*2.6. The Enhanced miR-941 Expression was Reduced in CD14*+ *Cells from PsA Patients after Successful Biologics Treatment*

To address whether the expression of miR-941 from circulating monocytes might serve as a potential biomarker, we examined whether the increased miR-941 expression in CD14+ monocytes from PsA patients was reduced after successful treatment. Among the 12 PsA patients that met the ACR20 achievement after 28 weeks of biologics treatment (etanercept, adalimumab, secukinumab, or ustekinumab) (Figure 5A), the enhanced expression of miR-941 in CD14+ monocytes returned to the level of NCs (Figure 5B).

**Figure 5.** Reduced miR-941 expression in CD14+ monocytes from PsA patients after successful biologics treatment. The expression levels of miR-941 were measured in CD14+ monocytes from HCs and PsA patients before and after successful biologic treatment. (**A**) The total number of tender and swollen joints in PsA patients were measured before and 28 weeks after biologics treatment. (**B**) The expression levels of miR-941 were measured in CD14+ monocytes from 31 NCs and 12 PsA patients before and after 28 weeks of biologics treatment using qRT-PCR. \* *p* < 0.05; \*\* *p* < 0.01.

#### **3. Discussion**

Our study investigated the overall expression of miRNAs from CD14+ monocytes of PsA patients. Expression of miR-941 was higher in CD14+ monocytes from PsA patients than that from PsO patients and HCs, and CD14+ monocytes from PsA patients tend to exhibit enhanced osteoclast differentiation potential and resorption activity. Further, miR-941 inhibition resumed the elevated osteoclast formation potential and resorption activity of PsA monocytes with a reciprocal upregulation of WNT16. The expression of miR-941 in CD14+ monocytes of PsA patients correlated with disease severity.

Pathological bone resorption in PsA results from increased numbers of osteoclast precursors [8]. Early diagnosis of PsA has been hindered by the lack of reliable biomarkers. Our study identifies miRNA-941 in CD14+ monocytes as an easily accessible marker for prompt computer-aided diagnosis of PsA (with an auROC value 0.79 by SVM model). In addition, we demonstrate that the increased miR-941 expression in CD14+ monocytes from PsA patients enhances osteoclast formation potential and active resorption activity. Our previous study reported that miR-146a-5p expression in CD14+ monocytes of PsA patients correlates with clinical efficacy and inducts osteoclast activation and bone resorption [11]. In this study, the overall microRNAs were measured using NGS study. The result showed that the increased expression of miR-941 from CD14+ monocytes could be used for detection of PsA. In addition, miR-941 and miR-146a-5p did not interact to activate osteoclastogenesis. In other disease, miR-941 has been indicated to be a severity marker. For example, in acute coronary syndrome, miR-941 expression was reported to be increased in the plasma of the patients [19]. Duttagupta et al. identify that miR-941 is associated with ulcerative colitis [20].

The pro-inflammatory cytokines, including TNF-α, IL-17, IL-33, and osteopontin, through activation of RANKL, induce osteoclast differentiation and activation in PsA [21–24]. Biologics selectively targeting these specific cytokines (TNF-α, IL-17 and IL-12/23) and/or intracellular signaling pathways effectively inhibit osteoclastogenesis. However, more than 40% of PsA patients show only a partial response or fail to respond to current biologics [25]. On the other hand, both enhanced osteoclast differentiation and bone

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resorption could be reversed by miR-941 inhibition in CD14+ monocytes from PsA patients in this study (*n* = 10), indicating that miR-941 may serve as both a potential biomarker and treatment target for PsA.

WNT16 is a strong anti-resorptive soluble factor acting on osteoclast precursors [26]. It can significantly inhibit RANKL-induced osteoclastogenesis of human CD14+ peripheral blood monocytes, indicating a direct action of WNT16 on osteoclast precursors. Reduced osteoclast formation by WNT16 was associated with a time- and dose-dependent blunted mRNA expression of osteoclast functional genes [18,27]. Our results provide miR-941 inhibitor increased the expression of WNT16 and inhibit osteoclastogenesis, indicating the potential involvement of miR-941-WNT in the active osteoclastogenesis in patients with PsA.

This study has several limitations. First, the case number was small. The results may be validated by large-scale studies to identify potential confounders. Second, the PsA patients recruited may have different intrinsic co-morbidities that also alter miRNA expression (e.g., diabetes mellitus, cerebrovascular disease, hypertension, etc.), making these confounders could potentially interfere the results. Third, the expression level of miR-941 in CD14+ monocytes from PsA patients before and after conventional synthetic disease-modifying antirheumatic drugs was not investigated. Fourth, although there was a reciprocal change of miR-941 and WNT16 expression in this study, whether WNT16 could be a direct target for miR-941 may require a luciferase assay.

In conclusion, the elevated expression of miR-941 in CD14+ monocytes from PsA patients enhances osteoclast activation and bone resorption activity through WNT16 repression. The activation of miR-941 and reciprocal changes of WNT16 in osteoclasts contribute to bone destruction in PsA.
