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

STAT3-mediated TH17 cell Differentiation was Related to the Carcinogenesis of Colitis Related Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Primary research STAT3-mediated T H 17 cell Differentiation was Related to the Carcinogenesis of Colitis Related Cancer Shiyong Lin, Qianwen Liu, Jing Wen, Kunhao Bai, Yandong Guo, Jing Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-23700/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract B ackground : Inflammation often induces regeneration to repair the tissue damage. However, chronic inflammation 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-inflammatory 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 flow 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 confirm 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 deficiency 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 inflammatory diseases. Cancer Biology miR-124 STAT3 TH17 cell colitis Colitis Related Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 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 inflammatory diseases, including IBD.Many published documents identified that miRs are essential regulators ofToll-like receptor signaling, which is important to trigger the intestinal inflammation.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 deficient 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 inflammation 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 inflammation 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 inflammation to cancer development. Yuan et al reviewed and listed that miRNAs were involved in inflammation 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 specifically 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 inflamed area and efficiently inhibited the Th17 polarization, causing the phenotype transition into Treg cells, which next inhibited the inflammatory response and promoted the mucosal regeneration, and finally 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. Reagents and antibodies MiR-124 mimic and inhibitor werepurchased from GenePharma. The following Flowcytometry antibodies were purchased from BDBiosciences(USA), and conjugated to FITC, PE, PECy5,PE-Cy7, PerCP-Cy5.5, PerCP-eFluor 710, eFluor450 or APC: CD45RB (C363.16A), CD4 (L3T4), CD25 (PC61.5), IL-17 (TC11-18H10), IFN-γ (XMG1.2), FOXP3(FJK-16S) and isotype controls. Antibodies for RORγ(B2D), Gr1 (RB6-8C5) and IL-4 (11B11) were purchased from eBioscience. 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. CD4 + T cell preparation and differentiation in vitro Naive CD4 + T cells (CD62L+CD44lo) were preparedby fluorescence-activated cell sorting from spleens andlymph nodes of C57BL/6 mice. The sorted cells wereprimed for 96 hrs with anti-CD3 (1 μg/ml; 145-2C11; BDBiosciences) and soluble anti-CD28 (2 μg/ml; 37.51; BDBiosciences). Cells stimulated under neutral conditionswere defined as TH0 cells. Cells were stimulated to differentiate into TH1 cells by supplementation with IL-12plus anti-IL-4 or into TH2 cells by supplementation withIL-4 and anti-IFN-γ. For TH17 cell differentiation, cells were stimulated with transforming growth factor-β1 (5 ng/ml), IL-6 (20 ng/ml) and IL-23 (10 ng/ml; all from R&DSystems) and into Treg cells by supplementation with transforming growth factor-β1 (15 ng/ml). Intracellular staining and flow cytometer For T cells, cells were stimulated with PMA andionomycin for 5 hrs in the presence of brefeldin A priorto intracellular staining. Cells were fixed 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. Flow cytometry was performed on aFACS Calibur (BD Biosciences). Cytokine ELISA Supernatants from cell cultures were collected after activation under various conditions and secreted cytokines in the supernatants were measured by ELISA kits withpurified coating and biotinylated detection antibodies:anti-IL-17, anti-IFN-γ (R & D systems), anti-IL-9, anti-IL-22 ,anti-IL-1β (e Bioscience) and anti-IL-4 (BDBioscience). 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. Briefly, purified 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 inflammation 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 significant. Results MiR-124 inhibited the T H 17 cell polarization To investigate the effects of miR-124 on adaptive immune cellfunction, we first 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 significantlysuppressed 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 flow 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 (figure 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 significantly 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 first 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 significantly 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. MiR-124 weaken the colitis in CD4 positive cell transfer model Previous reports have indicated a correlation betweenT H 17 activity and IBD pathogenesis. To further assessthe effects of miR-124 on T H 17 cell development in vivo, weperformed adoptive T cell transfer colitis experimentsusing CD4 + CD45Rb hi cells from C57BL/6 mice toinduce colitis in Rag1 -/- mice. Mice in the treatmentgroup received low dose miR-124 twice a week for 8 weekswhile the control group was treated with PBS. While theRag1 -/- mice reconstituted with naïve CD4 + T cells lostweight continuously, treatment with miR-124 significantly improved their