MiR-16-5p suppresses myofibroblast activation in systemic sclerosis by inhibiting NOTCH signaling

Systemic sclerosis (SSc) is a prototypic fibrotic disease characterized by localized or diffuse skin thickening and fibrosis. Tissue fibrosis is driven by myofibroblasts, and factors affecting myofibroblast activation may also be involved in the development of SSc. In this study, we examined molecular mechanisms underlying SSc by focusing on myofibroblast activation processes. Bioinformatics analysis conducted to identify differentially expressed miRNAs (DEMs) and genes (DEGs) revealed that microRNA-16-5p (miR-16-5p) was downregulated and NOTCH2 was upregulated in SSc patients. In vitro experiments confirmed that miR-16-5p was able to bind directly to NOTCH2 and inhibit myofibroblast activation. Moreover, miR-16-5p-dependent inhibition of NOTCH2 decreased collagen and α-SMA expression. MiR-16-5p downregulation and NOTCH2 upregulation was also confirmed in vivo in SSc patients, and NOTCH2 activation promoted fibrosis progression in vitro. These results indicate that miR-16-5p suppresses myofibroblast activation by suppressing NOTCH signaling.


INTRODUCTION
Systemic sclerosis (SSc), a complex and heterogeneous connective tissue disease, is observed mainly in female patients. The main characteristics of SSc include immune abnormalities, microvasculopathy, excessive extracellular deposition, and progressive fibrosis of the skin and other internal organs [1]. Currently, the mechanisms underlying SSc remain largely unknown, although infection, immune activation by cancer, and dysbiosis might trigger SSc on the genetic level [2,3]. In addition, serious complications of SSc, such as interstitial lung disease, pulmonary hypertension, and heart failure, can be life-threatening [4].
Fibroblasts play an important role in fibrosis and maintain profibrotic phenotypes in vitro when explanted from affected tissues, resulting in increased secretion of collagen, extra-cellular matrix proteins, and alphasmooth muscle actin (α-SMA), a crucial marker of myofibroblasts [5]. Additional in vitro studies are needed to investigate the underlying molecular and epigenetic mechanisms related to fibrosis [6]. For example, a recent study demonstrated that transforming growth factor beta (TGF-β) played a key role in SSc by regulating fibrosis progression [7]. microRNAs (miRNAs), a type of small non-coding RNA molecule approximately 22 nucleotides in length, AGING are present in plants, animals, and some viruses, and can both silence RNA expression and regulate gene expression post-transcriptionally [8]. miRNA expression has been associated with regulation of developmental processes and a variety of disease states [9]. Accumulating evidence suggests that miRNA plays a crucial role in many fibrotic conditions [10]. For example, miR-16-5p regulates epigenetic modification of the connective tissue and plays a key role in immune diseases [11]. miR-16-5p also modulates inflammatory cytokines and might be crucial for anti-inflammatory response [12].
It is well known that the NOTCH family of cell surface receptors is crucial for intercellular communication [13]. When activated by ligand binding, the NOTCH intracellular domain (NICD) is released, forms a transcriptional activator complex with RBPJ/RBPSUH, and activates genes in the enhancer of split locus [14]. It also affects the initiation of tissue differentiation, proliferation, and apoptotic programs [15]. It was previously demonstrated that NOTCH signaling is regulated by certain miRNAs, such as miR-164a, miR-34a, and miR-143-3p [16][17][18].
In this study, we examined miR-16-5p expression in SSc patients and found that miR-16-5p suppressed profibrotic activation of dermal fibroblasts in vitro. Furthermore, we show that this phenotype is driven by miR-16-5p-mediated NOTCH pathway suppression.

