| Background The malignant tumor has become a global health issue concerned by countries all over the world.Colorectal cancer is the third digestive tract malignant tumor with the third diagnosis rate in the world,although many early diagnosis and screening methods with comprehensive treatment for colorectal cancer have reduced the mortality rate related to colorectal cancer in recent years,while the therapeutic effect for patients with advanced colorectal cancer or even distant organ metastasis of colorectal cancer is still limited.Therefore,the discovery of new biomarkers and effective treatment targets for advanced target organ metastasis of colorectal cancer patients with more targeted treatment has become a research highlight in recent years.Cancer-associated fibroblasts(CAFs),as one of the main components of malignant tumor stromal cells,participate in formating of tumor microenvironment,regulate the biological behavior of malignant tumors,and induce normal fibroblasts(NFs)to differentiate into CAFs.However,the interaction mechanisms such as signal transduction and substance exchange between CAFs and cancer cells are not yet fully elucidated.MicroRNA(miRNA)is a type of endogenous non-codingRNA,which plays a role in regulating gene expression at the post-transcription level through its degradation of mRNA and translation inhibition.MiRNA-181 family is closely related to the proliferation,differentiation and metastasis of various tumors.One of the family members,miR-181 a,plays a vital role in the occurrence and metastasis of many neoplastic diseases,as well as in aiding in diagnosis and prognosis.Exosomes,also known as small extracellular vesicles,together with microvesicles and apoptotic bodies and other subgroups constitute extracellular vesicles(EVs).Because of its double-layered lipid membrane structure,it contains nucleic acid,protein,lipid and other biologically active molecules,which plays an important role in the process of cancer development.Previous studies have found that exosomal miRNA is particularly important in the progression of various diseases.Many studies have shown that exosomes can carry mRNA,DNA,miRNA,etc.into target cells to play a regulatory role and can be used as a biomarker for disease diagnosis,as a therapeutic target or as a vehicle to deliver specific substances to some target cells for regulating functions.TGF-β(transforming growth factor-beta)superfamily members regulate a variety of cell functions,including cell proliferation,adhesion,migration,invasion and participating in the fibrosis process of parenchymal organs,etc.The effect affects the occurrence and development process of malignant tumor cells.SMAD family protein(Sma-and Mad-related protein)is one of the important kinase substrates regulated by TGF-β receptor and downstream intracellular signal transduction,which is to make important solid organs such as kidney and liver as well as tissues fibrosis.This study focuses on how miRNA-181 a delivered by colon cancer CAFs exosomes regulates the proliferation and invasion ability of recipient tumor cells through the TGF-β/Smad signaling pathway and epithelial-mesenchymal transition(EMT)process with tumor cells.Aim To explore the regulation effect of the exosomes secreted by colon cancer CAFs carrying miRNA-181 a through the TGF-β/Smad signaling pathway on tumor cell proliferation,invasion and malignant tumor phenotypes,and through miRNA-181 a target gene Smad7 to explore the effect and possible mechanism of exosomal miRNA-181a/TGF-β/Smad7 on the regulation of epithelial-mesenchymal transition in colon cancer cells.Methods This study is divided into the following parts: the first part,resuscitation of CAFs preserved in our laboratory and culture then identification in vitro.The exosomes in CAFs conditioned medium were isolated by ultracentrifugation and their physicochemical characteristics were characterized.The uptake of CAFs exosome by colon cancer cells was observed by confocal microscope,and the expression of miR-181 a in human colon adenocarcinoma cell lines HCT116,HT-29,Lo Vo,Ca Co-2 and SW-480 was detected.In the second part,after regulating CAF-miR-181 a and co-culturing with HCT116 and HT-29 cell lines,the proliferation ability of cells in each group was detected by CCK-8 assay,the ability of migration and invasion was detected by wound-healing assay and Transwell assay.The tumor cells co-cultured with CAFs-exosomes apoptosis ratio was detected by flow cytometry,and the expression of TGF-β/Smad signal pathway related molecules,epithelial marker E-cadherin and mesenchymal markers Vimentin,N-cadherin were detected by q PCR and Western blot,immunofluorescence and ELISA assay.In the third part,the bioinformatics method was used to predict miRNA-181 a target gene,and the regulating relationship between miRNA-181 a and Smad7 was detected by q PCR method.Based on the regulation effect of miR-181 a,the up-regulation or down-regulation effects of Smad7 on the proliferation and invasion of colon cancer cells and the expression of TGF-β 1,Smad7,E-cadherin and Vimentin were observed by rescue test.In vivo experiment,the effect of regulating miR-181 a in CAFs on the proliferation of xenograft model in nude mice was confirmed.Results 1.In the first part of this experiment,CAFs was successfully resuscitated,cultured,and the CAFs phenotypes were identificated.The exosome derived from CAFs conditioned medium was successfully isolated by ultracentrifugation and its physicochemical characteristics were identified successfully.The colonic cancer cells absorb CAFs-derived exosomes after co-culturing were observed successfully by confocal microscope.After verifying the expression level of miR-181 a in five colon adenocarcinoma cell lines of HT-29,SW-480,Lo Vo,HCT116,Caco2,we decided to choose HT29 and HCT116 cell line for follow-up study.2.In the second part of this experiment,after overexpressing CAFs-miR-181 a,the expression of miR-181 a in CAFs-exosomes had a higher miR-181 a level compared with CAFs.It was found that by inhibiting of CAFs-miR-181 a could enhance the proliferation and wound healing ability of colon cancer cells and promote the cancer cells invasion and migration ability by Transwell assay.Flow cytometry showed that the proportion of apoptosis decreased,but the trend of up-regulation of miR-181 a of CAFs was opposite.The result of q RT-PCR,WB,immunofluorescence and ELISA assay showed that the expression of TGF-β/Smad pathway molecule and mesenchymal markers Vimentin and N-cadherin increased while the expression of epithelial marker E-cadherin decreased after suppressing CAFs exosomal miR-181 a. The expression trend of each molecule was opposite after up-regulation of exosomal miR-181 a derived from CAFs.3.In the third part of this experiment,the downstream target gene Smad7 of miR-181 a was predicted by using bioinformatics method.The negative regulating relationship between miR-181 a and Smad7 was confirmed by q PCR.The results showed that overexpression of Smad7 could promote the proliferation and invasion of colon cancer,up-regulate the expression of TGF-β1,Smad7 and Vimentin,and down-regulate the expression of E-cadherin.However,the trend of expression level of TGF-β1,Smad7,Vimentin and E-cadherin was opposite after down-regulation of Smad7.In vivo experiment,down-regulation of CAFs exosomal miR-181 a promotes the proliferation of xenograft model in nude mice.Conclusion In this study,through in vitro and in vivo experiments,the following conclusions of regulating role in colon cancer fibroblast exosomal miRNA-181 a were clarified:(1)CAFs secrete high-abundance exosomes,containing high concentrations of miR-181 a and can be taken up by colon cancer cells.(2)By regulating the miR-181 a of CAFs,it can affect the proliferation and invasion ability of colon cancer.The TGF-β/Smad pathway regulates the colon cancer cell epithelial-mesenchymal transition process.(3)By exploring the interaction relationship between regulating miR-181 a and the downstream target molecule Smad7,it is clear that overexpression of Smad7 can promote malignant biological processes in colon cancer,which may provide theoretical and practical basis for the clinical application of CAFs exosomal miRNA. |
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