MiR-222-3p Aggravates the Inflammatory Response by Targeting SOCS1 to Activate STAT3 Signaling in Ulcerative Colitis

Background: Ulcerative colitis is characterized by relapsing inflammation in the gastrointestinal tract with limited treatment options. The aim of the present study was to assess the anti-inflammatory effect of Suppressor of cytokine signaling (SOCS1) on lipopolysaccharide-stimulated RAW264.7 cells and to investigate its potential mechanisms. Methods: The in vitro ulcerative colitis model was established by using lipopolysaccharide-stimulated RAW264.7 cells. Western blotting was used to detect the protein expression levels of SOCS1, JAK2, STAT3, and VDR. Reverse transcription-quantitative polymerase chain reaction was used to measure the mRNA expression of SOCS1, miR-222-3p, and VDR. An enzyme-linked immunosorbent assay was performed to measure the levels of inflammatory cytokines. A luciferase assay assessed the binding of SOCS1 to miR-222-3p. A total of 15 patients with ulcerative colitis and 18 healthy controls were recruited. The expression levels of SOCS1 and miR-222-3p in the colonic mucosa tissues of patients with ulcerative colitis and healthy controls were determined by reverse transcription-quantitative polymerase chain reaction. Results: SOCS1 upregulation inhibited the lipopolysaccharide-induced inflammation in RAW264.7 cells. SOCS1 was confirmed to be targeted by miR-222-3p. Silencing SOCS1 significantly abolished the inhibitory effects of miR-222-3p downregulation on inflammation. MiR-222-3p activated STAT3 signaling and reduced VDR expression by targeting SOCS1 in lipopolysaccharide-treated RAW264.7 cells. Additionally, miR-222-3p expression was upregulated in ulcerative colitis patients (P = 5.16E−10), while SOCS1 (P = 2.75E−10) and VDR (P = 52.5E−9) expression was downregulated in ulcerative colitis patients. Endoscopic scores (UCEIS) revealed significant positive correlation with miR-222-3p and negative correlation with SOCS1 and VDR. Conclusion: MiR-222-3p targets SOCS1 to aggravate the inflammatory response by suppressing VDR and activating STAT3 signaling in ulcerative colitis.


INTRODUCTION
Inflammatory bowel disease (IBD) is an intestinal inflammatory disorder encompassing ulcerative colitis (UC) and Crohn's disease (CD). 1 The diagnosis rate and hospitalization rate of IBD patients worldwide have been increasing annually over the past decades. 2,3 Ulcerative colitis is characterized by rectal bleeding, mucosal damage, and ulceration. 4 Multiple pathogenic factors have been documented, including genetics, immune response, inflammatory factors, bacterial infection, and environmental factors, among which environmental and immunological factors play important roles in UC. 5 Highly activated monocytes and macrophages in UC patients have been reported. 6 Activated monocyte/macrophage releases pro-inflammatory cytokines, leading to epithelial barrier disruption and intestinal inflammation. 7,8 The severity and duration of diffuse mucosal inflammation of the colon are closely related to the risk of colorectal cancer formation. 9 Therefore, more effective diagnostic and therapeutic targets are needed for early clinical risk assessment and treatment.
Suppressors of cytokine signaling 1 (SOCS1) is a key negative regulator of inflammatory processes. 10 Depletion of SOCS1 in helper T cells suppresses Th17 differentiation by enhancing the antagonistic effect of IFN-γ. 11 The proinflammatory genetic background owing to SOCS1 deficiency causes dysbiosis of the gut microbiota, thereby generating a pro-colitogenic environment. 12 SOCS1 relieves dextran sulfate sodium 13 -induced colitis in mice by blocking IFN-γ/STAT1 pathway. 14 SOCS1 plays a key role in attenuating murine colitis by inhibiting cytokine signaling and controlling intestinal T cell activation. 15 These findings demonstrate the important role of SOCS1 in the pathogenesis of colitis. Additionally, SOCS1 is a critical negative modulator of JAK/STAT pathway and functions in inhibiting systemic autoimmunity mediated by dendritic cells. 16 Vitamin D receptor (VDR) belongs to the steroid receptor family and its protective effects against colitis have been reported. 17,18 It has been reported that STAT3 signaling contributes to downregulation of VDR. 19 MicroRNAs (miRNAs) are short non-coding RNAs, which control gene expression by binding to the 3'UTR of mRNAs. 20,21 Emerging reports have indicated that miR-NAs are associated with the procession of UC, such as miR-129-5p, miR-21, and miR-200, suggesting that miRNAs could be therapeutic targets for UC. [22][23][24] As previously reported, miR-222-3p expression is upregulated in doxorubicin-resistant colon cancer cells and induces doxorubicin resistance. 25 Additionally, miR-222-3p depletion alleviates liver inflammatory damage by elevating SOCS1 expression. 26 However, the regulatory mechanisms of the miR-222-3p/SOCS1 in UC are unclear.
In this study, we aimed to investigate the anti-inflammatory effect of SOCS1 on UC using Raw264.7cells inflammatory model induced by LPS. We also investigated its possible mechanism involving the regulation of JAK/ STAT3 signaling and the involvement of miR-222-3p. This study might provide some new potential research targets for molecular mechanisms of UC development.

