Knockdown of long non-coding RNA KCNQ1OT1 suppresses the progression of osteoarthritis by mediating the miR-211-5p/TCF4 axis in vitro

Abstract

Numerous studies have reported the critical roles of long non-coding RNAs (lncRNAs) in the regulation of osteoarthritis (OA) development. The present study aimed to assess the function and regulatory mechanism of a lncRNA, KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1), in OA in vitro. C28/I2 cells were treated with lipopolysaccharide (LPS) to generate an in vitro OA model. The relative expression levels of KCNQ1OT1, microRNA (miR)-211-5p and transcription factor 4 (TCF4) were determined via reverse transcription-quantitative polymerase chain reaction. The associations between KCNQ1OT1, miR-211-5p and TCF4 were confirmed using a dual-luciferase reporter assay. Furthermore, cell viability was assessed using the MTT assay. Inflammatory cytokine levels were measured using ELISA. The protein expression levels of matrix metalloproteinase-3/13, collagen II/X and TCF4 were detected by western blotting. KCNQ1OT1 and TCF4 were highly expressed in the cartilage tissues of patients with OA and C28/I2 cells treated with LPS (OA cells), whereas miR-211-5p was downregulated concomitantly in OA tissues and cells. Knockdown of KCNQ1OT1 stimulated cell viability, and suppressed the inflammation and degradation of the extracellular matrix (ECM) in OA cells. In addition, overexpression of miR-211-5p stimulated cell viability, and inhibited inflammation and degradation of the ECM in OA cells. Notably, miR-211-5p was revealed to be the target of, and was negatively regulated by, KCNQ1OT1. TCF4 was targeted and negatively modulated by miR-211-5p. Transfection of cells with the miR-211-5p inhibitor or pcDNA-TCF4 reversed the suppressive effects of short hairpin RNA (sh)-KCNQ1OT1 on inflammation and ECM degradation, as well as the promotive effect of sh-KCNQ1OT1 on viability in OA in vitro. Therefore, KCNQ1OT1 may regulate the miR-211-5p/TCF4 axis to ameliorate OA in vitro.

Keywords: long non-coding RNA KCNQ1OT1; miR-211-5p; microRNAs; osteoarthritis; transcription factor 4.

Figure 1

Silencing of KCNQ1OT1 alleviates chondrocyte injury caused by LPS in C28/I2 cells. (A) Relative expression level of KCNQ1OT1 in OA cartilage tissues and normal cartilage tissues was determined by RT-qPCR. ^**P<0.01 vs. normal. (B) Relative expression level of KCNQ1OT1 in C28/I2 cells was determined by RT-qPCR. ^**P<0.01 vs. normal. (C) Relative expression level of KCNQ1OT1 in C28/I2 cells was determined by RT-qPCR post-transfection with sh-KCNQ1OT1/NC or pcDNA-KCNQ1OT1/NC. ^**P<0.01 vs. blank. (D) Cell viability was assessed by MTT assay. ^**P<0.01 vs. control; ^##P<0.01 vs. LPS + sh-NC. Levels of (E) IL-6 (F) IL-1β and (G) TNF-α in culture medium were measured by ELISA. ^**P<0.01 vs. control; ^##P<0.01 vs. LPS + sh-NC. (H) Protein expression levels of MMP-3, MMP-13, collagen X and collagen II were detected by western blotting. ^**P<0.01 vs. control; ^##P<0.01 vs. LPS + sh-NC. LPS, lipopolysaccharide; OA, osteoarthritis; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; sh, short hairpin RNA; NC, negative control; KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; MMP, matrix metalloproteinase.

Figure 2

KCNQ1OT1 may serve as a competitive endogenous RNA of miR-211-5p. (A) Binding sequence between KCNQ1OT1 and miR-211-5p was predicted by lncBase Predicted v.2. (B) Relative expression level of miR-211-5p after transfection with sh-KCNQ1OT1/NC or pcDNA-KCNQ1OT1/NC into C28/I2 cells was detected by RT-qPCR. ^**P<0.01 vs. sh-NC. (C) Targeting association between KCNQ1OT1 and miR-211-5p was validated by dual-luciferase reporter assay. (D) Targeting association between KCNQ1OT1 and miR-211-5p was validated by RIP assay. ^**P<0.01 vs. miR-NC. miR, microRNA; sh, short hairpin RNA; NC, negative control; KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; wt, wild type; mut, mutant; RIP, RNA immunoprecipitation.

