miR-125 regulates PI3K/Akt/mTOR signaling pathway in rheumatoid arthritis rats via PARP2

Abstract

The present study aimed to explore miR-125 effects on rheumatoid arthritis (RA) development to provide a potential target for RA. Briefly, rat RA model was established (Model group) by injection of Freund's Complete Adjuvant into the left hind toe. Normal rats injected with saline in the same location were set as Normal group. All rats' secondary foot swelling degree, polyarthritis index score, spleen and thymus index were measured. Synovial tissues were subjected to Hematoxylin-Eosin (HE) staining and immunohistochemistry. Synovial cells of each group were isolated and named as Normal-C group and Model-C group, respectively. Synovial cells of Model-C group further underwent cotransfection with miR-125 mimics and PARP2-siRNA (mimics+siPARP2 group) or with miR-125 negative control (NC) and PARP2-siRNA NC (NC group). Quantitative reverse transcriptase PCR (qRT-PCR), Western blot, luciferase reporter assay, ELISA, and MTT assay were performed. As a result, compared with Normal group, rats of Model group showed significantly higher secondary foot swelling degree, polyarthritis index score, spleen and thymus index (P<0.01). Down-regulated miR-125 and up-regulated PARP2 was found in synovial tissues of Model group when compared with Normal group (P<0.01). Synovial tissues of Model-C group exhibited severe hyperplasia and inflammatory cell infiltration. Luciferase reporter assay indicated that PARP2 was directly inhibited by miR-125. Compared with NC group, cells of mimics+siPARP2 group had significantly lower IL-1β, MMP-1 and TIMP-1 levels, absorbance value, and p-PI3K, p-Akt and p-mTOR relative expression (P<0.01 or P<0.05). Thus, miR-125 might attenuate RA development by regulating PI3K/Akt/mTOR signaling pathway via directly inhibiting PARP2 expression.

Keywords: PARP2; PI3K/Akt/mTOR signaling pathway; miR-125; rheumatoid arthritis.

Figure 1. The group flow chart of the entire article

Figure 2. Rat RA model was successfully constructed

(A) Rats of model group exhibited much higher secondary foot swelling degree than the Normal group. (B) Significantly increased polyarthritis index was found in rats of model group when compared with Normal group. (C) The spleen and thymus index of rats of model group was much higher than that of Normal group. (D) HE staining indicated that the synovial tissues of rats in Normal group were much thinner with one to two layers of synovial cells, whereas the synovial tissues of rats in Model group occurred severe inflammatory cell infiltration with three to five layers of synovial cells. **P<0.01 when compared with Normal group.

Figure 3. Down-regulation of miR-125 and up-regulation of PARP2 in rats’ synovial tissues and cells

(A) miR-125 expression in synovial tissues of Model group declined when compared with Normal group. (B) Immunohistochemistry results showed that the number of PARP2 positive cells in synovial tissue of Model group was significantly higher than that of Normal group. (C) miR-125 expression in synovial cells of Model-C group was markedly lower than that of Normal-C group. (D) Significantly increased PARP2 protein expression was observed in synovial cells of Model-C group when compared with Normal-C group. **P<0.01 when compared with Normal group or Normal-C group.

Figure 4. PARP2 was a target gene of miR-125

(A) TargetScan predicted that the 3′-UTR region was the target binding domain of PARP2 and miR-125. (B) Significantly decreased luciferase activity intensity was found in WT+mimics group when compared with WT+NC group, indicating that PARP2 was directly inhibited by miR-125. **P<0.01 when compared with WT+NC group.

Figure 5. Co-transfection of miR-125 mimics and PARP2-siRNA inhibited IL-1β, MMP-1, and TIMP-1 levels in synovial cells of RA rats

(A–C) According to ELISA, IL-1β, MMP-1, and TIMP-1 levels in synovial cells of Model-C group were significantly higher than those of Normal-C group. Compared with NC group, much decreased IL-1β, MMP-1, and TIMP-1 levels was found in synovial cells of mimics+siPARP2 group. **P<0.01 or *P< 0.05 when compared with Normal-C group. ^##P<0.01 or ^#P<0.05 when compared with NC group.

Figure 6. Co-transfection of miR-125 mimics and PARP2-siRNA inhibited synovial cells proliferation of RA rats

At 48–96 h, the OD₄₉₅ values of synovial cells in Model-C group were significantly higher than that in Normal-C group. Synovial cells OD₄₉₅ values in mimics+siPARP2 group were significantly decreased when compared with NC group. *P<0.05 when compared with Normal-C group. ^#P<0.05 when compared with NC group.

Figure 7. Co-transfection of miR-125 mimics and PARP2-siRNA inhibited PI3K/Akt/mTOR signaling pathway activity in synovial cells of RA rats

(A–C) The relative expression of p-PI3K, p-Akt, and p-mTOR in synovial cells of Model-C group was significantly higher than that of Normal-C group. Compared with NC group, significantly decreased p-PI3K, p-Akt, and p-mTOR relative expression was found in synovial cells of mimics+siPARP2 group. *P<0.05 when compared with Normal-C group. ^#P<0.05 when compared with NC group.