miR-143-3p regulates cell proliferation and apoptosis by targeting IGF1R and IGFBP5 and regulating the Ras/p38 MAPK signaling pathway in rheumatoid arthritis

Abstract

It has been demonstrated that the deregulation of microRNAs (miRNAs) affects the development of rheumatoid arthritis (RA). The primary objective of the current study was to determine the role of miR-143-3p in the progression of RA. The expression of miR-143-3p in synovium taken from patients with RA was assessed by reverse transcription-quantitative polymerase chain reaction. The expression of miR-143-3p was higher in synovium tissues of RA than that of osteoarthritis (OA). The decreased expression of miR-143-3p suppressed cell proliferation and promoted apoptosis in vitro. In addition, inhibition of miR-143-3p decreased levels of inflammatory cytokines, as determined by an enzyme-linked immunosorbent assay. IGF1R and IGFBP5 were found to be the target genes of miR-143-3p, and it was demonstrated that miR-143-3p regulated the proliferation and apoptosis of MH7A cells by targeting IGF1R and IGFBP5. Furthermore, TNF-α treatment stimulated the Ras/p38 mitogen activated protein kinase (MAPK) signaling pathway, whereas miR-143-3p inhibition suppressed it. The results of the current study indicate that miR-143-3p may regulate cell proliferation and apoptosis by targeting IGF1R and IGFBP5 expression and regulating the Ras/p38 MAPK signaling pathways. Therefore, miR-143-3p may be a novel therapeutic target in RA.

Keywords: insulin-like growth factor 1 receptor; insulin-like growth factor binding protein 5; microRNA-143-3p; rheumatoid arthritis; synovial fibroblasts.

Figure 1.

The expression of miR-143-3p in RA tissue and cells. (A) The expression of miR-143-3p in patients with RA compared with patients with OA and a healthy control. (B) The expression of miR-143-3p in a model of RA induced by treatment with TNF-α. Data are presented as the mean ± standard deviation from at least three independent experiments. *P<0.05 and **P<0.01 vs. control. miR, microRNA; NS, non-significant; OA, osteoarthritis. RA, rheumatoid arthritis; TNF, tumor necrosis factor.

Figure 2.

Inhibition of miR-143-3p reduces cell proliferation and promotes cell apoptosis. (A) Transfection of miR-143-3p inhibitor in MH7A cells. **P<0.01 vs. control. (B) Effect of TNF-α treatment and transfection with miR-143-3p inhibitor on cell proliferation and (C and D) apoptosis. (E and F) Effect of TNF-α treatment and miR-143-3p inhibition on the expression of Bax, Bcl-2, pro-caspase-3 and active caspase-3. Data are presented as the mean ± standard deviation from at least three independent experiments. *P<0.05 and **P<0.01 vs. control; ^#P<0.05 vs. inhibitor NC group. TNF, tumor necrosis factor; miR, microRNA; OD, optical density; NC, negative control.

Figure 3.

Inhibition of miR-143-3p suppresses the expression of inflammatory factors. Levels of (A) IL-1β, (B) IL-6, (C) IL-8, (D) MMP-1 (E) and MMP-13. Data are presented as the mean ± standard deviation from at least three independent experiments. *P<0.05 and **P<0.01. miR, microRNA; NC, negative control; IL, interleukin; MMP, matrix metalloproteinase.

Figure 4.

The prediction and examination of the targeting effects of miR-143-3p. (A) IGF1R and (B) IGFBP5 are target genes of miR-143-3p. Relative luciferase activity of (C) IGF1R and (D) IGFBP5. (E) The expression of IGF1R and IGFBP5 mRNA following transfection with miR-143-3p mimic and inhibitor. (F) The expression of IGF1R and IGFBP5 protein following transfection with miR-143-3p mimic and inhibitor. (G) The expression of IGF1R and IGFBP5 mRNA following treatment with TNF-α. (H) The expression of IGF1R and IGFBP5 protein following treatment with TNF-α. Data are presented as the mean ± standard deviation from at least three independent experiments. *P<0.05 and **P<0.01. UTR, untranslated region; miR, microRNA; TNF, tumor necrosis factor; NC, negative control; IGF1R, insulin-like growth factor 1 receptor; IGFBP5, insulin-like growth factor-binding protein 5.

Figure 5.

Effect of IGF1R or IGFBP5 suppression on cell growth and apoptosis. (A-D) Transfection efficiency of si-IGF1R and si-IGFBP5. *P<0.05, **P<0.01 and ***P<0.001. (E-H) Effect of silencing IGF1R and IGFBP5 expression on cell proliferation and apoptosis. Data are presented as the mean ± standard deviation from at least three independent experiments. *P<0.05; **P<0.01 and ***P<0.001 vs. the inhibitor NC group. ^&P<0.05 and ^&&P<0.01 vs. the si-NC group; ^#P<0.05 and ^##P<0.01 vs. the si-IGF1R group or si-IGFBP5 group. miR, microRNA; si, small interfering; NC, negative control; IGF1R, insulin-like growth factor 1 receptor; IGFBP5, insulin-like growth factor-binding protein 5; OD, optical density.

Figure 6.

The effects of TNF-α treatment and miR-143-3p inhibition on the Ras/p38 MAPK signaling pathway. (A) Levels of Ras and p-p38/p38 mRNA. (B) Levels of Ras and p-p38/p38 protein. Data are presented as the mean ± standard deviation from at least three independent experiments. **P<0.01 and ***P<0.001. p-, phosphorylated; miR, microRNA; TNF, tumor necrosis factor; NC, negative control.