*2.5. MicroRNA-941 Inhibits WNT16 Expression in CD14*+ *Monocyte, and then Contributes to Active Osteoclastogenesis Independent of miR-146a-5p*

Our previous study demonstrated that higher expression of miR-146a-5p in CD14+ monocytes of psoriatic arthritis than that in HCs. We then asked whether miR-941 and miR-146a-5p interact to activate osteoclastogenesis. Peripheral CD14+ monocytes were obtained from five PsA patients. Some cells were cultured with medium only while other cells were treated with M-CSF for 72 h. We then asked whether miR-941 regulated miR-146a-5p expression or vice versa. Therefore, four treatment groups were designed—one transfected with a control microRNA inhibitor (mock transfection control), one with a miR-941 inhibitor, one with miR-146a-5p inhibitor, and one without transfection. After transfection, cultures were treated with TNF-α and RANKL every three days for nine days to induce osteoclast formation. At day 13, expressions of miR-941 and miR-146a-5p in monocytes and monocyte-derived osteoclasts were measured. As anticipated, the results showed that the expressions of miR-941 and miR-146a-5p increased after osteoclastogenesis (Figure 4A,B). However, blocking miR-941 did not alter the expressions of miR-146a-5p. Conversely, blocking of miR-146a-5p did not alter the expressions of miR-941, either (Figure 4A,B). The results indicated that the two miRNA pathways may not directly interact with each other in the osteoclastogenesis of PsA. We next asked what regulatory pathways mediate the miR-941-induced osteoclastogenesis and bone resorption. Both hedgehog-signaling and insulin-signaling pathways were reported as the potential target genes of miR-941, including *SMO, SUFU, GLI, WNT16, IRS1, mTOR*, and *PPP1CA* [17]. Notably, specifically in the process of osteoclastrogenesis, Movérare-Skrtic et al. reported that WNT16 represses human osteoclastogenesis and prevents cortical bone fragility fractures through direct effects on osteoclast progenitors [18]. Hence, we examined whether miR-941 could negatively regulate WNT16 expression, leading to active osteoclastogenesis. To address that, we investigated if miR-941 inhibition could reverse the expression of WNT16 in CD14+ monocytes of PsA patients, PsO patients, and HCs. In addition, we also measured the expression of mTOR as an internal control in the same experimental setting. The result showed that WNT16 expression was significantly lower in monocytes, monocytes derived osteoclasts with/without mock microRNA transfection of PsA patients compared to those from PsO patients or HCs (*p* < 0.05). The expression of WNT16 in osteoclast with miR-941 inhibitor from PsA patients is similar to that from PsO patients or HCs (Figure 4C). In contrast, the expression of mTOR did not change with or without miR-941 inhibition (Figure 4D).

**Figure 4.** miR-941 inhibits osteoclastogenesis and negatively regulates WNT16 independent of miR-146a-5p. The CD14+ monocytes obtained from five PsA patients, five PsO patients, and five HCs were treated with medium only or subsequently activated into osteoclasts with treatment of M-CSF for 72 h. The cells with M-CSF treatment were either nontransfected or transfected with the control-microRNA inhibitor, miR-941 inhibitor or miR-146a-5p followed by TNF-α and RANKL every three days for nine days to study their ability to form osteoclasts and their activity of bone resorption. (**A**,**B**) At day 13, the expressions of miR-941 and miR-146a-5p in the monocytes and osteoclasts were measured by qRTPCR to evaluate the effect of miR-941 and miR-146a-5p inhibitors transfection. (**C**,**D**) The protein expression of WNT16 and mTOR, the target genes of miR-941, were measured by western blot. \* *p* < 0.05 and \*\* *p* < 0.01.