condition (Figure 4A,B). Parallel histologicstudies of colonic sections from Rag1 -/- mice treated withmiR-124 revealed fewer inflammatory cell infiltration andsignificantly lower pathological scorescompared tomice treated with PBS (Figure 4C). And, miR-124 expression also was confirmed in the treatment of miR-124 mimic (Figure 4D). In addition, micetreated with miR-124 had significantly 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 inflammatory 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 significantly 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 identified 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 inflammation impaired the epithelial microenvironment and the TH17 cells function, resulting in development of inflammation 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 inflammation 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 inflammatory 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 sacrificed 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 significantly 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 inflammatory bowel disease (IBD) with high risk of colorectal cancer, which included two key pathophysiological features: dysregulation of immune system and impaired mucosal repair[16]（这句和前面重复了）. Limited therapies available at present remains UC and colitis-related colon cancer a challenging problemfor the clinician. In our study, we confirmed 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 inflammation and colon cancer occurrence in murine colitis model. Thus miR-124 could inhibit Th17 cell differentiation and pro-inflammatory cytokines induction, thereby promoting the mucosal repair and suppressing the development of colitis associated carcinoma. We concluded thatmiR-124 is a critical regulator in control of intestinal immune function and epithelial regeneration, andtargeting miR-124 can beselected as a “smart” therapeutic strategy for UC and colitis-related colon cancer. 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-inflammatory 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 infiltrating 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 inflammation were protective, excessive inflammation was deleterious and was at the basis ofinflammatory bowel disease (IBD) and inflammation-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 verified 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-inflammatory 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 significantly 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 firmly 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 inflammatory diseases. Conclusions Our study highlight the potential role of miR-124 in the control of immune responses and pathogenesis of inflammatory diseases. It is important that miR-124 mimic suppressed the TH17 deffierentiation during colitis process and delayed or inhibited the development of colitis-related cancer. miR-124 maybe could acted as biologic therapy point in colitis. Abbreviations CRC : Colitis Related Cancer miR-124 : microRNA-124 qPCR : quantitative real-time PCR DSS : Dextran Sulfate sodium salt AOM : azoxymethane Declarations Ethics approval and consent to participate The animal study protocols were approved by the Institutional Animal Care and Use ethical Committee of Sun Yat-sen University Cancer Center. Consent for publication All authors reached the agreement for the publication Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request Competing interests The authors declare that they have no competing interests. Funding This work was in part supported by Open Project Fund of Guangdong Provincial Key Laboratory of Gastroenterology(2017),and Foundation of President of Nanfang Hospital (2016Z015). Authors' contributions Conceptualization and methodology: SX and JW; Formal analysis and data collection: JW ,YG and KB; Writing—original draft preparation and Writing—review and editing: SX and JW; Approval of final manuscript: all authors. All authors read and approved the final manuscript. Acknowledgements Not applicable. References Zhong W, Jiang ZY, Zhang L, et al. Role of LAP(+)CD4(+) T cells in the tumor microenvironment of colorectal cancer. World journal of gastroenterology. 2017 Jan 21;23(3):455-463. doi: 10.3748/wjg.v23.i3.455. PubMed PMID: 28210081; PubMed Central PMCID: PMC5291850. Razi S, Baradaran Noveiry B, Keshavarz-Fathi M, et al. IL-17 and colorectal cancer: From carcinogenesis to treatment. Cytokine. 2019 Apr;116:7-12. doi: 10.1016/j.cyto.2018.12.021. PubMed PMID: 30684916. 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IRF4 regulates IL-17A promoter activity and controls RORgammat-dependent Th17 colitis in vivo. Inflammatory bowel diseases. 2011 Jun;17(6):1343-58. doi: 10.1002/ibd.21476. PubMed PMID: 21305677. Tong Z, Yan H, Liu W. Interleukin-17F attenuates H2O2-induced cell cycle arrest. Cellular immunology. 2014 Feb;287(2):74-7. doi: 10.1016/j.cellimm.2013.12.007. PubMed PMID: 24423465. Cai WL, Huang WD, Li B, et al. microRNA-124 inhibits bone metastasis of breast cancer by repressing Interleukin-11. Molecular cancer. 2018 Jan 17;17(1):9. doi: 10.1186/s12943-017-0746-0. PubMed PMID: 29343249; PubMed Central PMCID: PMC5773190. Taniguchi K, Sugito N, Kumazaki M, et al. MicroRNA-124 inhibits cancer cell growth through PTB1/PKM1/PKM2 feedback cascade in colorectal cancer. Cancer letters. 