Circular RNA CSNK1G1 promotes the progression of osteoarthritis by targeting the miR‑4428/FUT2 axis

Abstract

Osteoarthritis (OA) is a chronic disease that results in chronic arthralgia and functional disability of the affected joint. To date, there is no effective treatment available for this disease. Circular RNAs (circRNAs) are a type of intracellular stable RNA that can regulate the development and progression of OA. However, the function of circCSNK1G1 in OA has not yet been investigated. In the present study, it was found that circCSNK1G1 was upregulated in OA cartilage tissues. The upregulation of circCSNK1G1 was associated with extracellular matrix (ECM) degradation and chondrocyte apoptosis. Moreover, the expression of miR‑4428 was downregulated and that of fucosyltransferase 2 (FUT2) was upregulated in OA‑affected cartilage tissues. Dual‑luciferase reporter assay and RNA immunoprecipitation confirmed that miR‑4428 targeted FUT2 mRNA to inhibit FUT2 expression. circCSNK1G1 and FUT2 induced ECM degradation and chondrocyte apoptosis. The negative effects of circCSNK1G1 and FUT2 were reversed by miR‑4428. On the whole, the present study demonstrates that circCSNK1G1 promotes the development of OA by targeting the miR‑4428/FUT2 axis. Thus, the circCSNK1G1/miR‑4428/FUT2 axis may present a novel target for the treatment of OA in the clinical setting.

Keywords: circular RNA CSNK1G1; miR-4428; osteoarthritis; chondrocyte; fucosyltransferase 2.

Figure 1

circCSNK1G1 is upregulated in OA-affected cartilage. (A) Expression of circCSNK1G1 in cartilage tissue from patients with OA and normal human cartilage. (B) Expression of circCSNK1G1 in rat cartilage. Data are shown as the means ± SD (n=4, Student's t-test, ^**P<0.01, ^***P<0.001). OA, osteoarthritis.

Figure 2

circCSNK1G1 knockdown inhibits inflammation in OA. (A) circCSNK1G1 expression detected by RT-qPCR. (B-D) IL-6, TNF-α and LDH expres-sion in rat serum detected by ELISA. (E-G) IL-6, TNF-α and LDH expression in rat articular cartilage detected by RT-qPCR assay. (H) Cleaved caspase-3 and cleaved-caspase-9 expression in rat cartilage were detected by western blot analysis. (I) TUNEL staining of rat cartilage. (J) Evaluation of cartilage morphology by hematoxylin and eosin staining. (K) Morphological changes in articular cartilage evaluated by saffron solid green staining. Data are shown as the means ± SD (n=4, ANOVA followed by Tukey's multiple comparison test, ^*P<0.05, ^**P<0.01, ^***P<0.001). OA, osteoarthritis; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; LDH, lactate dehydrogenase.

Figure 3

circCSNK1G1 promotes chondrocyte apoptosis by sponging miR-4428. (A) Predicted binding sites of miR-4428 in circCSNK1G1. (B) Detection of the expression of miR-4428 in the rat osteoarthritis (OA) model by RT-qPCR (n=4, ANOVA followed by Tukey's multiple comparison test, ^**P<0.01). (C) Expression of miR-4428 in cultured chondrocytes detected by RT-qPCR (n=4, ANOVA followed by Tukey's multiple comparison test, ^**P<0.01). (D) Correlation analysis between miR-4428 and circCSNK1G1 expression in cartilage tissue from patients with OA (n=10, r=−0.6621, P<0.05). (E) Proliferation assays of cultured chondrocytes. (F and G) Percentage of apoptotic cells detected by flow cytometry. (H-L) Expression of Bax, Bcl-2, Cyt-c, MMP-13, collagen II and aggrecan in cultured chondrocytes was detected by western blot analysis or RT-qPCR (ANOVA followed by Tukey's multiple comparison test, ^*P<0.05, ^**P<0.01, ^***P<0.001). Cyt-c, cytochrome c; MMP-13, matrix metalloproteinase 13.

Figure 4

miR-4428 protects chondrocytes in OA. (A) miR-4428 expression in rat cartilage detected by RT-qPCR. (B) Expression of miR-4428 in cultured chondrocytes detected by RT-qPCR. (C) Proliferation assays of cultured chondrocytes. (D and E) Percentage of apoptotic cells detected by flow cytometry. (F-J) Expression of Bax, Bcl-2, Cyt-c, MMP-13, collagen II and aggrecan in cultured chondrocytes was detected by western blot analysis or RT-qPCR (n=4, ANOVA followed by Tukey's multiple comparison test, ^*P<0.05, ^**P<0.01, ^***P<0.001). OA, osteoarthritis; Cyt-c, cytochrome c; MMP-13, matrix metalloproteinase 13.

Figure 5

miR-4428 targets FUT2 in OA. (A) Predicted binding sites of miR-4428 and FUT2 mRNA. (B) FUT2 expression following circCSNK1G1 knock-down in the rat model of OA. (C) Detection of luciferase activity to confirm the association between miR-4428 and FUT2. (D) Expression of FUT2 in cultured chondrocytes. ^*P<0.05, ^**P<0.01. (E) Correlation analysis between miR-4428 and FUT2 mRNA expression in patients with OA (n=10, r=−0.7597). OA, osteoarthritis; FUT2, fucosyltransferase 2.

Figure 6

miR-4428 inhibits chondrocyte apoptosis by targeting FUT2. (A) Expression of FUT2 in cultured chondrocytes. (B) Expression of FUT2 following FUT2 siRNA knockdown. (C) Proliferation assays of cultured chondrocytes. (D and E) Percentage of apoptotic cells detected by flow cytometry assay. (F-J) Expression of Bax, Bcl-2, Cyt-c, MMP-13, collagen II and aggrecan in cultured chondrocytes was detected by western blot analysis or RT-qPCR ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001). OA, osteoarthritis; Cyt-c, cytochrome c; MMP-13, matrix metalloproteinase 13.