STAT3-mediated TH17 cell Differentiation was Related to the Carcinogenesis of Colitis Related Cancer

Background: Inammation often induces regeneration to repair the tissue damage. However, chronic inammation can transform temporary hyperplasia into a fertile ground for tumorigenesis. Here, we demonstrate that the miR-124 acts as a safeguard to inhibit the pro-inammatory production and reparative regeneration. Methods: The expression levels of miR-124 and IL-17, IFN-γ were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). T H 17 or T H 1 cells were detected by ow cytometer, respectively. the binding of STAT3 to the promoter region of IL-17 gene was analyzed by Chip assay . miR-124 binding to the 3’UTR of STAT3 gene was detected by reported plasmid construction and luciferase assay. Furthermore, DSS-induced colitis mice model and T cell transfer model were used to conrm the function of miR-124 in vivo. The related gene expression was analyzed by ELISA and western blot experiments. Results: The results indicated that miR-124a deciency leads to colon tumorigenesis after Citrobacter rodentium infection and AOM/DSS induced colon cancer murine model. In molecular mechanism, miR-124 targets STAT3 to suppress T helper 17 (T H 17) cell differentiation and expansion, and keep T H 17 polarization in colonic microenvironment. Conclusions: Our study highlights highlight the potential role of miR-124 in the control of immune responses and pathogenesis of inammatory diseases. by regulating the expression and phosphorylation of STAT3, but special cell type was not involved[14]. The previous study also demonstrated that miR-124 depression was related to carcinogenesis, and development by targeting different gene[23, 24, 25].These observations suggested that miR-124 played an key role in colitis and sporadic colon cancer.In our present study, we focused on the miR-124 function in Th17 cell and found that miR-124 could inhibit the polarization of Th17 cell and promote the transition of Th17 to treg in colitis and colitis related colon cancer by targeting stat3 gene.These results are consistent with downregulation of miR-124 developingintestinal failure with M1 macrophage phenotype by targeting stat3 and acetylcholinesterase (AChE). We believe that in the absence of miR-124 signaling cascade, the presence of intestinal commensal bacteria will drive intestinal CD4 + T helper cells toward Th17 cell polarization, resulting in a hyper-inammatory response with associated tissue damage and pathogenesis. Thus, our studies demonstrate that miR-124 expressed in Th17 cell modulates shaping of Th17 phenotype. We suggest a novel mechanism for the effect of miR-124 targeting STAT3 in the modulation of Th17 cell differentiation. Treatment with miR-124 mimic signicantly suppressed Th17 cell activation by inhibiting the expression of STAT3and IL-17 signature genes in vitro and ameliorated colitis and colon cancer development in vivo. Taken together, the results rmly establish miR-124 plays an important role in the modulation of Th17 cell activation and highlight the potential role of miR-124 in the control of immune responses and pathogenesis of inammatory diseases. study potential role of miR-124 in the control of immune responses and pathogenesis of inammatory diseases. It is important that miR-124 mimic suppressed the TH17 deerentiation during colitis process and delayed or inhibited the development of colitis-related cancer. miR-124 maybe could acted as biologic therapy point in colitis.