2015 Jul 10;363(1):17-27. doi: 10.1016/j.canlet.2015.03.026. PubMed PMID: 25818238. Liu K, Yao H, Lei S, et al. The miR-124-p63 feedback loop modulates colorectal cancer growth. Oncotarget. 2017 Apr 25;8(17):29101-29115. doi: 10.18632/oncotarget.16248. PubMed PMID: 28418858; PubMed Central PMCID: PMC5438716. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-23700","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Primary research","associatedPublications":[],"authors":[{"id":563978,"identity":"5ca93fe4-1843-48e4-bd43-5d86e324dc45","order_by":1,"name":"Shiyong Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIie3RMQrCQBBA0VlWUq1Ju0FQjzASCAi5iU3SbGefIuBCYC3TegzBC0QGrAIewCbeIAewMFaWmVJwfz2vmBkAn+8HC0FaGFAvIympZ5EAhBWnMkviY2CQTaTqTHG+q7XmEV04PXckLqQAocp2PBI7kinN2x5uZm8nyWIkG0dBSmGOwhKTFI5UUivUfNJ2RqNkk9Wz3toyQ03jkXPOLpGi6+OF+tA0RP1QZdNkbPZ9R84Y/yQH5qDP5/P9a2+Fdjx/GHDXNgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3881-6422","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shiyong","middleName":"","lastName":"Lin","suffix":""},{"id":563979,"identity":"5a1a2fdb-6ffe-4e53-a8ea-05449b4dc40f","order_by":2,"name":"Qianwen Liu","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qianwen","middleName":"","lastName":"Liu","suffix":""},{"id":563980,"identity":"3d842370-c5d7-467b-aeb6-3dc961f957cc","order_by":3,"name":"Jing Wen","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wen","suffix":""},{"id":563981,"identity":"42bb177c-2138-402f-8ed3-1b7b4cfecc6d","order_by":4,"name":"Kunhao Bai","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kunhao","middleName":"","lastName":"Bai","suffix":""},{"id":563982,"identity":"429a0c77-a673-40c9-b0b8-f2996077e069","order_by":5,"name":"Yandong Guo","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yandong","middleName":"","lastName":"Guo","suffix":""},{"id":563983,"identity":"bf6ebe33-6948-41de-ac96-fb15947ed6fe","order_by":6,"name":"Jing Wang","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2020-04-18 10:50:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-23700/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-23700/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":1099173,"identity":"bbda8230-490d-46b7-bc0f-526606288a4b","added_by":"auto","created_at":"2020-05-14 15:03:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91346,"visible":true,"origin":"","legend":"miR-124 selectively suppresses TH17 and TH1 cell differentiation. A. miR-124 expression in TH0,TH17,TH1 and treg polarizing conditions by qPCR assay .Naïve CD4+ T cells from C57BL/6 mice were differentiated under TH17 and TH1 polarizing conditions respectively in the presence of 4.8nmol miR-124 mimic for 3 days and analyzed through qPCR assay(B,C) , ELISA (D,E), and flow cytometry (F). G. Naïve CD4+ T cells from C57BL/6 mice were differntiated under TH17 polarizing conditions respectively in the presence of 4.8nmol miR-124 mimic for 3 days and analyzd through flow cytometry. H. CD4+ cell proliferation was detected by CSFE. G. Naïve CD4+ T cells from C57BL/6 mice were differntiated under TH17 polarizing conditions respectively in the presence of 4.8nmol miR-124 mimic for 3 days , and stained by Annexin-V ,analyzd through flow cytometry. *p \u003c 0.05, **p \u003c 0 .01, ***p \u003c 0.001 versus cells cultured, all tests were performed three time.","description":"","filename":"figure1.JPG","url":"https://assets-eu.researchsquare.com/files/rs-23700/v1/figure1.JPG"},{"id":1099174,"identity":"9d9dc74b-14c7-4391-93a6-ac370f9f9b7e","added_by":"auto","created_at":"2020-05-14 15:03:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35892,"visible":true,"origin":"","legend":"miR-124 suppresses TH17 activation by targeting STAT3. Naïve CD4+ T cells from C57BL/6 mice were differentiated under TH17 and TH1 polarizing conditions with IL-6 (10 ng/ml) and TGFb (5 ng/ml) for 24 or 72 hrs in the presence of 4.8nmol miR-124 mimic. (A), miR-124 expression was detected by qPCR, U6 as control. (B,C).The whole cell lysates were prepared and western blotting was performed for the analysis of protein expression. b-actin expression serves as a control. (D) El4 cell was stimulated with CD3 and CD28 antibody and primed with IL-6 and TGFb in the presence of miR-124 mimic, Chip assay was performed with anti-STAT3 antibody, IgG antibody as negative control. *, P\u003c0.05; **, P\u003c0.01. The results are representative of three independent experiments.","description":"","filename":"figure2.JPG","url":"https://assets-eu.researchsquare.com/files/rs-23700/v1/figure2.JPG"},{"id":1099175,"identity":"3483e9bc-6902-480a-a1fe-582cbb2e6077","added_by":"auto","created_at":"2020-05-14 15:03:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58647,"visible":true,"origin":"","legend":"STAT3 is the target of miR-124\n(A).EL4 cells was primed with IL-6 and TGFb in the presence of miR-124 mimic, STAT3 protein level was evaluated by western blotting. (B) Schematic representation of wild-type (wt) and mutant (mut) STAT3 3`UTR luciferase reporter constructs of predicted miR-124 target gene. The miR-124 binding region is indicated. (C). EL4 cells was co-transfected with miR-124 mimic and STAT3 3`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. The immunoprecipitated RNA was purified 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.","description":"","filename":"figure3.JPG","url":"https://assets-eu.researchsquare.com/files/rs-23700/v1/figure3.JPG"},{"id":1099176,"identity":"b6710262-8985-40ae-8834-791a2c37dc43","added_by":"auto","created_at":"2020-05-14 