**Upregulation of miR-941 in Circulating CD14**+ **Monocytes Enhances Osteoclast Activation via WNT16 Inhibition in Patients with Psoriatic Arthritis**

**Shang-Hung Lin 1,2,3, Ji-Chen Ho 1, Sung-Chou Li 4, Yu-Wen Cheng 1, Yi-Chien Yang 1, Jia-Feng Chen 5, Chung-Yuan Hsu 5, Toshiaki Nakano 2,6, Feng-Sheng Wang 7, Ming-Yu Yang 2,8, Chih-Hung Lee 1,\* and Chang-Chun Hsiao 2,9,\***


Received: 11 May 2020; Accepted: 11 June 2020; Published: 17 June 2020

**Abstract:** Psoriatic arthritis (PsA) is a destructive joint disease mediated by osteoclasts. MicroRNAs (miRNAs) regulate several important pathways in osteoclastogenesis. We profiled the expression of miRNAs in CD14+ monocytes from PsA patients and investigated how candidate microRNAs regulate the pathophysiology in osteoclastogenesis. The RNA from circulatory CD14+ monocytes was isolated from PsA patients, psoriasis patients without arthritis (PsO), and healthy controls (HCs). The miRNAs were initially profiled by next-generation sequencing (NGS). The candidate miRNAs revealed by NGS were validated by PCR in 40 PsA patients, 40 PsO patients, and 40 HCs. The osteoclast differentiation and its functional resorption activity were measured with or without RNA interference against the candidate miRNA. The microRNA-941 was selectively upregulated in CD14+ monocytes from PsA patients. Osteoclast development and resorption ability were increased in CD14+ monocytes from PsA patients. Inhibition of miR-941 abrogated the osteoclast development and function while increased the expression of WNT16. After successful treatment, the increased miR-941 expression in CD14+ monocytes from PsA patients was revoked. The expression of miR-941 in CD14+ monocytes is associated with PsA disease activity. MiR-941 enhances osteoclastogenesis in PsA via WNT16 repression. The miR-941 could be a potential biomarker and treatment target for PsA.

**Keywords:** psoriatic arthritis; osteoclastogenesis; miR-941; WNT16

### **1. Introduction**

Psoriatic arthritis (PsA) is a chronic and indolent inflammatory disease involving progressive arthropathy in approximately 30% of patients with psoriasis. Skin manifestations usually precede the onset of PsA by an average of 10 years. [1]. The diagnosis of PsA is based on the recognition of clinical and imaging features [2]. The most widely used diagnostic criteria for PsA is the Classification Criteria for Psoriatic Arthritis (CASPAR) [3]. PsA is easily overlooked and missed with incorrect diagnosis or messed up with delayed diagnosis, both of which could lead to poor radiographic and devastating functional outcome [4]. In fact, Haroon et al. reported that a diagnostic delay of more than six months contributes to poor radiographic and functional outcome in PsA [5].

PsA is featured with bone erosions mediated by activated osteoclasts. Osteoclasts, the multinucleated giant cells with a monocyte/macrophage lineage, are the main cells responsible for bone resorption [6,7]. It was reported that numbers of osteoclast precursors are increased in PsA patients as compared with those from healthy controls [8]. Consistently, we have demonstrated that circulating CD14+ monocytes from patients with PsA have active osteoclastogenesis and active resorption activity.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate the expression of protein-coding genes. Emerging evidence suggests that miRNA-mediated regulation represents a fundamental layer of epigenetic control over diverse physiological and pathological processes [9,10].

With a hypothesis-driven approach, we previously investigated the role of three common osteoclast activation microRNAs (miR-146a/b and miR-155) in CD14+ monocytes in PsA. We demonstrated miR-146a-5p in CD14+ monocytes from PsA patients correlates with its disease severity in vivo and active bone resorption in vitro [11]. However, in that study, only three microRNAs are investigated in osteoclasts of PsA patients. A general expression profile of various miRNAs in PsA is required to investigate whether there were other important miRNAs critical in the pathogenesis of PsA.

Next-generation sequencing (NGS) provides a high-throughput sequencing platform that performs much better than the traditional Sanger sequencing. NGS facilitates the discovery of genes and regulatory elements associated with disease [12] so that we could determine miRNA expression profile of the pooled RNA libraries from osteoclasts of PsA patients, psoriasis patients without arthritis (PsO), and healthy controls (HC). We adopted MiSeq platform (Illumina) for large scale profiling. The RNA libraries are first prepared with TruSeq Small RNA Sample Preparation protocol (Illumina) followed by sequencing with MiSeq platform. The generated NGS data is further analyzed with miRSeq for evaluating sequencing quality and determining miRNA expression profile.

Circulating monocytes in the blood are appropriate and readily accessible for bone-related studies and are a good source of osteoclast precursors to study [13,14]. Therefore, in this study, we utilized circulating monocytes to investigate the functional activation osteoclasts in individual subjects. In addition, the overall expression of miRNAs from osteoclast precursors, the CD14+ monocytes, has not been profiled independently. The present study aimed to identify whether specific miRNAs (through NGS) from CD14+ monocytes could serve as diagnostic biomarkers and treatment targets for PsA (through clinical subject categories). This study also addressed the mechanisms by which specific miRNAs contribute to active osteoclastogenesis and functional activity in PsA (through RNA interference and bone resorption assay).

#### **2. Results**

#### *2.1. Subject Demographics*

Forty patients with PsA (Female/Male: 12/28, average age: 47.6 years old), 40 PsO patients (Female/Male: 9/31, average: 43.8 years old), and 40 HCs (Female/male: 11/29, average age: 44.1 years old) were recruited (Table 1). Most PsA patients had severe psoriasis (average PASI of 14.2), and all of them had peripheral arthritis, including 35% with axial arthritis, 35% with dactylitis, and 45% with enthesitis.


**Table 1.** Demographics of psoriatic arthritis patients (PsA), psoriatic patients without arthritis (PsO), and healthy controls (HCs).

\* PASI: Psoriasis Area and Severity Index.