Figure 3

Overexpression of miR-211-5p reduces chondrocyte injury caused by LPS in C28/I2 cells. (A) Relative expression level of miR-211-5p in OA cartilage tissues and normal cartilage tissues was determined by RT-qPCR. ^**P<0.01 vs. normal. (B) Relative expression level of miR-211-5p in C28/I2 cells was determined by RT-qPCR. ^**P<0.01 vs. control. (C) Relative expression level of miR-211-5p in C28/I2 cells was determined by RT-qPCR following transfection with miR-211-5p mimics. ^**P<0.01 vs. blank. (D) Cell viability was assessed by MTT assay. ^**P<0.01 vs. LPS + miR-NC. Levels of (E) IL-6, (F) IL-1β and (G) TNF-α in culture medium were measured by ELISA. ^**P<0.01 vs. LPS + miR-NC. (H) Protein expression levels of MMP-3 MMP-13, collagen X and collagen II were detected by western blotting. ^**P<0.01 vs. LPS + miR-NC. miR, microRNA; LPS, lipopolysaccharide; OA, osteoarthritis; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control; MMP, matrix metalloproteinase.

Figure 4

TCF4 is a target gene of miR-211-5p. (A) Binding sequence between miR-211-5p and TCF4 was predicted by TargetScan and miRDB. (B) Relative protein expression level of TCF4 was detected by western blotting. ^**P<0.01 vs. miR-NC. (C) Relative protein expression level of TCF4 was detected by western blotting following transfection of C28/I2 cells with pcDNA-KCNQ1OT1. ^**P<0.01 vs. pcDNA-NC. (D) Targeting association between miR-211-5p and TCF4 was validated by dual-luciferase reporter assay. ^**P<0.01, vs. miR-NC. (E) Targeting association between miR-211-5p and TCF4 was validated by RIP assay. ^**P<0.01, vs. miR-NC. miR, microRNA; NC, negative control; KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; TCF4, transcription factor 4; wt, wild type; mut, mutant; RIP, RNA immunoprecipitation.

Figure 5

KCNQ1OT1 indirectly regulates expression of TCF4 by competitively binding to miR-211-5p. (A) Relative expression level of TCF4 in OA cartilage tissues and normal cartilage tissues was determined by RT-qPCR. ^**P<0.01 vs. normal. (B) Relative protein expression level of TCF4 in C28/I2 cells was determined by western blotting. ^**P<0.01 vs. control. (C) Expression levels of miR-211-5p and TCF4 were detected by RT-qPCR. ^**P<0.01 vs. inhibitor NC or pcDNA-NC. (D) Relative protein expression level of TCF4 in transfected C28/I2 cells were determined by western blotting. ^**P<0.01 vs. LPS + sh-NC; ^##P<0.01 vs. LPS + sh-KCNQ1OT1. (E) Cell viability was assessed by MTT assay. ^**P<0.01 vs. LPS + sh-NC; ^##P<0.01 vs. LPS + sh-KCNQ1OT1. Levels of (F) IL-6, (G) IL-1β and (H) TNF-α in culture medium were measured by ELISA. ^**P<0.01 vs. LPS + sh-NC; ^##P<0.01 vs. LPS + sh-KCNQ1OT1. (I) Protein expression levels of MMP-3, MMP-13, collagen X and collagen II were detected by western blotting. ^**P<0.01 vs. LPS + sh-NC; ^##P<0.01 vs. LPS + sh-KCNQ1OT1. miR, microRNA; OA, osteoarthritis; LPS, lipopolysaccharide; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; sh, short hairpin RNA; NC, negative control; KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; TCF4, transcription factor 4; MMP, matrix metalloproteinase.