Identification of DEGs and DEMs
The GSE137472 (miRNA) and GSE145120 (mRNA) gene expression profiles were obtained from the GEO database. The data were analyzed using the GEO2R online tool. A total of 28 upregulated and 29 downregulated DEMs, including miR-16-5p, were identified in SSc patients as compared to normal controls. 392 upregulated DEGs, including NOTCH2, and 348 downregulated DEGs were identified in SSc patients compared to controls. The top 30 up-regulated and top 30 down-regulated miRNAs and mRNAs are shown in Figures 1 and 2, respectively.

miRNA-targeted gene prediction
miR-16-5p, which was down-regulated in SSc, has been reported as an epigenetic regulator of the connective tissue as well as a key regulator in immune diseases [11]. We therefore searched TargetScanHiman to identify potential downstream targets of miR16-5p; 1515 potential targeted genes were identified. Interestingly, NOTCH2 was both listed as a potential target gene of miR-16-5p and was among the DEGs identified in SSc patients, and the Notch family of cell surface receptors is crucial for intercellular communication [13]. These results suggest that the effects of miR-16-5p may be in mediated in part by downstream NOTCH2 activity; in vitro and in vivo experiments supported this hypothesis. A schematic diagram of the target gene prediction process is shown in Figure 3.

GO and KEGG enrichment analysis
GO term analysis and KEGG pathway enrichment analyses were performed using the DAVID bioinformatics tool. DEGs in SSc patients were significantly enriched in the following categories and processes: in the biological process category, defense response to virus, response to virus, and type I interferon; in the cellular component category, membrane, cytosol, and Golgi apparatus; in the molecular function category, protein binding, double-stranded RNA binding, and phosphatase activity ( Table 1 and Figure 4). The top 5 KEGG pathways associated with DEGs in SSc patients were influenza A, herpes simplex infection, measles, protein processing in endoplasmic reticulum, and hepatitis C (Table 2 and Figure 5).

miR-16-5p and NOTCH2 expression in SSc patients and NOTCH2 overexpression-induced myofibroblast activation in HSFs
qRT-PCR and western blot analyses of serum samples collected from SSc patients and healthy controls suggested that miR-16-5p was downregulated and NOTCH2 was upregulated in SSc patients ( Figure 7A-7B and Supplementary Figures 5 and 6). Transfection of a NOTCH2 overexpression plasmid resulted in the expected increase in NOTCH2 levels in HSFs according to qRT-PCR and western blot analysis ( Figure 7C and Supplementary Figure 7). We then measured the expression of profibrotic markers in NOTCH2 overexpressing fibroblasts. NOTCH2 overexpression in HSFs increased levels of Col 1A1, Col 1A2, α-SMA, and CTGF transcript, and miR-16-5p inhibition partially reversed these increases ( Figure  7C-7F). Furthermore, MMP-1 and MMP-8 expression    Top 2 terms were selected according to P-value when more than 2 terms enriched terms were identified in each category.  Top 5 terms were selected according to P-value when more than 5 terms enriched terms were identified in each category.

