FGD5-AS1 Inhibits Osteoarthritis Development by Modulating miR-302d-3p/TGFBR2 Axis

Abstract

Objective: Osteoarthritis (OA) is a common joint disorder, accompanied by extracellular matrix (ECM) degradation. Reportedly, long noncoding RNAs (lncRNAs) are involved in OA pathogenesis. However, the role of lncRNA FYVE, RhoGEF, and PH domain containing 5 antisense RNA 1 (FGD5-AS1) in OA development is still not fully clarified. This study was aimed to clarify the role of FGD5-AS1 in OA.

Methods: FGD5-AS1 and miR-302d-3p expression levels were determined in cartilage tissues and chondrocytes by quantitative real-time polymerase chain reaction (qRT-PCR). Chondrocytes (C20/A4 cells) were stimulated with interleukin 1β (IL-1β) to mimic the inflammatory environment of OA. Cell viability was detected by cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays. Cell apoptosis was measured by the caspase-3 activity assay and flow cytometry. Transforming growth factor beta receptors II (TGFBR2), matrix metalloproteinase 13 (MMP-13), and ADAM metallopeptidase with thrombospondin type 1 motif 5 expression levels were examined by qRT-PCR or Western blot. The regulatory relationships among FGD5-AS1, miR-302d-3p, and TGFBR2 were predicted by the StarBase v2.0, miRanda, miRDB, and TargetScan databases, and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay.

Results: FGD5-AS1 and TGFBR2 expression levels were downregulated while miR-302d-3p expression was increased in cartilage tissues of OA patients. Knocking down FGD5-AS1 inhibited the viability of C20/A4 cells but induced apoptosis and ECM degradation, while FGD5-AS1 overexpression exerted opposite effects. MiR-302d-3p was identified as a target of FGD5-AS1, and TGFBR2 was identified as a target of miR-302d-3p. FGD5-AS1 positively regulated TGFBR2 expression by repressing miR-302d-3p expression, and miR-302d-3p inhibition or TGFBR2 restoration reversed the changes of cell viability, apoptosis, and ECM degradation induced by FGD5-AS1 knockdown.

Conclusion: FGD5-AS1 can probably inhibit OA progression by regulating miR-302d-3p/TGFBR2 axis.

Keywords: FGD5-AS1; MiR-302d-3p; TGFBR2; osteoarthritis.

Figure 1.

FGD5-AS1 expression in OA and normal cartilage tissues. (A) FGD5-AS1 expression was detected by qRT-PCR in the cartilage tissues from patients with OA or healthy donors (n = 35 in each group). (B) FGD5-AS1 siRNAs (si-FGD5-AS1-1 or -2) or the control siRNA were transfected into C20/A4 cells. qRT-PCR was utilized for analyzing FGD5-AS1 expression in C20/A4 cells after the transfection. OA = osteoarthritis; qRT-PCR = quantitative real-time polymerase chain reaction; si-NC = siRNA negative control; si-FGD5-AS1 = siRNA targeting FGD5-AS1. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2.

Impacts of FGD5-AS1 overexpression on chondrocyte proliferation, apoptosis, and ECM degradation after IL-1β treatment. (A) pcDNA-FGD5-AS1 or pcDNA-NC was transfected into C20/A4 cells, and qRT-PCR was conducted for determining FGD5-AS1 expression. (B) C20/A4 cells were stimulated with IL-1β (0, 1, 5, and 10 ng/mL) for 48 hours, and qRT-PCR was conducted for determining FGD5-AS1 expression. (C-E) The viability, proliferation, and apoptosis of C20/A4 cells were detected by CCK-8 assay (C), EdU assay (D), and flow cytometry analysis (E), respectively. Il-1β = interleukin-1β; qRT-PCR = quantitative real-time polymerase chain reaction. **P < 0.01 and ***P < 0.001.

Figure 3.

FGD5-AS1 repressed miR-302d-3p expression as a ceRNA. (A) StarBase 2.0 database was employed for predicting the interaction between FGD5-AS1 and miR-302d-3p. (B) MiR-302d-3p inhibitors (anti-miR-302d-3p) or mimics (miR-302d-3p) and the controls (anti-NC or miR-NC) were transfected into C20/A4 cells, and qRT-PCR was conducted for detecting miR-302d-3p expression. (C and D) In C20/A4 cells co-transfected with miRNAs and luciferase reporter vectors, luciferase activity was detected to validate the predicted targeting relationship. (E) RIP assay was utilized for analyzing the direct interaction between FGD5-AS1 and miR-302d-3p. (F) MiR-302d-3p expression was examined in C20/A4 cells transfected with pcDNA-FGD5-AS1 or si-FGD5-AS1-1. OA = osteoarthritis; WT = wide-type; MUT = mutant-type; miR-NC = mimics negative control; miR-302d-3p = miR-302d-3p mimics; anti-NC = inhibitors negative control; anti-miR-302d-3p = miR-302d-3p inhibitors; si-NC = siRNA negative control; si-FGD5-AS1 = siRNA targeting FGD5-AS1. *P < 0.05 and ***P < 0.001.

Figure 4.

MiR-302d-3p targeted TGFBR2. (A) MiRanda, miRDB, and TargetScan were applied for predicting miR-302d-3p’s potential targets, and TGFBR2 was among the candidates. (B and C) In C20/A4 cells co-transfected with miRNAs and luciferase reporter vectors, luciferase activity was detected to validate the predicted targeting relationship. (D and E) MiR-302d-3p inhibitors (anti-miR-302d-3p) or mimics (miR-302d-3p) and the controls (anti-NC or miR-NC) were transfected into C20/A4 cells. qRT-PCR and Western blot were utilized for examining TGFBR2 expression in C20/A4 cells at mRNA and protein level, respectively. WT = wide-type; MUT = mutant-type; miR-NC = mimics negative control; miR-302d-3p = miR-302d-3p mimics; anti-NC = inhibitors negative control; anti-miR-302d-3p = miR-302d-3p inhibitors. *P < 0.05 and ***P < 0.001.