MiR-192-5p/RB1-mediated Epithelial-mesenchymal Transition Modulates IL-10 Secretion In Gastric Cancer To Affect Regulatory T Cell Differentiation

Regulatory T(Treg)cells,one of the most important components of the tumor microenvironment(TME),are closely related to the development and metastasis of malignant tumors.Treg cells are differentiated from CD4~+T cells.CD4~+T cells could respond to a variety of factors(TGF-β,IL-10)produced by tumor cells and differentiate into Treg cells,which have immunosuppressive function.Tumor cells undergoing epithelial-mesenchymal transition(EMT)play vital roles in promoting metastasis and immune escape of malignant tumors.It has been reported that EMT tumor cells can secrete various mediators acting on immune cells in TME to remodel the immunosuppressive TME and promote tumor metastasis.However,the specific mechanisms by which EMT tumour cells regulate Treg cells in GC have not yet been evaluated.Micro RNA(miRNA)is a small non-coding RNA that can bind to the m RNA of target genes to regulate their expression.The aberrantly expressed miRNAs in tumors could induce Treg cell differentiation in TME by secreting multiple mediators,thereby promoting tumor metastasis.It has been reported that miR-192-5p is abnormally overexpressed in malignant tumors and recognized as an oncogene in the EMT,invasion,and metastasis of malignant tumors.Mi R-192-5p was reported to promote the secretion of TGF-β,suggesting a potentially important role of miR-192-5p in tumor EMT and Treg cell differentiation.However,whether miR-192-5p-mediated EMT can regulate the secretion of cytokines and affect Treg cell differentiation in GC remains unclear.RB1 is a tumour suppressor that involved in regulating tumor development and the EMT procession.The deletion of RB1 activates the STAT3,EMT-related transcription factors(Slug and ZEB1),and NF-κB pathway to promote tumor EMT and metastasis.NF-κB and STAT3 signaling pathways have important roles in inducing IL-10 secretion,suggesting that RB1 may be involved in tumor EMT and Treg cell differentiation.However,whether RB1 can affect Treg cell differentiation by regulating tumor EMT in the TME is still unclear.Based on the above research,we evaluated the miR-192-5p expression in GC tissues and investigated the prognostic value of miR-192-5p in GC patients.Bioinformatics analysis was performed to analyze the target genes of miR-192-5p and their potential functions.We further explored the specific mechanism of miR-192-5p regulate RB1 to affect tumor cell proliferation,EMT,migration,and invasion of GC.Then a co-culture model was conducted to explore the mechanism via which miR-192-5p/RB1 regulates tumor EMT and induces the IL-10 secretion to affect the Treg cell differentiation in GC.This study mainly includes the following four parts:Part I.Mi R-192-5p is correlated with clinicopathological characteristics and poor prognosis in gastric cancer patients.Background Bioinformatics analysis and the evaluation of GC tissue revealed the expression and prognostic value of miR-192-5p and its target genes.Methods The miRNA expression was identified by examining three Gene Expression Omnibus(GEO)GC miRNA expression datasets.30 paired of GC tissues and adjacent normal tissue(ANT)was used to evaluated the expression of miR-192-5p and RB1.RT-PCR,Western blot,and immumohistochemical(IHC)staining assay were used to detected the RB1 expression.The correlations between the miR-192-5p and clinicopathologic features,prognosis of GC patients were analyzed.Results Based on the three GEO database,miR-1925p was identified that was elevated in tumors in all three datasets.RT-PCR result demonstrated that miR-192-5p expression was considerably elevated in GC compared to ANT and was associated with N stage.Prognostic analysis showed the overexpression of miR-192-5p in tumor tissues was correlated with poor overall survival(OS)of GC.Univariate and multivariate analysis showed that the miR-192-5p was an independent prognostic factor for poor OS in GC patients.Gene set enrichment analysis(GSEA)was performed on predicted genes and found that the predicted genes were enriched in cell cycle,EMT pathways,and Th17cell differentiation.RB1 expression was substantially lower in GC and decreased RB1expression was vastly associated with poor OS in GC.In addition,miR-192-5p expression was inversely linked with RB1 expression.Conclusions Mi R-192-5p was markedly highly expressed in GC samples.Mi R-192-5p was significantly correlated with the clinicopathological characteristics of GC patients,and was an independent prognostic factor of GC.The predicted target gene RB1 was lowly expressed in GC samples and was significantly negatively correlated with miR-192-5p expression.Part II.The mechanism study of mi R-192-5p regulating RB1 to promote the proliferation,migration,and invasion of gastric cancer.Background The specific regulatory mechanism of mi R-192-5p on the target