15:03:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80739,"visible":true,"origin":"","legend":"MiR-124 weaken the colitis in CD4 positive cell transfer model in vivo. CD4+CD45Rbhi T cells were purified from C57BL/6mice and 5 x 105cells were injected (i.p.) into recipient Rag1-/-mice. Mice were treated with control or miR-124 mimic (at 10 nm/mouse) every three days. (A). Morphology of intestines; (B), disease scores, *p \u003c 0.05 versus recipients of control group (n = 5-6 mice per group) , (C). sections of colons with colitis from Rag1-/- mice (n = 5-6 mice in each group) 8 weeks after naïve T cell transfer as described above. Scale bar, 100 μM. (D,E). The percentage of IL-17 -producing cells from mesenteric lymph nodes and LPL of Rag1-/- mice in control and miR-124 mimic treated group. **p \u003c 0.01 versus recipients of control treated group.","description":"","filename":"figure4.JPG","url":"https://assets-eu.researchsquare.com/files/rs-23700/v1/figure4.JPG"},{"id":1099177,"identity":"df7003f7-7ac3-4121-93ad-dc334ce04f3b","added_by":"auto","created_at":"2020-05-14 15:03:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85177,"visible":true,"origin":"","legend":"miR-124 suppressed the development of colitis associated carcinoma by inhibiting the TH17 differentiation. (A) Schematic of AOM/DSS administration. Mice were injected with 12.5 mg/kg AOM and subjected to three 7-day cycles of 2.5% DSS respectively in the presence of control or miR-124 mimic , (B ,C) Morphology of intestines and representative H\u0026E-stained sections and PAS after DSS or AOM/DSS treatment .Scale bar:100μm. (D).Total RNA was extracted and mRNA was detected by qPCR, b-actin as control, *, P\u003c0.05; **, P\u003c0.01. The results are representative of three independent experiments, (E). The percentage of IL-17 -producing cells from mesenteric lymph nodes of mice in control and miR-124 mimic treated group. *p \u003c 0.05 versus recipients of control treated group.","description":"","filename":"figure5.JPG","url":"https://assets-eu.researchsquare.com/files/rs-23700/v1/figure5.JPG"},{"id":1099178,"identity":"8fb02e1d-ef95-4fd4-b4b5-9f0fe081b177","added_by":"auto","created_at":"2020-05-14 15:03:04","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":69046,"visible":true,"origin":"","legend":"MiR-124 depressed the colon carcinogenesis in C. rodentium infection colitis related cancer murine model. 8-week-old wild-type mice (WT; n = 12) infected orally with 2 × 109 colony-forming units (CFU) of C. rodentium in the presence of control (n=6) or miR-124 mimic (n=6).(A). Morphology of intestines and representative H\u0026E-stained sections and PAS after C. rodentium infection .Scale bar:100μm. (D),Total RNA was extracted and mRNA was detected by qPCR, b-actin as control, *, P\u003c0.05; (E), he percentage of IL-17 -producing cells from mesenteric lymph nodes of mice in control and miR-124 mimic treated group. *p \u003c 0.05 versus recipients of control treated group.","description":"","filename":"figure6.JPG","url":"https://assets-eu.researchsquare.com/files/rs-23700/v1/figure6.JPG"},{"id":15666987,"identity":"02e06e64-cc95-4411-be2f-c74a7eaac9aa","added_by":"auto","created_at":"2021-11-18 13:39:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":821789,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-23700/v1/0c57c978-a949-438a-93f7-6eb6d2c705e4.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSTAT3-mediated T\u003csub\u003eH\u003c/sub\u003e17 cell Differentiation was Related to the Carcinogenesis of Colitis Related Cancer\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eCRC 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-\u0026beta;, CXCR3, CC chemokine receptor 6 (CCR6) and IL-6and also suppress CD8\u003csup\u003e+\u003c/sup\u003e T cells which have anti-tumor activity. Moreover, it has been shown that the number of IFN-\u0026gamma; producingCD8\u003csup\u003e+\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003eMicroRNAs (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 inflammatory diseases, including IBD.Many published documents identified that miRs are essential regulators ofToll-like receptor signaling, which is important to trigger the intestinal inflammation.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 deficient 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 inflammation 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 inflammation 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 inflammation to cancer development. Yuan et al reviewed and listed that miRNAs were involved in inflammation 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 specifically 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.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;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 inflamed area and efficiently inhibited the Th17 polarization, causing the phenotype transition into Treg cells, which next inhibited the inflammatory response and promoted the mucosal regeneration, and finally decreased the colitis-related colonic cancer development. Therefore, we expect targeting miR-124 would offer a novel therapeutic strategy for colitis-related colonic cancer.