Background CRC progression is a process that involves interactions between thetumor and the host cellular immunity in the tumor microenvironment.Neoplastic cells secrete pro-inflammatory mediators and immune cellsproduce cytokines which all lead to tumor development. Studieshave shown that the number of Th17 cells are significantly higher inCRC tissues [1,2]. Th17 cells induce immune suppressive mediatorssuch as TGF-β, CXCR3, CC chemokine receptor 6 (CCR6) and IL-6and also suppress CD8 + T cells which have anti-tumor activity. Moreover, it has been shown that the number of IFN-γ producingCD8 + T cells is increased in the IL-17 deficient mice [1,3].IL-17 is produced by Th17 cells and it is an important cytokine in various immune responses such as type2 immune response. In pro-inflammatoryresponses, IL-17 plays an important role in activation andrecruitment of neutrophils. Neutrophils are the main sources of cytokinesrelated to Th2-type immune response that induce negative feedback,suppress neutrophil, decrease tissue destruction, and also induceIL-17 production [4].Published data demonstrated that the level of IL-17 was significantlyhigher in most of CRC tissues. The up-regulation of IL-17 begins from adenoma stage andits level is higher at the cancer stage but it is not associated with TNMparameters of the tumor [5,6]. Therefore, IL-17 is involved in CRC tumorigenesisthrough several pathways and underwater molecular mechanism.
MicroRNAs (miRs) are small non-coding RNA oligonucleotides that can regulatethe expression of a large number of genes and have been involved in different humandiseases [7]. MiRs are centrally involved in the pathogenesis of different human in ammatory diseases, including IBD.Many published documents identi ed that miRs are essential regulators ofToll-like receptor signaling, which is important to trigger the intestinal in ammation.For example, miR-146b induced by IL-10-IL-10R signaling regulated the Toll-likereceptor 4 (TLR4) by negative feedback in human monocytes [8],and miR-146b de cient mice were easier to develop colitis by targeting IRF5 [9], which was regarded as a regulator of TLRs in LPS-driven TLR signaling.Moreover, many evidences have indicated that in ammation may contribute to carcinogenesis and IBD can increase the risk of colorectal cancer. However, it is still elusive to elucidate the details of the relationships between IBD and CRC [10]. Josse et al summarized the recognized molecular mediators which linked in ammation to colorectal cancer, including cytokines, growth factors, Toll-like receptors, PI3K/MAPK signaling, NF-kB/STAT3signaling, Wnt signaling way [11]. In addition, more recent studies have indicated that miRNAs can target above signaling molecules and connect in ammation to cancer development. Yuan et al reviewed and listed that miRNAs were involved in in ammation to cancer. For example, miR-126 could directly target the CXCR4 or PI3K/AKT signaling pathway on tumor suppression [12,13].Among those, miR-124 is deregulated speci cally in pediatric patients withactive UC, leading to increased levels of STAT3 expression and the transcriptionalactivation of its downstream targets. Moreover, in active pediatric-UC the miR124/STAT3pathway is epigenetically regulated, suggesting the involvement of epigenetic-transcription regulatory circuits in the pathogenesis of pediatric-UC [14]. But, it is still unclear whether miR-124 can mediate the colitis related colon cancer progression.
In this study, we observed that miR-124 could inhibit the th17 cell proliferation and was down-regulated in th17 cell differentiation. The miR-124 mimic would retain in the in amed area and e ciently inhibited the Th17 polarization, causing the phenotype transition into Treg cells, which next inhibited the in ammatory response and promoted the mucosal regeneration, and nally decreased the colitis-related colonic cancer development. Therefore, we expect targeting miR-124 would offer a novel therapeutic strategy for colitis-related colonic cancer.

Methods
Mice C57BL/6J and Rag1 -/mice were obtained from Model Animal Research Center of Nanjing University and maintained in the barrier facility at Guangzhou Medical University. To induce MC38 tumor-bearing mice model, 5 x 10 5 MC38 cells were injected subcutaneously into mice. The animal study protocols were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center. FITC Annexin V Apoptosis DetectionKit I (2293683) was purchased from BD Pharmingen.Anti-RORγt, anti-T-bet (MBL), anti-STAT3, anti-pSTAT3 (CellSignaling), and anti-β-actin (Sigma) antibodies for western blotting were usedaccording to the manufacturers' instructions. Secondaryantibodies were from Santa Cruz Biotechnology, Inc.

Intracellular staining and ow cytometer
For T cells, cells were stimulated with PMA andionomycin for 5 hrs in the presence of brefeldin A priorto intracellular staining. Cells were xed with IC FixationBuffer (BD Bioscience), incubated with permeabilizationbuffer, and stained with antibodies. For macrophages,bone marrow derived macrophages were activated with LPS (200 ng/ml) plus IFN-g (10 ng/ml) overnightand brefeldin A was added to the culture for 5 hrs prior tointracellular staining. RNA isolation and quantitative real-time RT-PCR (qPCR) Total RNA was extracted using an RNeasy pluskit (QIAGEN, Valencia, CA) and cDNA was generatedwith an oligo (dT) primer and the Superscript II system(Invitrogen, USA) followed by analysis using iCycler PCRwith SYBR Green PCR master Mix (Applied Biosystems).Results were normalized based on the expression ofubiquitin. The sequences of primers are shown inSupplementary Table 1.
T cell-transfer colitis T cell transfer colitis was performed as previouslydescribed. Brie y, puri ed CD4 + CD45RB hi T cellsfrom WT mice were injected intraperitoneally into Rag1 -/recipients (5 x 10 5 cells per mouse in 200 μl sterile PBSper injection) [15]. Mice were weighed every week throughoutthe course of experiments. The degree of in ammation inthe epithelium, submucosa and submuscularis propria wasscored separately as described previously [9].

