Knockdown of LINC01138 protects human chondrocytes against IL-1β-induced damage by regulating the hsa-miR-1207-5p/KIAA0101 axis

Abstract

Introduction: Long intergenic non-protein coding RNA 1138 (LINC01138) plays a vital role in human cancers. In this study, we aimed to investigate the effect of LINC01138 on the progression of osteoarthritis (OA) and explore its potential mechanism of action.

Methods: The expression of LINC01138, hsa-miR-1207-5p, and KIAA0101 in OA tissues and normal tissues was analyzed using GSEA datasets and confirmed in human specimens. Human chondrocytes were treated with interleukin (IL)-1β to establish an OA cell model. Quantitative real time PCR(qRT-PCR), enzyme-linked immunosorbent assay, and western blotting analyses were performed to evaluate the role of LINC01138, hsa-miR-1207-5p, and KIAA0101 during extracellular matrix (ECM) protein degeneration and cellular inflammatory response. The target relationship was predicted using DIANA-TarBase and TargetScan. The binding effects were verified by dual-luciferase reporter assay.

Results: LINC01138 expression was higher in OA tissues than in normal controls. LINC01138 levels increased in chondrocytes treated with IL-1β. Silencing of LINC01138 attenuated the IL-1β-induced decrease in Col2α1, aggrecan, and sulphated glycosaminoglycan (sGAG), and inhibited the IL-1β-induced increase in matrix metalloproteinase (MMP)-13, IL-6, and tumor necrosis factor (TNF)-α. miR-1207-5p is weakly expressed in OA tissues and cell models. The inhibition of hsa-miR-1207-5p, a target of LINC01138, attenuated the effects of LINC01138 silencing on chondrocyte ECM degeneration and inflammatory responses. Silencing KIAA0101, a target of hsa-miR-1207-5p, alleviated the effect of hsa-miR-1207-5p on chondrocyte ECM degeneration and inflammatory responses. Furthermore, silencing of KIAA0101 inhibited the JAK/STAT and Wnt signaling pathways.

Conclusion: Silencing LINC01138 protected chondrocytes from IL-1β-induced damage, possibly by regulating the hsa-miR-1207-5p/KIAA0101 axis.

Keywords: IL-1β; KIAA0101; LINC01138; hsa-miR-1207-5p; osteoarthritis.

Figure 1

LINC01138 was highly expressed in OA tissues. (A) LINC01138 level in GSE105027 data set was analyzed in GEO database. (B) Expression level of LINC01138 in OA patient tissue was determined by qRT‐PCR. (C) Expression level of LINC01138 in chondrocytes treated with IL‐1β was detected by qRT‐PCR. *p < .05 compare with normal (NC) group. ns, no significant; qRT‐PCR, Quantitative real time PCR.

Figure 2

Silencing of LINC01138 protected chondrocytes from matrix degradation and inflammation caused by IL‐1β. (A) Expression level of LINC01138 in cells was determined by qRT‐PCR. (B) Level of Col2α1 and (C) aggrecan in cells was determined by qRT‐PCR. (D) Level of sGAG in cells was determined by sGAG analysis. (E) Levels of MMP‐13, IL‐6, and TNF‐α in cell supernatant were determined by ELISA. (F) Protein levels of MMP‐13, IL‐6, and TNF‐α in cells were determined by western blot. *p < .05 as compared with the indicated group. ELISA, enzyme‐linked immunosorbent assay; IL, interleukin; MMP, matrix metalloproteinase; qRT‐PCR, Quantitative real time PCR; sGAG, sulphated glycosaminoglycans; TNF, tumor necrosis factor.

Figure 3

LINC01138 targeted hsa‐miR‐1207‐5p. (A) The differentially expressed miRNAs in GSE105027 data set was analyzed and target genes of the LINC01138 were searched using the DIANA‐TarBase. hsa‐miR‐1207‐5p was screened out as an overlapped gene. (B) Expression level of hsa‐miR‐1207‐5p in OA patient tissue was determined by qRT‐PCR. (C) Expression level of hsa‐miR‐1207‐5p in chondrocytes treated with IL‐1β was detected by qRT‐PCR. (D) Binding sites between LINC01138 and miR‐1207‐5p were predicted by DIANA‐TarBase. (E) Expression level of miR‐1207‐5p was detected by qRT‐PCR. (F) Luciferase activity was detected by Dual‐luciferase reporter assay. (G) Transfection efficient was determined by qRT‐PCR. (H) Levels of Col2α1 and (I) aggrecan in cells were determined by qRT‐PCR. (J) Level of sGAG in cells was determined by sGAG analysis. (K) The levels of MMP‐13, IL‐6, and TNF‐α in cells were determined by ELISA. (L) Protein levels of MMP‐13, IL‐6, and TNF‐α in cells were determined by western blot analysis. ^* p < .05 as compared with the indicated group. ELISA, enzyme‐linked immunosorbent assay; IL, interleukin; miRNA, microRNA; qRT‐PCR, Quantitative real time PCR; sGAG, sulphated glycosaminoglycans; TNF, tumor necrosis factor.

Figure 4

Hsa‐miR‐1207‐5p targeted KIAA0101. (A) Differential expressed genes in GSE1919 data set were searched and target genes of hsa‐miR‐1207‐5p were searched using Targetscan. KIAA0101 was screened out as an overlapped gene. (B) KIAA0101 level in GSE1919 data set was analyzed. (C) Expression level of KIAA0101 in OA patient tissues was determined by qRT‐PCR. (D) Expression level of KIAA0101 in chondrocytes treated with IL‐1β was detected by qRT‐PCR. (E) Binding sites between KIAA0101 and miR‐1207‐5p were predicted by Targetscan. (F) Expression level of KIAA0101 was detected by qRT‐PCR. (G) Luciferase activity was detected by Dual‐luciferase reporter assay. (H) Transfection efficient was determined by qRT‐PCR. (I) Expression levels of Col2α1 and (J) aggrecan in cells were determined by qRT‐PCR. (K) Level of sGAG in cells was determined by sGAG analysis. (L) The levels MMP‐13, IL‐6, and TNF‐α in cells were determined by ELISA. (M) Protein levels of MMP‐13, IL‐6, and TNF‐α in cells were determined by western blot analysis. ^* p < .05 as compared with the indicated group. ELISA, enzyme‐linked immunosorbent assay; IL, interleukin; qRT‐PCR, Quantitative real time PCR; sGAG, sulphated glycosaminoglycans; TNF, tumor necrosis factor.

Figure 5

Silencing of KIAA0101 inhibits the JAK/STAT and Wnt signaling pathways. (A, C) Pathways activated by KIAA0101 were obtained by GSEA analysis. (B, D) Protein levels of JAK1, p‐JAK1, STAT3, p‐STAT3, Wnt1, and β‐catenin were measured by western blotting.