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6J and Rag1\u003csup\u003e-/- \u003c/sup\u003emice 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\u003csup\u003e5\u003c/sup\u003eMC38 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReagents and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMiR-124 mimic and inhibitor werepurchased from GenePharma. The following Flowcytometry antibodies were purchased from BDBiosciences(USA), and conjugated to FITC, PE, PECy5,PE-Cy7, PerCP-Cy5.5, PerCP-eFluor 710, eFluor450 or APC: CD45RB (C363.16A), CD4 (L3T4), CD25 (PC61.5), IL-17 (TC11-18H10), IFN-\u0026gamma; (XMG1.2), FOXP3(FJK-16S) and isotype controls. Antibodies for ROR\u0026gamma;(B2D), Gr1 (RB6-8C5) and IL-4 (11B11) were purchased from eBioscience. FITC Annexin V Apoptosis DetectionKit I (2293683) was purchased from BD Pharmingen.Anti-ROR\u0026gamma;t, anti-T-bet (MBL), anti-STAT3, anti-pSTAT3 (CellSignaling), and anti-\u0026beta;-actin (Sigma) antibodies for western blotting were usedaccording to the manufacturers\u0026rsquo; instructions. Secondaryantibodies were from Santa Cruz Biotechnology, Inc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD4\u003csup\u003e+ \u003c/sup\u003eT cell preparation and differentiation in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNaive CD4\u003csup\u003e+\u003c/sup\u003e T cells (CD62L+CD44lo) were preparedby fluorescence-activated cell sorting from spleens andlymph nodes of C57BL/6 mice. The sorted cells wereprimed for 96 hrs with anti-CD3 (1 \u0026mu;g/ml; 145-2C11; BDBiosciences) and soluble anti-CD28 (2 \u0026mu;g/ml; 37.51; BDBiosciences). Cells stimulated under neutral conditionswere defined as TH0 cells. Cells were stimulated to differentiate into TH1 cells by supplementation with IL-12plus anti-IL-4 or into TH2 cells by supplementation withIL-4 and anti-IFN-\u0026gamma;. For TH17 cell differentiation, cells were stimulated with transforming growth factor-\u0026beta;1 (5 ng/ml), IL-6 (20 ng/ml) and IL-23 (10 ng/ml; all from R\u0026amp;DSystems) and into Treg cells by supplementation with transforming growth factor-\u0026beta;1 (15 ng/ml).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular staining and flow cytometer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor T cells, cells were stimulated with PMA andionomycin for 5 hrs in the presence of brefeldin A priorto intracellular staining. Cells were fixed 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. Flow cytometry was performed on aFACS Calibur (BD Biosciences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupernatants from cell cultures were collected after activation under various conditions and secreted cytokines in the supernatants were measured by ELISA kits withpurified coating and biotinylated detection antibodies:anti-IL-17, anti-IFN-\u0026gamma; (R \u0026amp; D systems), anti-IL-9, anti-IL-22 ,anti-IL-1\u0026beta; (e Bioscience) and anti-IL-4 (BDBioscience).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and quantitative real-time RT-PCR (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT cell-transfer colitis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT cell transfer colitis was performed as previouslydescribed. Briefly, purified CD4\u003csup\u003e+\u003c/sup\u003eCD45RB\u003csup\u003ehi\u003c/sup\u003e T cellsfrom WT mice were injected intraperitoneally into Rag1\u003csup\u003e-/-\u003c/sup\u003e recipients (5 x 10\u003csup\u003e5\u003c/sup\u003e cells per mouse in 200 \u0026mu;l sterile PBSper injection)[\u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e]. Mice were weighed every week throughoutthe course of experiments. The degree of inflammation inthe epithelium, submucosa and submuscularis propria wasscored separately as described previously[\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results are shown as means \u0026plusmn; SD and statisticalanalysis was performed using Student\u0026rsquo;s t-Test. Wheremore than two groups were compared, one way- ANOVAwith Bonferroni`s correction were performed. P \u0026lt; 0.05was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMiR-124 inhibited the T\u003csub\u003eH\u003c/sub\u003e17 cell polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of miR-124 on adaptive immune cellfunction, we first focused on T helper cells. Na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e Tcells from C57BL/6 mice were primed in vitro for 3 daysunder T\u003csub\u003eH\u003c/sub\u003e0, T\u003csub\u003eH\u003c/sub\u003e17, Treg and T\u003csub\u003eH\u003c/sub\u003e1 conditionsand miR-124 expression was evaluated by qPCR. We found that miR-124 expression was obviously decreased in T\u003csub\u003eH\u003c/sub\u003e17 cell rather than TH1 (Figure 1A). qPCR and ELISA experiments showed that miR-124 mimic significantlysuppressed expression of T\u003csub\u003eH\u003c/sub\u003e17 or T\u003csub\u003eH\u003c/sub\u003e1-associatedgenes including IL-17, IFN-\u0026gamma;(Figure1B,C,D,E). We next detect whether the T\u003csub\u003eH\u003c/sub\u003e17 and T\u003csub\u003eH\u003c/sub\u003e1 cell differentiation was affectedin the presence of miR-124 mimic.These observations correlatedwith reduced IL-17 and IFN-\u0026gamma; production by T\u003csub\u003eH\u003c/sub\u003e17 or T\u003csub\u003eH\u003c/sub\u003e1 cells treated with miR-124 mimic as determined by flow cytometer (Figure1F).The results showed that the frequency of IL-17- and IFN-\u0026gamma;-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\u003csup\u003e+\u003c/sup\u003e 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\u003csup\u003e+\u003c/sup\u003e cell