DISCUSSION
Myofibroblasts play a key role in tissue fibrosis, and α-SMA expression is a significant marker of myo-fibroblast activation [19]. The proportion of myofibroblasts in the overall fibroblast population is closely associated with the severity of fibrosis-related diseases [20]. For an example, a recent study indicated that α-SMA expression was increased in fibroblasts cultured from SSc skin biopsies, which is consistent with the transforming growth factor-β (TGF-β)induced increase in α-SMA levels observed in vitro. AGING Furthermore, a recent single-cell RNA-sequencing analysis identified different subgroups of fibroblasts in human skin samples [21]. In this study, we found that α-SMA levels were elevated both in antagomiR-16-5p-treated and NOTCH2 overexpression plasmid-treated HSFs, suggesting that both miR-16-5p inhibition and NOTCH2 overexpression can activate myofibroblasts in HSFs. Our bioinformatics analyses also indicated that miR-16-5p is a regulatory factor for priming myofibroblasts. miR-16-5p expression was reduced in SSc patients compared to healthy volunteers, and miR-16-5p knockdown induced α-SMA expression in HSFs. Additionally, miR-16-5p has previously been reported to regulate epigenetic changes in connective tissue and to play a key role in immune diseases [11]. We therefore hypothesized that miR-16-5p expression would affect collagen secretion in HSFs and performed additional in vitro and in vivo assays to confirm the role of miR-16-5p in fibrosis.
Matrix metallopeptidases (MMPs) are known to degrade a variety of extra cellular matrix components and play significant roles in tissue fibrosis processes [22]. The collagenases MMP1 and MMP8 initiate the cleavage of fibrillar collagen, the digested products of which are further degraded by other MMPs [23]. In this study, we found that inhibition of miR-16-5p reduced MMP-1 and MMP-8 expression in HSFs, indicating that miR-16-5p has direct antifibrotic effects. However, the potential molecular mechanisms underlying those effects should be investigated further in future studies.
Prior studies reported that NOTCH signaling, which is responsible for profibrotic phenotypes in vitro and tissue fibrosis in vivo, is activated in fibroblasts extracted from SSc patients' skin [24]. Accumulating evidence also indicates that miR-16-5p is an important regulator of NOTCH signaling [25][26]. The bioinformatics results of this study indicate that NOTCH2 is enriched in SSc patients, and qRT-PCR also demonstrated that NOTCH2 levels were elevated in the serum of SSc patients. We therefore verified the association between miR-16-5p and NOTCH2 in vitro and found that miR-16-5p partially reversed NOTCH2dependent myofibroblast activation. These results suggest that miR-16-5p affects myofibroblast activation by regulating NOTCH signaling.
In summary, our data show that miR-16-5p is downregulated in SSc patients compared to healthy controls, and this downregulation is crucial for NOTCH2 signaling-induced myofibroblast activation. Additionally, our findings suggest that drugs that inhibit miR-16-5p overexpression might be a promising clinical treatment for SSc.

Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analyses
The online Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources version 6.8 tool was used to perform GO term and KEGG pathway enrichment analysis of the DEGs. Results were visualized by GOplot, and the volcano plot was generated using GraphPad Prism 8.0.

MiRNA target gene prediction
The online prediction tool TargetScanHuman (http://www.targetscan.org/vert_72/) was used to identify target genes of the differentially expressed miRNAs. The results of target gene analysis were visualized using Cytoscape 3.7.2.

Ethical statement
The study protocols were approved by the Ethics Committee of Affiliated Zhoupu Hospital, Shanghai University of Medicine and Health Sciences, and informed consent was obtained from all participants.

Blood collection
Peripheral blood samples were collected from patients at our Hospital. (30 healthy volunteers, 30 SSc patients) from May 2018 to March 2020 for measurement of miR-16-5p and NOTCH2 mRNA expression.

Cell culture and transfection
High-glucose Dulbecco's modified eagle medium (Gibco BRL, Grand Island, USA) was used to culture human skin fibroblasts (HSFs, FuHeng Biology, Shanghai, China). HSFs were cultured at 37° C with 5% CO2 and 95% humidity. The agomiR and antagomiR transfection constructs were purchased from GenePharma (Shanghai), and transfection was performed according to the manufacturer's protocol using a concentration of 200 mM. RNA interference was used for endogenous NOTCH2 knockdown. siRNA oligonucleotides were purchased from Life Technologies, and transfection was performed using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's protocol. The NOTCH2 expression plasmid was purchased from the Public Protein/Plasmid Library (PPL, #BC071562).

qRT-PCR analysis
First, total RNA was isolated from cells and serum samples using TRIzol® reagent (Thermo Fisher Scientific). Next, the reverse-transcription was performed using ReverTra Ace® qPCR RT Master Mix (Toyobo Life Science) to obtain cDNA based on the manufacturer's protocol. Relative miRNA levels were normalized to GAPDH levels (the internal control) and were calculated using the 2 -ΔΔCt approach. All experiments were conducted in triplicate; miRNA and mRNA primer sequences are listed in Table 3.

Statistical analysis
GraphPad Prism 8.0 was used to perform statistical analyses. Results are shown as mean ± SD. Student's ttests were used to compare two groups, while one-way analysis of variance with Tukey's post hoc test was used to compare more than two groups. A significance threshold of p < 0.05 was used.

Ethical approval
The