 in diabetic nephropathy mice and (B) the high concentration glucose (30 mM)-induced MES13 cells. ^##P<0.01 vs. the db/m control group; **P<0.01 vs. the 5.5 mM glucose treated MES13 cells group. circRNA, circular RNA.

Figure 3.

Circ_0000491 silencing inhibits the extracellular matrix accumulation and fibrosis-associated protein synthesis in MES13 cells. RT-qPCR revealed the expression levels of E-cad, Vim, FN, α-SMA, Col. I, Col. III and Col. IV in (A) diabetic nephropathy mice and (B) MES13 cells treated with high glucose. (C) The expression levels of circ_0000491 in MES13 cells transfected using siRNA. (D) Western blotting showing the protein expression levels of (E) E-cad, (F) Vim, (G) FN, (H) α-SMA, (I) Col. I, (J) Col. III and (K) Col. IV in MES13 cells with high glucose treatment and transfected with si_circ_0000491. *P<0.05, **P<0.01 vs. the corresponding control group; ^#P<0.05, ^##P<0.01 vs. the Si_Circ_NC group. Col., collagen; circRNA, circular RNA; E-cad, E-cadherin; FN, fibronectin; si, small interfering; SMA, smooth muscle actin; Vim, vimentin.

Figure 4.

Circ_0000491 targets miRNA-101b. (A) The miR-101b mRNA expression levels in MES13 cells transfected with miR-101b mimics or miR-101b inhibitor was confirmed using RT-qPCR. (B) Circ_0000491 expression was examined in the transfected MES13 cells. (C) Diagram of the miR-101b putative binding sites in circ_0000491. (D) miR-101b expression in db/db mice and high glucose treated MES13 cells was measured using RT-qPCR. (E) Luciferase reporter assay showed the binding activity of miR-101b and circRNA_0000491. (F) miR-101b mRNA expression levels in MES13 cells transfected with si_circ_0000491 was measured using RT-qPCR. *P<0.05, **P<0.01 vs. the corresponding control group. circRNA, circular RNA; miR/miRNA, microRNA; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering.

Figure 5.

miRNA-101b targets TGFβRI. (A) Diagram of the binding sites between TGFβRI and miRNA-101b. Red text represents the mutant sequence of TGFβRI (B) Relative luciferase activity of the wild-type and 3′-untranslated region mutant constructs of TGFβRI co-transfected with miR-101b mimics and miRNA control. (C) The mRNA expression levels of TGFβRI in db/db mice. (D) The mRNA expression levels of TGFβRI in the high glucose treated MES13 cells. **P<0.01 Control mimics + wild-type TGFβRI group vs. miR-101b mimics + wild-type TGFβ RI group; ▲P<0.05 db/db group vs. Db/dm group; ^##P<0.01 5.5 mM glucose treated group vs. 30 mM glucose treated group. miR/miRNA, microRNA; TGFβR1, TGFβ receptor 1.

Figure 6.

Effect of circ_0000491 and miRNA-101b inhibition on extracellular matrix and fibrosis-associated protein expression via targeting TGFβRI. (A) Western blotting showed that miR-101b inhibitor reversed the effect of si_circRNA_0000491 on the protein expression levels of (B) TGFβ1, (C) TGFβRI, (D) pSmad3/Smad3, (E) E-cad, (F) Vim, (G) FN, (H) α-SMA, (I) Col. I, (J) Col. III and (K) Col. IV. *P<0.05, **P<0.01. Col., collagen; circRNA, circular RNA; E-cad, E-cadherin; FN, fibronectin; miR/miRNA, microRNA; p, phosphorylated; si, small interfering; SMA, smooth muscle actin; TGFβR1, TGFβ receptor 1; Vim, vimentin.