proliferation (figure G,H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-124 alters DNA binding activity in T\u003csub\u003eH\u003c/sub\u003e17 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, the evidence prompted us to probe for the molecular basis for howmiR-124 modulated T\u003csub\u003eH\u003c/sub\u003e17 cell differentiation. Since many studies have shown that several transcription factors including ROR\u0026gamma;t, STAT3, and AHR are important for T\u003csub\u003eH\u003c/sub\u003e17 cell differentiation, we hypothesized that miR-124 might affect the expression of these transcription factors. To address this, na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells from C57BL/6 mice were primed in vitro for 3 days under T\u003csub\u003eH\u003c/sub\u003e0 or T\u003csub\u003eH\u003c/sub\u003e17 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 ROR\u0026gamma;t 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 significantly 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTAT3 is the target of miR-124\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe 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\u0026rsquo;UTR of STAT3 gene, and inhibit the protein translation.To verify that miR-124 really targets STAT3,we first 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\u0026rsquo;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 significantly 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.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-124 weaken the colitis in CD4 positive cell transfer model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious reports have indicated a correlation betweenT\u003csub\u003eH\u003c/sub\u003e17 activity and IBD pathogenesis. To further assessthe effects of miR-124 on T\u003csub\u003eH\u003c/sub\u003e17 cell development in vivo, weperformed adoptive T cell transfer colitis experimentsusing CD4\u003csup\u003e+\u003c/sup\u003eCD45Rb\u003csup\u003ehi \u003c/sup\u003ecells from C57BL/6 mice toinduce colitis in Rag1\u003csup\u003e-/-\u003c/sup\u003e mice. Mice in the treatmentgroup received low dose miR-124 twice a week for 8 weekswhile the control group was treated with PBS. While theRag1\u003csup\u003e-/-\u003c/sup\u003e mice reconstituted with na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells lostweight continuously, treatment with miR-124 significantly improved their condition (Figure 4A,B). Parallel histologicstudies of colonic sections from Rag1\u003csup\u003e-/-\u003c/sup\u003e mice treated withmiR-124 revealed fewer inflammatory cell infiltration andsignificantly lower pathological scorescompared tomice treated with PBS (Figure 4C). And, miR-124 expression also was confirmed in the treatment of miR-124 mimic (Figure 4D). In addition, micetreated with miR-124 had significantly lower percentagesof IL-17 cells and lower expression of T\u003csub\u003eH\u003c/sub\u003e17 and T\u003csub\u003eH\u003c/sub\u003e1 signature gene expression than PBS treated mice (Figure4E,F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-124 suppressed the development of colitis associated carcinoma by inhibiting the T\u003csub\u003eH\u003c/sub\u003e17 differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUlcerative colitis (UC) is a subcategory of inflammatory 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\u003csub\u003eH\u003c/sub\u003e17 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 significantly 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 identified 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 inflammation impaired the epithelial microenvironment and the TH17 cells function, resulting in development of inflammation 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 \u0026ldquo;the second hit\u0026rdquo; of cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-124 depressed the colon carcinogenesis in \u003cem\u003eC. rodentium\u003c/em\u003e infection colitis colon cancer murinemodel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrobial dysbiosis causes chronic inflammation associated with CRC .\u003cem\u003eC. Rodentium\u003c/em\u003e 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. \u003cem\u003eC. Rodentium\u003c/em\u003e has been used as a model to study mucosal immunology, including intestinal inflammatory responses during bacteria-induced colitis and colon tumorigenesis. \u003cem\u003eC. rodentium\u003c/em\u003e infection increases the number of colonic adenomas in Apc\u003csup\u003eMin\u003c/sup\u003e mice but does not cause adenoma formation in wild-type mice.\u003c/p\u003e\n\u003cp\u003eAfter \u003cem\u003eC. Rodentium\u003c/em\u003e (2 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e 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 sacrificed 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\u003csup\u003e+\u003c/sup\u003eT cells from \u003cem\u003eC. Rodentium\u003c/em\u003e-infected colon cancer tissue and analyzed the relative abundance of T\u003csub\u003eH\u003c/sub\u003e17 subpopulations according to their associated expression of IL-17. IL-17 was significantly decreasedafter miR-124 treatment (Figure6 D,E). So, miR-124 mimic treatmentdepressed the T\u003csub\u003eH\u003c/sub\u003e17 cell differentiation in the colon post \u003cem\u003eC. Rodentium\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUlcerative colitis (UC) is a subcategory of inflammatory bowel disease (IBD) with high risk of