Statistical analysis
The results are shown as means ± SD and statisticalanalysis was performed using Student's t-Test. Wheremore than two groups were compared, one way-ANOVAwith Bonferroni`s correction were performed. P < 0.05was considered statistically signi cant.

MiR-124 inhibited the T H 17 cell polarization
To investigate the effects of miR-124 on adaptive immune cellfunction, we rst focused on T helper cells. Naïve CD4 + Tcells from C57BL/6 mice were primed in vitro for 3 daysunder T H 0, T H 17, Treg and T H 1 conditionsand miR-124 expression was evaluated by qPCR. We found that miR-124 expression was obviously decreased in T H 17 cell rather than TH1 ( Figure 1A). qPCR and ELISA experiments showed that miR-124 mimic signi cantlysuppressed expression of T H 17 or T H 1-associatedgenes including IL-17, IFN-γ(Figure1B,C,D,E). We next detect whether the T H 17 and T H 1 cell differentiation was affectedin the presence of miR-124 mimic.These observations correlatedwith reduced IL-17 and IFN-γ production by T H 17 or T H 1 cells treated with miR-124 mimic as determined by ow cytometer (Figure1F).The results showed that the frequency of IL-17-and IFN-γ-producingcells decreased following miR-124 mimic treatment. To rule out the possibility that the reduced TH17 and TH1 cell differentiation was due to abnormal cell apoptosiscaused by miR-124, we isolated and analyzed CD4 + T cells from spleensas well as lymph nodes of C57BL/6 mice by Annexin V and PI staining or CSFE staining, The results showed that miR-124 mimic administration did not increase the T cell apoptosis but affected the CD4 + cell proliferation ( gure G,H).

MiR-124 alters DNA binding activity in T H 17 cells
Furthermore, the evidence prompted us to probe for the molecular basis for howmiR-124 modulated T H 17 cell differentiation. Since many studies have shown that several transcription factors including RORγt, STAT3, and AHR are important for T H 17 cell differentiation, we hypothesized that miR-124 might affect the expression of these transcription factors. To address this, naïve CD4 + T cells from C57BL/6 mice were primed in vitro for 3 days under T H 0 or T H 17 polarizing conditions.IL-17 mRNA was detected in TH17 polarization in the presence of miR-124 mimic andthe results showed that IL-17 was inhibited by miR-124 mimic ( Figure 2A). However,the levels of RORγt and AHR protein were comparable in the presence of miR-124 compared with control (Figure2B), but STAT3 expression decreased after miR-124 mimic treatment (Figure2C).In addition, ChIP analysis demonstrated that the binding of STAT3 to the promoter region of IL-17 gene was signi cantly reduced ( Figure 2D). The data suggested that miR-124 could suppress the STAT3 expression and subsequently affect the IL-17 expression in Th17 cell differentiation condition.

STAT3 is the target of miR-124
We further investigated how miR-124 affect the STAT3 in Th17 cell differentiation. The previous document and biomatinformatio analysis revealed that miR-124 could bind the 3'UTR of STAT3 gene, and inhibit the protein translation.To verify that miR-124 really targets STAT3,we rst detected that STAT3 was affected in the presence of miR-124 mimic, andthe results suggested that miR-124 mimic decreased the STAT3 expressionin EL4 cell line ( Figure 3A). Next, we constructed the luciferase reporter plasmids of the STAT3 3'UTR of mouse (including WT and miR-124 bind site mutant plasmids). The plasmids were then respectively co-transfected with the miR-124 mimic to El4 or HEK293T cells. Interestingly, transfection of miR-124 mimic signi cantly decreased the luciferase activity in group transfected with STAT3 plasmid; however, miR-124 mimic had no effect on luciferase activity in group transfected with STAT3 mutant plasmid ( Figure 3B,C,D). RNA immunoprecipitation (RIP) experiments demonstrated that the miR-124 and STAT3 mRNA were in the same miRNA-induced silencing complex (miRISC) ( Figure 3E). Taken together, the results suggest miR-124 modulates TH17 differentiation by targeting STAT3.  Figure 4A,B). Parallel histologicstudies of colonic sections from Rag1 -/mice treated withmiR-124 revealed fewer in ammatory cell in ltration andsigni cantly lower pathological scorescompared tomice treated with PBS ( Figure 4C). And, miR-124 expression also was con rmed in the treatment of miR-124 mimic ( Figure 4D). In addition, micetreated with miR-124 had signi cantly lower percentagesof IL-17 cells and lower expression of T H 17 and T H 1 signature gene expression than PBS treated mice (Figure4E,F).

