circ_0000389 inhibits intervertebral disc degeneration by targeting the miR-346/KLF7 axis

Abstract

Intervertebral disc degeneration (IVDD) is a major cause of low back pain. An increasing number of studies have demonstrated that circRNAs regulate the progression of IVDD. However, the specific role of circ_0000389 in the progression of IVDD is not clear. In this study, circ_0000389 is selected by bioinformatics analysis of the GSE67566 dataset. RT-qPCR is performed to detect the expressions of circ_0000389, miR-346 and KLF7 in nucleus pulposus (NP) tissues. The proliferative capacity of nucleus pulposus cells (NPCs) is examined via CCK-8 assay. Western blot analysis of extracellular matrix (ECM) catabolism is performed in NPCs. Dual-luciferase reporter gene assays and RNA immunoprecipitation (RIP) confirm the interaction of circ_0000389 with miR-346 and KLF7. The expression of circ_0000389 is significantly downregulated in degenerating NP tissues. Functionally, circ_0000389 inhibits ECM catabolism. Mechanistically, we identify miR-346 and KLF7 as downstream target genes of circ_0000389 and miR-346, respectively. miR-346 overexpression reverses the effect of circ_0000389 on NPCs, and KLF7 overexpression reverses the effect of miR-346 on NPCs, indicating that circ_0000389 alleviates IVDD progression by regulating the miR-346/KLF7 axis. This study may provide a new therapeutic target for the treatment of IVDD.

Keywords: KLF7; circ_0000389; intervertebral disc degeneration; miR-346.

Figure 1

circ_0000389, miR-346 and KLF7 are closely related in the process of intervertebral disc degeneration (A) Differential expression of circ_RNAs in GSE67566 was shown by heatmap. (B) Differential expression of circ_RNAs in GSE67566 was shown by Volcano map. (C) Relative expression of circ_0000389 was detected by RT-qPCR. (D) The efficiency of pcDNA4.0-circ_0000389 (pcDNA4.0-389) and shRNA-circ_0000389 (sh389) was confirmed by RT-qPCR. (E) Representative western blots of ACAN, COL2A1, MMP-13 and ADAMTS-5 proteins in NP cells. (F) Quantitative analysis of ACAN, COL2A1, MMP-13 and ADAMTS-5 protein expression levels. (G) Binding site between circ_0000389, miR-346 and KLF7 was predicted in online database (https://rnasysu.com/encori/). (H) Relative expression of miR-346 was detected by RT-qPCR in NP cells after upregulation or downregulation of circ_0000389. (I) Relative expression of KLF7 mRNA was detected by RT-qPCR in NP cells after upregulation or downregulation of circ_0000389. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

The expression of miR-346 is negatively correlated with KLF7 (A) Relative expression of miR-346 in normal and IVDD tissues was detected by RT-qPCR. (B) Relative expression of KLF7 in normal and IVDD tissues was detected by RT-qPCR. (C) The expression of miR-346 is negatively correlated with KLF7. (D) Representative western blots of COL2A1, MMP-13 and KLF7 proteins in normal and IVDD tissues. (E) Quantitative analysis of COL2A1, MMP-13 and KLF7 expression levels. (F) KLF7, COL2A1 and MMP-13 proteins in normal and IVDD tissues were detected by IHC. Scale bar = 50 μm. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

miR-346 regulates the expressions of KLF7, MMP-13, ADAMTS-5, ACAN and COL2A1 in NPCs (A) Toluidine blue staining results of NPCs. Bar = 50 μm. (B) MiR-346 was overexpressed/knocked down in NPCs. (C) The expression of KLF7 with different transfection. (D) MiR-346 upregulation/silencing affected the cellular viability of NPCs. (E) Relative expressions of MMP-13, ACAN, COL2A1 and ADAMTS-5 was detected by RT-qPCR. (F) Representative western blots of COL2A1, ACAN, MMP-13, ADAMTS-5 and KLF7 proteins. (G) Quantitative analysis of COL2A1, ACAN, MMP-13, ADAMTS-5 and KLF7 expression levels. (H) ACAN protein was determined by immunofluorescence staining. (I) The expression of circ_0000389 with different transfection. Scale bar = 50 μm. n=3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Mimics of miR-346 reverses the role of KLF7 induced by circ_0000389 NP cells transfected with pcDNA4.0 or pcDNA4.0-389 were treated mimics-NC or mimics. (A) Relative expression of KLF7 in NPCs was determined by RT-qPCR. (B) NPCs viability was detected by CCK-8 assay. (C) Relative mRNA expression of MMP-13, ACAN, COL2A1, ADAMTS-5 and KLF7 in NP cells was determined by RT-qPCR. (D) KLF7 protein was determined by immunofluorescence staining. Scale bar = 50 μm. (E) Relative protein expression of MMP-13, ACAN, COL2A1, ADAMTS-5 and KLF7 was detected by Western blot. (F) Quantitative analysis of MMP-13, ACAN, COL2A1, ADAMTS-5 and KLF7 expression levels. (G) ACAN protein was determined by immunofluorescence. Scale bar = 50 μm. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

KLF7 reversed the biological function of miR-346 in NPCs. NPCs treated with mimics or miR-inhibitor were transfected with pcDNA4.0-KLF7 or shKLF7 (A) The expression level of KLF7 mRNA in different transfection group was obtained. (B) KLF7 levels altered the effects of miR-346 on cell viability. (C) Relative mRNA expression of MMP-13, ACAN, COL2A1 and ADAMTS-5 was detected by RT-qPCR. (D) KLF7 protein was determined by immunofluorescence. Scale bar = 50 μm. (E) Representative western blots of COL2A1, ACAN, MMP-13, ADAMTS-5 and KLF7 proteins. (F) Quantitative analysis of COL2A1, ACAN, MMP-13, ADAMTS-5 and KLF7 expression levels. (G) COL2A1 protein was determined by immunofluorescence staining. Scale bar = 50 μm. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

circ_0000389 acts as a sponge of miR-346 to regulate KLF7 expression in NPCs (A–C) Dual-luciferase gene reporter assays proved the relationship between circ_0000389, miR-346 and KLF7. (D) Pull-down experiment was conducted to verify the interaction between circ_0000389 and miR-346. (E) RIP was performed to verify the interaction between circ_0000389 and AGO2. (F) RIP was performed to verify the interaction between miR-346 and KLF7. (G) RT-qPCR results of the RIP assay of circ_0000389, miR-346 and KLF7. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Upregulation of circ_0000389 attenuates IVDD of mice (A) Disc height of L5/6 was measured by micro-CT. (B) HE was used to evaluate the disc structure of L5/6. (C–E) ADAMTS-5, KLF7 and COL2A1 protein levels in intervertebral disc of L5/6 were determined by IHC. Scale bars = 200 μm for 10×, 100 μm for 20×, and 25 μm for 80×.