colorectal cancer, which included two key pathophysiological features: dysregulation of immune system and impaired mucosal repair[16]（这句和前面重复了）. Limited therapies available at present remains UC and colitis-related colon cancer a challenging problemfor the clinician. In our study, we confirmed 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 inflammation and colon cancer occurrence in murine colitis model. Thus miR-124 could inhibit Th17 cell differentiation and pro-inflammatory cytokines induction, thereby promoting the mucosal repair and suppressing the development of colitis associated carcinoma. We concluded thatmiR-124 is a critical regulator in control of intestinal immune function and epithelial regeneration, andtargeting miR-124 can beselected as a \u0026ldquo;smart\u0026rdquo; therapeutic strategy for UC and colitis-related colon cancer.\u003c/p\u003e\n\u003cp\u003eAfter 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-inflammatory cytokine, which isassociated with cancer progression[18, 19]. The main source of IL-17 is a subpopulation of CD4\u003csup\u003e+\u003c/sup\u003e T cells known as T-helper17 (Th17) cells . Tumor infiltrating 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 inflammation were protective, excessive inflammation was deleterious and was at the basis ofinflammatory bowel disease (IBD) and inflammation-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 verified to be related with colon cancer development. But how to regulate the Th17 cell differentiation still was elusive.\u003c/p\u003e\n\u003cp\u003eSeveral agents are required for differentiation and stabilization, such as transforming growth factor beta (TGF-\u0026beta;), interleukin-6 (IL-6), IL-21, IL-23 and IL-1b. Also, retinoic orphan receptor-\u0026gamma; (ROR\u0026gamma;) 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\u003csup\u003e+\u003c/sup\u003e T helper cells toward Th17 cell polarization, resulting in a hyper-inflammatory response with associated tissue damage and pathogenesis.\u003c/p\u003e\n\u003cp\u003eThus, 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 significantly 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 firmly 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 inflammatory diseases.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study highlight the potential role of miR-124 in the control of immune responses and pathogenesis of inflammatory diseases. It is important that miR-124 mimic suppressed the TH17 deffierentiation during colitis process and delayed or inhibited the development of colitis-related cancer. miR-124 maybe could acted as biologic therapy point in colitis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eCRC\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eColitis Related Cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-124\u003c/strong\u003e: microRNA-124\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqPCR : \u003c/strong\u003equantitative real-time PCR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDSS\u003c/strong\u003e: Dextran Sulfate sodium salt\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAOM\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eazoxymethane\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study protocols were approved by the Institutional Animal Care and Use \u003cstrong\u003eethical\u003c/strong\u003e Committee of Sun Yat-sen University Cancer Center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors reached the agreement for the publication\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was in part supported by Open Project Fund of Guangdong Provincial Key Laboratory of Gastroenterology(2017),and Foundation of\u0026nbsp;President\u0026nbsp;of\u0026nbsp;Nanfang Hospital\u0026nbsp;(2016Z015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization and methodology: SX and JW; Formal analysis and data collection: JW ,YG and KB; Writing\u0026mdash;original draft preparation and Writing\u0026mdash;review and editing: SX and JW; Approval of final manuscript: all authors. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhong W, Jiang ZY, Zhang L, et al. Role of LAP(+)CD4(+) T cells in the tumor microenvironment of colorectal cancer. World journal of gastroenterology. 2017 Jan 21;23(3):455-463. doi: 10.3748/wjg.v23.i3.455. PubMed PMID: 28210081; PubMed Central PMCID: PMC5291850.\u003c/li\u003e\n\u003cli\u003eRazi S, Baradaran Noveiry B, Keshavarz-Fathi M, et al. IL-17 and colorectal cancer: From carcinogenesis to treatment. Cytokine. 2019 Apr;116:7-12. doi: 10.1016/j.cyto.2018.12.021. PubMed PMID: 30684916.\u003c/li\u003e\n\u003cli\u003eMa C, Dong X. Colorectal cancer-derived Foxp3(+) IL-17(+) T cells suppress tumour-specific CD8+ T cells. Scandinavian journal of immunology. 2011 Jul;74(1):47-51. doi: 10.1111/j.1365-3083.2011.02539.x. 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MicroRNAs, intestinal inflammatory and tumor. Bioorganic \u0026amp; medicinal chemistry letters. 2019 Jun 12. doi: 10.1016/j.bmcl.2019.06.013. PubMed PMID: 31213403.\u003c/li\u003e\n\u003cli\u003eJosse C, Bours V. MicroRNAs and Inflammation in Colorectal Cancer. Advances in experimental medicine and biology. 2016;937:53-69. doi: 10.1007/978-3-319-42059-2_3. PubMed PMID: 27573894.\u003c/li\u003e\n\u003cli\u003eChen T, Xue H, Lin R, et al. MiR-126 impairs the intestinal barrier function via inhibiting S1PR2 mediated activation of PI3K/AKT signaling pathway. Biochemical and biophysical research communications. 