MiR-124 suppressed the development of colitis associated carcinoma by inhibiting the T H 17 differentiation
Ulcerative colitis (UC) is a subcategory of in ammatory bowel disease (IBD) with high risk of colorectal cancer. 2 key pathophysiological features of this disease are dysregulation of immune system and impaired mucosal repair. As previously reported, T H 17 cell differentiation was closely related to CAC occurrence.After the third cycle of CAC regimen suspended, we had successfully created a colitis associated carcinoma model and observed palpable tumors near the rectum of mice. Interestingly, miR-124 mimic treatment group developed signi cantly smaller tumor numbers and tumor areas than controlledgroup (Figure5B). In addition, histological assessments showed that the colonic mucosa in miR-124 mimic treatment mice presented with low-grade dysplasia, but the tumors of controlledgroup were usually identi ed as high-grade dysplasia (Figure5C). Strikingly, in the process of acute mucosal injury, miR-124 mimic treated mice exhibited better epithelium structures concomitant with down-regulation of IL-17.Whileduring the CAC, IL-17 expression in colonic mucosa of miR-124 mimic treated mice was lower than that in controlledgroup (Figure5D,E), suggesting that in the background of AOM induced missense mutations, chronic in ammation impaired the epithelial microenvironment and the TH17 cells function, resulting in development of in ammation induced cancer, and Th17 cells recruited around the neoplastic epithelial cells in regeneration, and the concomitant alteration was also observed in STAT3. Therefore, miR-124 suppressed the development of CAC by attenuated the "the second hit" of cancer.

MiR-124 depressed the colon carcinogenesis in C. rodentium infection colitis colon cancer murinemodel
Microbial dysbiosis causes chronic in ammation associated with CRC .C. Rodentium is a mouse mucosal pathogen that shares pathogenic mechanisms and 67% of its genes with enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), which are two clinically important human gastrointestinal pathogens. C. Rodentium has been used as a model to study mucosal immunology, including intestinal in ammatory responses during bacteria-induced colitis and colon tumorigenesis. C. rodentium infection increases the number of colonic adenomas in Apc Min mice but does not cause adenoma formation in wild-type mice.
After C. Rodentium (2 × 10 9 CFU) infection, wild-type micedeveloped diarrhea and weight loss within 2 weeks, and werethendivided into two groups: miR control and miR-124 mimic. Until 6 months, all mice were sacri ced and performed the histologic staining analysis. Microscopic sections from WTcontrol mice were free of dysplastic and neoplastic changes at six-month time points following infection. In miR-124 mimic treatment mice, less dysplasia or early neoplasia was present at this time point, whereas 16/20 (wt control) vs 5/20 (miR-124 mimic) mice had microscopic changes ranging from dysplasia to adenocarcinoma (Figure6 A,B,C).We further isolated CD4 + T cells from C. Rodentium-infected colon cancer tissue and analyzed the relative abundance of T H 17 subpopulations according to their associated expression of IL-17. IL-17 was signi cantly decreasedafter miR-124 treatment (Figure6 D,E). So, miR-124 mimic treatmentdepressed the T H 17 cell differentiation in the colon post C. Rodentium infection.

