MiR-195-5p/NOTCH2-mediated Epithelial-mesenchymal Transition Modulates IL-4 Secretion In Colorectal Cancer To Affect Tumor-associated Macrophages Alternative Polarization

Metastasis is an important step in the development of colorectal cancer and is closely related to poor prognosis.Epithelial–mesenchymal transition(EMT),as a major actor modulating tumor metastasis,may be involved in the interaction between the tumor cells and tumor microenvironment(TME).EMT tumor cells secrete large amounts of inflammatory mediators to remodel TME.Tumor-associated macrophages(TAMs),as the most abundant immune cells in TME,responded to various factors produced by tumor cells in TME.EMT-programmed tumor cells could secrete amounts of inflammatory mediators to polarize TAMs to the M2 phenotype,thereby facilitating tumor progression and metastasis.Recent studies have shown that the Notch pathway is involved in tumor EMT.NOTCH2,as one of the NOTCH receptors,induces the release of the intracellular domain of NOTCH2(N2ICD,Ad-NOTCH2)after ligands activation.N2 ICD is then translocated to the nucleus,binding to the transcription factor RBPJ,resulting in complex activation of EMT-related target genes.Furthermore,activation of the Notch pathway is able to mediate IL-4 secretion induced by GATA3 expression(an important M2-type TAMs polarization factor).This suggests that the Notch pathway may be involved in tumor EMT and macrophage alternative polarization.However,whether the Notch pathway promotes EMT of CRC and affects TAMs polarization is not fully understood.As a member of the miR-15/107 family,miR-195-5p has been widely studied in various cancers and is considered to be a tumor suppressor that inhibits CRC proliferation and enhances chemosensitivity.We have demonstrated that miR-195-5p inhibits colorectal cancer EMT by inhibiting YAP1.However,how miR-195-5p-mediated EMT modulates cytokine secretion in CRC to affect TAMs polarization is unclear.Taking into account that NOTCH2 may be a target of miR-195-5p,we hypothesized that miR-195-5p /NOTCH2 may influence CRC EMT status and modulate IL-4 mediated M2-like TAMs polarization.This topic consists of three parts as following:Part 1.Integrated analysis of nine datasets to analyze the expression and pathway of hsa-mir-195-5pBackground Bioinformatics analysis revealed the expression of mir-195-5p in colorectal cancer and verified its prognostic value.Methods Micro RNA sequencing data from primary tumor tissues and paired adjacent normal tissue samples(PANT,same patients)from colorectal cancer patients were used for integrated analysis and identification of miRNAs expression,and the expression profiles of miR-195-5p and NOTCH2 were verified by the TCGA-COAD(colon adenocarcinoma)survival data from TCGA.Results we integrated nine polycentric miRNA datasets of colorectal cancer,analyzed miRNAs expression and used public TCGA-COAD colorectal cancer patients miR-195-5 p and NOTCH2 m RNA expression data to find that miR-195-5 p was negatively correlated with prognosis in patients with colorectal cancer and NOTCH2 was positive related.Using the GO analysis and gene enrichment of collection(GESA),we found that the P53,NOTCH and EMT related signaling pathway is closely with CRC.Conclusion miR-195-5 p was negatively correlated with prognosis in patients with colorectal cancer and NOTCH2 was positive related.MiR-195-5 p and NOTCH2 may plays an important role in the development of CRC.Part 2.Study on the mechanism of miR-195-5p /NOTCH2 in regulating EMT status in colorectal cancer cellsBackground: Verify the effect of miR-195-5p and NOTCH2 on the proliferation,invasion,migration and EMT status of colorectal cancer cells.Furtherly explore how miR-195-5p regulated the target gene NOTCH2.Methods Functional assays,including the Ed U,clone formation,wound healing,and transwell assays,were used to determine the anticancer role of miR-195-5p in human CRC progression.Furthermore,RNA immunoprecipitation(RIP),RNA decay,and dual-luciferase reporter assays were used to determine the mechanism of miR-195-p in NOTCH2 in CRC progression.Results Altered miR-195-5p in colon cancer cells led to distinct changes of proliferation,migration,invasion,and EMT.Mechanistically,miR-195-5p regulated NOTCH2 expression in a post-transcriptional manner by directly binding to 3’-UTR of the Notch2 m RNA.After knocking down NOTCH2,the proliferation,migration,invasion and EMT stasus of colorectal cancer cell were inhibited significantly.After overexpressed NOTCH2,these effects were reversed.The dual luciferase reporter assay found that miR-195-5p binded directly to the 3-UTR of NOTCH2 m RNA.The combined miR-195-5p did not affect the decay of m RNA,but inhibited the expression of NOTCH2 protein in a post-transcriptional manner.Conclusion MiR-195-5p inhibited the EMT and proliferation,migration and invasion of colon cancer cells in vitro and in vivo.Altered miR-195-5p affects colon cancer cell EMT by modulating NOTCH2 expression in a post-transcriptional manner.Part 3.Mechanism of CRC EMT status on regulating TAMs alternative polarizationBackground How EMT-programmed tumor cells affect M2-like TAMs still need further exploration.Methods Co-culture,migration,and ELISA assays were applied to determine the role of miR-195-5p in macrophage recruitment and alternative polarization.Xenograft mouse models were used to determine the role of miR-195-5p in CRC tumorigenicity and TAMs polarization in vivo.Results Decreasing the level of miR-195-5p in the tumor or over-expressing NOTCH2 can induce EMT in colorectal cancer cells.Tumor cells in the EMT state are able to express high levels of GATA3 and IL-4,resulting in the recruitment and polarization of M2-like TAMs in vitro and in vivo.Silencing NOTCH2 and inhibiting IL-4 or neutralizing IL-4 reduced M2-like TAMs recruitment and polarization.Conclusion miR-195-5p inhibits M2-like TAMs polarization by suppressing the NOTCH2/GATA3/IL-4 pathway in CRC cells.