gene RB1 was investigated by performing in vitro experiments,as well as exploring the effect of mi R-192-5p/RB1 on tumor cell proliferation,EMT,migration,and invasion of gastric cancer.Methods Mi R-192-5p mimic and inhibitor were used for mi R-192-5p overexpression and knockdown transfection.Oe-RB1 plasmid and si-RB1 were used for RB1 overexpression and knockdown transfection.RT-PCR and Western blot assays were applied to detect the expression of RB1 and EMT-related genes.RNA immunoprecipitation assay(RIP)and dual luciferase reporter assay were used to verify the mechanism of mi R-192-5p regulating RB1.The effects of mi R-192-5p/RB1 on biological functions of gastric cancer cells were assessed using Ed U,wound healing assay,transwell migration,and transwell invasion assays.Results Mi R-192-5p significantly promoted the expression level of Vimentin and decreased the expression level of RB1 and E-cadherin in gastric cancer cells.RIP and dual luciferase reporter assays confirmed that mi R-192-5p could bind to the 3’-UTR of RB1 m RNA and regulate RB1 m RNA expression.In vitro assays showed that mi R-192-5p overexpression significantly promoted the proliferation,migration,and invasion of gastric cancer cells,while knockdown of mi R-192-5p showed the opposite effects.Besides,the proliferation,migration,and invasion of gastric cancer cells were significantly inhibited by RB1 overexpression,while RB1 knockdown reversed these effects.Conclusions Mi R-192-5p promoted the proliferation,EMT,migration,and invasion of gastric cancer cells by targeting the 3’-UTR of RB1 m RNA.Part III.Mechanism of GC cell EMT on regulating Treg cell differentiation.Background To investigate the specific molecular mechanisms by which mi R-192-5p/RB1 in EMT-programmed tumor cells regulates Treg cell differentiation in the TME.Methods The correlation of mi R-192-5p/RB1 with TH17 cells and Treg cells was examined using GC samples.Human Peripheral Blood Mononuclear Cell(PBMC)was co-cultured with gastric cancer cells to conduct an in vitro co-culture model,and the ratio of Treg cell differentiation was detected by flow cytometry.A series of cytological and molecular biology experiments were applied to investigate the role of mi R-192-5p/RB1 on regulating Treg cell differentiation by affecting the EMT-mediated IL-10 secretion.Results Mi R-192-5p/RB1 was significantly associated with Treg cell infiltration in GC samples.Mi R-192-5p overexpression or RB1 knockdown induced EMT in gastric cancer cells and activated NF-κBp65 pathway,thereby mediating IL-10 secretion,which promoted Treg cell differentiation in TME and PDL1 expression on the surface of gastric cancer cells.Inhibition of NF-κBp65 pathway or neutralized IL-10 were able to decrease the Treg cell differentiation.RB1 could bind to NF-κBp65 in the nucleus and inhibit NF-κBp65 transcriptional activity,thereby suppressing the transcriptional expression of IL-10.While knockdown of RB1 could promote NF-κBp65 transcriptional activity.Activated NF-κBp65 bound to the mi R-192-5p promoter region and promoted the transcription of mi R-192-5p,thus forming a positive feedback loop.Conclusions Mi R-192-5p/RB1 promoted Treg cell differentiation by regulating the NF-κBp65/IL-10 pathway in the TME.NF-κBp65 increased mi R-192-5p transcription and formed a positive feedback loop.Part IV.Silencing mi R-192-5p inhibits the Treg cell differentiation and tumor growth in vivoBackground Further in vivo experiments were used to validate the regulatory role of mi R-192-5p/RB1 on promoting tumor development through mediating EMT in tumor cells and regulating Treg cell differentiation in the TME.Methods A subcutaneous tumor formation model was constructed in nude mice,and mi R-192-5p inhibitors as well as PBMC cells were injected intratumorally to determine the effect of mi R-192-5p on tumor growth.The effect of mi R-192-5p on Treg cell differentiation was analyzed by RT-PCR,Western blot,and IHC.Then a subcutaneous tumor formation model was constructed in normal mice.Mice were injected intraperitoneally with CD25 antibodies to deplete Treg cells or injected with IL-10 neutralizing antibodies.The effects of mi R-192-5p on Treg cell differentiation and tumor growth were analyzed.Results In vivo experiments in nude mice showed that decreased mi R-192-5p in tumors promoted the expression of RB1,inhibited tumor EMT,and reduced the expression of IL-10,which led to a decrease in the proportion of Treg cell differentiation.Both the depletion of Treg cells and inhibition of Treg cell differentiation by IL-10 antibody could inhibit tumor growth in mice.Furthermore,reduced mi R-192-5p expression combined with depletion of Treg cells or injection of IL-10 antibody significantly inhibited tumor growth.Conclusions Knockdown of MiR-192-5p in tumors significantly inhibited tumor EMT and decreased IL-10 secretion,which in turn inhibited Treg cell differentiation and tumor growth of gastric cancer.