Discussion
Ulcerative colitis (UC) is a subcategory of in ammatory bowel disease (IBD) with high risk of colorectal cancer, which included two key pathophysiological features: dysregulation of immune system and impaired mucosal repair [16] . Limited therapies available at present remains UC and colitis-related colon cancer a challenging problemfor the clinician. In our study, we con rmed the miR-124 was a key regulator for Th17 cell differentiation in UC and CAC, and performed the miR-124 mimic to effectively inhibit the in ammation and colon cancer occurrence in murine colitis model. Thus miR-124 could inhibit Th17 cell differentiation and pro-in ammatory cytokines induction, thereby promoting the mucosal repair and suppressing the development of colitis After tumor formation in colon, immune system reacts againstneoplastic cells. Immune responses include immune cells proliferation,phenotype alteration, synthesis and release of cytokine [17], such as interleukin-17 (IL-17). IL-17 is a pro-in ammatory cytokine, which isassociated with cancer progression [18,19].
The main source of IL-17 is a subpopulation of CD4 + T cells known as T-helper17 (Th17) cells . Tumor in ltrating Th17 cells were found in many types of cancers [19,20]. In colon cancer, published document showed that Th17 was involved in colitis and colitis related cancer [3,21];Th17 cells was related to tumor angiogenesis [22],and also could mediate the activity of CTLs in colon cancer development [3]. Although physiologic levels of in ammation were protective, excessive in ammation was deleterious and was at the basis o n ammatory bowel disease (IBD) and in ammation-promotedcolorectal cancer.Production of cytokinestogether with that of matrix-degrading enzymes, growth factors, and reactive oxygen species promote tumorigenesis bycreating a microenvironment favoring intestinal epithelial cellproliferation, cell survival, and invasiveness.In our study, Th17 also was veri ed to be related with colon cancer development. But how to regulate the Th17 cell differentiation still was elusive.
Several agents are required for differentiation and stabilization, such as transforming growth factor beta (TGF-β), interleukin-6 (IL-6), IL-21, IL-23 and IL-1b. Also, retinoic orphan receptor-γ (RORγ) and signal transducer and activator of transcription 3 (STAT3)are the transcription factors responsible for Th17 differentiation andstabilization.Gerogios et al demonstrated that miR-124 could promote the children UC and pathogenesis by regulating the expression and phosphorylation of STAT3, but special cell type was not involved [14]. The previous study also demonstrated that miR-124 depression was related to carcinogenesis, and development by targeting different gene[23, 24, 25].These observations suggested that miR-124 played an key role in colitis and sporadic colon cancer.In our present study, we focused on the miR-124 function in Th17 cell and found that miR-124 could inhibit the polarization of Th17 cell and promote the transition of Th17 to treg in colitis and colitis related colon cancer by targeting stat3 gene.These results are consistent with downregulation of miR-124 developingintestinal failure with M1 macrophage phenotype by targeting stat3 and acetylcholinesterase (AChE). We believe that in the absence of miR-124 signaling cascade, the presence of intestinal commensal bacteria will drive intestinal CD4 + T helper cells toward Th17 cell polarization, resulting in a hyper-in ammatory response with associated tissue damage and pathogenesis.
Thus, our studies demonstrate that miR-124 expressed in Th17 cell modulates shaping of Th17 phenotype. We suggest a novel mechanism for the effect of miR-124 targeting STAT3 in the modulation of Th17 cell differentiation. Treatment with miR-124 mimic signi cantly suppressed Th17 cell activation by inhibiting the expression of STAT3and IL-17 signature genes in vitro and ameliorated colitis and colon cancer development in vivo. Taken together, the results rmly establish miR-124 plays an important role in the modulation of Th17 cell activation and highlight the potential role of miR-124 in the control of immune responses and pathogenesis of in ammatory diseases.

Conclusions
Our study highlight the potential role of miR-124 in the control of immune responses and pathogenesis of in ammatory diseases. It is important that miR-124 mimic suppressed the TH17 de erentiation during colitis process and delayed or inhibited the development of colitis-related cancer. miR-124 maybe could acted as biologic therapy point in colitis.     UTRluc if eraserep or terconstructsofpredictedmiR -124tar ≥ t ≥ ≠ . ThemiR -124b ∈ d ∈ gregionis ∈ dicated. (C). EL4cellswasco -tra UTR luciferase reporter plasmid after priming with IL-6 and TGFb. (D) 293 T cells were co-transfected with either WT or mutant STAT3 luciferase reporter plasmids together the miR-124 mimic for 48 hrs. The cell lysates were prepared and luciferase activity was determined (Data represent mean ±s.d).(E). EL4 cells was primed with IL-6 and TGFb in the presence of miR-124 mimic for 24 hrs. Total RNA was extracted and immunoprecipitated with anti-Ago2 antibody.

Abbreviations
The immunoprecipitated RNA was puri ed and qPCR was performed for the analysis of miR-124 and STAT3 mRNA expression (Data represent mean ±s.d).
The results are representative of three independent experiments (Data represent mean ±s.d). The results are representative of two independent experiments.