MATERIALS AND METHODS Tissue Samples
Colonic mucosa tissues were collected from the sigmoid colon of 15 patients with active UC and 18 healthy controls undergoing screening colonoscopies between June 2015 and July 2016 at The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing, Jiangsu, China. Informed consent was obtained from all study participants. The inclusion criteria included: active stage of UC, but no prior treatment at the time of serum sampling. Exclusion criteria included: history of autoimmune diseases and previous treatment (immunomodulators, steroids, antibiotics, 5-aminosalicylic acid, blood cell apheresis, biologics, or surgery) at the time of serum sampling. The biopsies were immediately snap-frozen and stored at −80°C after pinching. The study was approved by the Ethics Committee of The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing, Jiangsu, China.

Transfection
Small interfering RNA targeting SOCS1 (Si-SOCS1) and Si-NC were constructed by Ribobio (Guangzhou, China). Anti-miR-222-3p and miR-NC were also obtained from Ribobio. Full-length SOCS1 was amplified and then digested with XbaI to obtain nucleotides of SOCS1 cDNA sequence and inserted into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) between EcoRI and EcoRV cleavage sites as the backbone. LPScultured cells were transfected with the above vectors using Lipofectamine 2000 (Invitrogen). After 24 hours, the transfection was tested by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR).

Reverse Transcription-Quantitative Polymerase Chain Reaction
Total RNA was extracted from LPS-cultured RAW264.7 cells or specimens utilizing TRIzol (Invitrogen). The extracted RNA was kept at −80°C for later use. Next, cDNA was obtained using a High Capacity cDNA Reverse Transcription kit (Takara, Japan were synthesized by GenePharma (Shanghai, China), and U6 or GAPDH was as an internal reference. Reverse transcription-quantitative polymerase chain reaction was conducted using a TaqMan MicroRNA Assay kit (Applied Biosystems, Foster City, CA, USA) for miR2223p and a Power SYBR Green qPCR Master Mix (Takara) for SOCS1, VDR, TNF-α, IL-6, and IL-8 following the manufacturer's instructions. The expression was evaluated using the 2 −ΔΔCt . The mouse primers used are shown in Table 1.

Statistical Analysis
Each assay was conducted at least 3 times to prevent errors. GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analyses. Values are expressed as mean ± standard deviation. The Student's t-test was used for data analysis of pairwise comparison. Comparison between 3 or more groups was analyzed by one-way ANOVA. Correlations between the UCEIS and gene expression were analyzed using nonparametric Spearman's correlation. P < .05 was considered statistically significant.

SOCS1 Knockdown Eliminates the Effects of miR-222-3p Inhibition in LPS-Treated RAW264.7 Cells
Reverse transcription-quantitative polymerase chain reaction and western blotting revealed that SOCS1 mRNA (0.24 ± 0.04; P = 3.54E−4) and protein (0.44 ± 0.04; P = .001) expression was decreased after transfection with Si-SOCS1 ( Figure 4A and B). RAW264.7 cells were then transfected with the indicated plasmids and exposed to 1 μg/mL LPS for 24 hours. The mRNA expression and concentrations of cytokines in the culture supernatant were measured. In Figure 4C