2017 Dec 16;494(3-4):427-432. doi: 10.1016/j.bbrc.2017.03.043. PubMed PMID: 28302479.\u003c/li\u003e\n\u003cli\u003eLiu Y, Zhou Y, Feng X, et al. MicroRNA-126 functions as a tumor suppressor in colorectal cancer cells by targeting CXCR4 via the AKT and ERK1/2 signaling pathways. International journal of oncology. 2014 Jan;44(1):203-10. doi: 10.3892/ijo.2013.2168. PubMed PMID: 24189753.\u003c/li\u003e\n\u003cli\u003eKoukos G, Polytarchou C, Kaplan JL, et al. MicroRNA-124 regulates STAT3 expression and is down-regulated in colon tissues of pediatric patients with ulcerative colitis. Gastroenterology. 2013 Oct;145(4):842-52 e2. doi: 10.1053/j.gastro.2013.07.001. PubMed PMID: 23856509; PubMed Central PMCID: PMC4427058.\u003c/li\u003e\n\u003cli\u003eOuyang X, Zhang R, Yang J, et al. Transcription factor IRF8 directs a silencing programme for TH17 cell differentiation. Nature communications. 2011;2:314. doi: 10.1038/ncomms1311. PubMed PMID: 21587231; PubMed Central PMCID: PMC3112536.\u003c/li\u003e\n\u003cli\u003eDeng F, He S, Cui S, et al. A Molecular Targeted Immunotherapeutic Strategy for Ulcerative Colitis via Dual-targeting Nanoparticles Delivering miR-146b to Intestinal Macrophages. Journal of Crohn's \u0026amp; colitis. 2019 Mar 30;13(4):482-494. doi: 10.1093/ecco-jcc/jjy181. 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PubMed PMID: 28454345; PubMed Central PMCID: PMC5403242.\u003c/li\u003e\n\u003cli\u003eChiba T, Marusawa H, Ushijima T. Inflammation-associated cancer development in digestive organs: mechanisms and roles for genetic and epigenetic modulation. Gastroenterology. 2012 Sep;143(3):550-563. doi: 10.1053/j.gastro.2012.07.009. PubMed PMID: 22796521.\u003c/li\u003e\n\u003cli\u003eMudter J, Yu J, Zufferey C, et al. IRF4 regulates IL-17A promoter activity and controls RORgammat-dependent Th17 colitis in vivo. Inflammatory bowel diseases. 2011 Jun;17(6):1343-58. doi: 10.1002/ibd.21476. PubMed PMID: 21305677.\u003c/li\u003e\n\u003cli\u003eTong Z, Yan H, Liu W. Interleukin-17F attenuates H2O2-induced cell cycle arrest. Cellular immunology. 2014 Feb;287(2):74-7. doi: 10.1016/j.cellimm.2013.12.007. PubMed PMID: 24423465.\u003c/li\u003e\n\u003cli\u003eCai WL, Huang WD, Li B, et al. microRNA-124 inhibits bone metastasis of breast cancer by repressing Interleukin-11. Molecular cancer. 2018 Jan 17;17(1):9. doi: 10.1186/s12943-017-0746-0. PubMed PMID: 29343249; PubMed Central PMCID: PMC5773190.\u003c/li\u003e\n\u003cli\u003eTaniguchi K, Sugito N, Kumazaki M, et al. MicroRNA-124 inhibits cancer cell growth through PTB1/PKM1/PKM2 feedback cascade in colorectal cancer. Cancer letters. 2015 Jul 10;363(1):17-27. doi: 10.1016/j.canlet.2015.03.026. PubMed PMID: 25818238.\u003c/li\u003e\n\u003cli\u003eLiu K, Yao H, Lei S, et al. The miR-124-p63 feedback loop modulates colorectal cancer growth. Oncotarget. 2017 Apr 25;8(17):29101-29115. doi: 10.18632/oncotarget.16248. PubMed PMID: 28418858; PubMed Central PMCID: PMC5438716.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"miR-124, STAT3, TH17 cell, colitis, Colitis Related Cancer","lastPublishedDoi":"10.21203/rs.3.rs-23700/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-23700/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eB\u003cstrong\u003eackground\u003c/strong\u003e: Inflammation often induces regeneration to repair the tissue damage. However, chronic inflammation 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-inflammatory production and reparative regeneration.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe expression levels of\u0026nbsp;miR-124 and IL-17, IFN-γ were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). T\u003csub\u003eH\u003c/sub\u003e17 or T\u003csub\u003eH\u003c/sub\u003e1 cells were detected by flow 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 confirm the function of miR-124 \u003cem\u003ein vivo\u003c/em\u003e. The related gene expression was analyzed by ELISA and western blot experiments.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe results indicated that miR-124a deficiency leads to colon tumorigenesis after \u003cem\u003eCitrobacter rodentium\u003c/em\u003e infection and AOM/DSS induced colon cancer murine model. In molecular mechanism, miR-124 targets STAT3 to suppress T helper 17 (T\u003csub\u003eH\u003c/sub\u003e17) cell differentiation and expansion, and keep T\u003csub\u003eH\u003c/sub\u003e17 polarization in colonic microenvironment.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur study highlights highlight the potential role of miR-124 in the control of immune responses and pathogenesis of inflammatory diseases.\u003c/p\u003e","manuscriptTitle":"STAT3-mediated TH17 cell Differentiation was Related to the Carcinogenesis of Colitis Related Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-05-14 15:03:02","doi":"10.21203/rs.3.rs-23700/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"359b9c22-0fb5-43ed-9ac2-f0bc5986e228","owner":[],"postedDate":"May 14th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":100323,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2020-05-29T03:01:13+00:00","versionOfRecord":[],"versionCreatedAt":"2020-05-14 15:03:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-23700","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-23700","identity":"rs-23700","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}