| BackgroundEsophageal cancer is one of the common gastrointestinal tumors currently,Which can be divided into two types according to its pathological diagnosis:esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).ESCC has a high incidence rate in China,especially in Henan in China.At present,the clinical treatment methods for ESCC are mainly surgery,radiotherapy,chemotherapy,and targeted therapy.However,the 5-year overall survival rate of patients is less than 20%.Therefore,it is necessary to further explore the pathogenesis of ESCC,so as to improve treatment options,and improve the 5-year survival rate of patients.Tumors are in a complex environment in vivo,and their occurrence and development are the products of multiple interactions and are affected by the tumor microenvironment(TME).TME is composed of tumor cells and many other types of cells and extracellular matrix.Through direct or indirect contact between cells and cells,a regulatory network that promotes or inhibits each other is formed to regulate the internal immune state of the tumor and promote tumor progression.Tumor associated macrophages(TAMs)of M2 type play an important role in TME.Its high expression of CD 163 can inhibit CD8+T cells to kill tumor cells by secreting cytokines such as IL10 and TGF?.What's more,CCL2 interacts with C-C motif chemokine receptor 2(CCR2)to mediate chemotaxis.of monocytes and polarize them into M2-type TAMs,thereby contributing to the formation of the tumor microenvironment and promoting the progression of cancer.The Musashi(MSI)family is a class of highly evolutionary conserved RNA binding proteins(RBP)encoded by the Musashi gene,including Musashi 1(MSI1)and Musashi2(MSI2).MSI1 were originally discovered in the maintenance of neural stem cells and played an important role in cell differentiation.Subsequent experiments found that MSI1 was related to tumor proliferation and apoptosis.MSI1 found that it can promote the inflammatory response ability and tumor cell invasion ability through the AKT-mTOR signaling pathway,but its role in the tumor microenvironment is unclear.Macrophages play an important role in TME,and exploring the effects of MSI1 on macrophages can help us understand their role in TME.ObjectiveTo investigate the effect of tumor cell expression of MSI1 on macrophages and provide new ideas for the treatment of esophageal squamous cell carcinoma.MethodsThe TCGA database of esophageal cancer was divided into MSI1 high expression group and MSI1 low expression group according to the median expression value of MSI1 to analyze the correlation between MSI1 expression and clinical parameters and prognosis of patients;The TIMER website was used to analyzed the correlation between MSI1 expression and tumor infiltrating cells;qPCR was used to detect the mRNA expression of MSI1 in esophageal squamous cell carcinoma cell lines;TE1 and KYSE70 cells was transfected with empty vector as Negative control(siNC)group,and TE1 and KYSE70 cells was transfected with siMSIl?1 sequence as siMSI1#1 group,qPCR and Western blot was used to detect the mRNA and protein expression level of MSI1 gene in the siNC and siMSI1#1 group of TE1 and KYSE70 cells respectively;qPCR and chemokine kit were used to detecte the mRNA and protein expression levels of 13 chemokines in the siNC and siMSI1#1 group of TE1 and KYSE70 cells respectively;the GEPIA website was used to analyze the correlation between MSI1 and CCL2;The GEO datebase of esophageal squamous cell carcinoma was used to rank the samples according to the level of MSI 1 expression,10 high-expression samples and 10 low-expression samples was used to screen for differentially expressed genes,then the differential genes were used for enrichment analysis and GSEA enrichment analysis;Western blot was used to verify the expression of AKT,mTOR,CCL2 in the siNC and siMSI1#1 group of TE1 and KYSE70 cells respectively;The transwell assay was used to analyze the recruitment of monocytes in the supernatants of the siNC and siMSI1#1 group of TE1 and KYSE70 cells,and the flow cytometry was used to detect the proportion of CCR2 on recruited monocytes in the siNC and siMSI1?1 group of TE1 and KYSE70 cells;The transwell assay was used to analyze the recruitment of CCR2 inhibitor pre-treatment monocytes in the supernatants of the siNC and siMSI1?1 group of TE1 and KYSE70 cells;The supernatant of siNC and siMSI1#1 group in TE1 cells was used to culture the sorted monocytes for 7 days.The morphology of the cells was observed with a microscope,the proportion of CD163+cells was observed with immunofluorescence,and the expression of surface markers(CD 163,IL10,TGF?)of M2 macrophages was detected by qPCR,The expression of CD 163 on polarized monocytes was detected by flow cytometry.Results1.The results of TCGA data show that the expression of MSI1 in patients is related to the clinical TNM stage and occurrence site.The overall survival of patients with low MSI1 expression is longer.TIME website predicts that MSI1 is related to macrophages.2.The results of qPCR and Western blot showed that the sequence of siMSI#1 could effectively silence the expression of MSI 1 gene on TE1 and KYSE70 cells.3.The results of qPCR and multi-factor detection showed that compared to the siNC group of TE1 and KYSE70 cells,the siMSI1#1 group expressed lower levels of CCL2,and the GEPIA website predicted that the expression of MSI1 and CCL2 had a positive correlation.4.The results of GEO database showed that compared to tumor cells with low MSI1 expression,the differential genes of tumor cells with high MSI1 expression are mainly concentrated in the AKT-mTOR signaling pathway,Western blot further verified that the siMSI1#1 group of TE1 and KYSE70 cell lines could reduce the secretion of CCL2 through the AKT-mTOR signaling pathway.5.The results of transwell assay proved that compared to the siNC group of TE1 and KYSE70 cells,the supernatant of siMSI1#1 group in TE1 and KYSE70 cells could promote the recruitment of monocytes.At the same time,the results of cell flow cytometry showed that the proportion of CCR2 on the siMSI1#1 group recruited monocytes reduced.After suppressing the expression of CCR2 on monocytes,the supernatant of the siNC group of TE1 and KYSE70 cells reduced the recruitment of monocytes.6.Immunofluorescence,qPCR and cell flow cytometry assay showed that the supernatant of siMSI1?1 group in the TE1 cells was difficult to polarize the recruited monocytes into M2-type macrophages.Conclusion1.In patients with esophageal cancer,the expression of MSI1 in patients is related to the clinical TNM stage,occurrence site and the overall survival.2.The MSI1 on esophageal squamous cell carcinoma cell lines promotes the secretion of CCL2 through the AKT-mTOR signaling pathway,and recruits monocytes and polarizes them into M2-type macrophages. |
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