DISCUSSION
SOCS1 was found downregulated in colonic mucosa tissue samples of UC and TNF-α-stimulated human intestinal epithelial cells. 28 Here, we demonstrated that SOCS1 expression showed a decreased trend in murine macrophage cells after LPS treatment. However, a study indicated that the expression of SOCS1 was seen to increase in the active UC population. 29 We hypothesize that different population of samples may turn out differently. Although UC can be triggered by bacteria, viruses, and other environmental factors, the inflammatory process of the intestinal mucosa is ultimately induced by soluble inflammatory mediators. 30 The inflammatory mediators such as IL-1beta and TNF-α play leading roles in the development of UC. 31 Therefore, the effective reduction of inflammatory mediators in the serum  and colon tissues is a reasonable modality for UC treatment. 32,33 Additionally, the anti-inflammatory effects of SOCS1 in immune disease have been uncovered. SOCS1 inhibits its downstream toll-like receptor-mediated inflammatory pathways in RAW264.7 macrophages. 34 SOCS1 production is induced by resveratrol in LPS-cultured RAW264.7 macrophages to inhibit the release of TNF-α and IL-6. 35 Moreover, silencing SOCS1 in the cell population that contributes to mucosal damage in IBD increases IL-6 and IL-8 production. 36 SOCS1 was also shown involved in the inflammatory mechanisms in the colonic mucosa of UC. 37 In this study, our experiments further demonstrated that SOCS1 inhibited the release of inflammatory cytokines in LPS-stimulated RAW264.7 macrophages. Moreover, SOCS1 expression was downregulated in UC patients.
Emerging evidence has demonstrated that miRNAs are implicated in the pathogenesis of UC. Loss of miR-24-3p promotes epithelial cell apoptosis and impairs the recovery from intestinal inflammation. 38 MiR-200b-3p alleviates TNF-α-induced apoptosis and inflammation of intestinal epithelial cells and ulcerative colitis progression in rats. 39 Here, we identified that miR-222-3p has a binding site for SOCS1 by bioinformatics analysis and confirmed their targeted relationship. As reported, miR-222-3p is overexpressed in inflammatory breast cancer as well as in children with mycoplasma pneumonia, acting as a promising biomarker for the diagnosis of diseases. 40,41 The role of miR-222-3p in UC is unknown. Here, we showed high expression of miR-222-3p in LPS-treated RAW264.7 macrophages and in UC patients. Moreover, miR-222-3p silencing showed inhibitory effects on LPS-stimulated inflammation, while these effects were reversed by SOCS1 depletion, suggesting the effects of the miR-222-3p/SOCS1 axis in UC.
JAK/STAT components could be activated by a series of cytokines related to receptor families, including the gp130 receptor family, such as IL-6. 42 STAT3 activation leads to pro-inflammatory activities including Th17 differentiation in lymphocytes, 43,44 suggesting the strong plasticity of JAK/STAT pathway in the regulation of inflammatory responses. Moreover, JAK/STAT signaling activation shows a strong inflammatory involvement in the pathogenesis of UC. 45 It has been indicated that assessment of specific genotypes of JAK/STAT inhibition could be an effective treatment idea for UC. 46 The JAK/ STAT inhibitor tofacitinib has been recently approved for the treatment of active UC. 45 Therefore, investigation of JAK/STAT in UC may contribute to a better understanding of UC pathogenesis. SOCS1 is a negative modulator of JAK/STAT signaling through degradation of JAK2 or suppression of JAK1 and JAK2. 47,48 We examined the effects of the miR-222-3p/SOCS1 axis on STAT3 signaling under inflammatory conditions and found that silencing miR-222-3p suppressed STAT3 signaling by upregulating SOCS1. Additionally, dysfunction of VDR contributes to the etiology of IBD by regulating autophagy, immune response, and mucosal permeability. 49 VDR is shown to have a protective effect on the onset or progression of IBD. 50 It was reported that STAT signaling contributes to the downregulation of VDR. 19 In this investigation, miR-222-3p depletion downregulated VDR in LPS-stimulated RAW264.7 cells by upregulating SOCS1.
In conclusion, we revealed that miR-222-3p expression was high in UC patients, while SOCS1 and VDR expression was low in UC patients. In addition, miR-222-3p targeted SOCS1 to aggravate the inflammatory response by suppressing VDR and activating STAT3 signaling in UC. It may provide a new regulatory mechanism for SOCS1 in the investigation of UC progression. However, there were some limitations in the present study. These included the small number of samples and specimens from the same area. The overall results were statistically significant, but whether miR-222-3p/SOCS1 can be used as a reliable diagnostic indicator of UC and can be used clinically requires further research and verification. These findings should be confirmed and evaluated in prospective, large-sample, multicenter, randomized clinical trials.