| Objective: Colorectal cancer(CRC)is one of the most common malignant tumors of the digestive system in the world.Data from the global Oncology annals in 2023 show that the incidence of colorectal cancer ranks in the third in terms of cancer-related incidence,and its mortality also rank in the third in terms of cancer-related death.Although comprehensive treatment strategies such as radical surgery,chemotherapy,radiotherapy,targeted therapy,and immunotherapy are currently used for colorectal cancer,some patients experience tumor recurrence or distant metastasis during or within a short period of time after treatment,leading to treatment failure.Deeply exploring the pathogenesis of colorectal cancer can contribute to the early diagnosis of the disease and the improvement of treatment strategies,ultimately improving the prognosis of patients with colorectal cancer.Tumor microenvironment(TME)refers to the local environment in which tumor cells or tumor stem cells exist.Cancer associated fibroblasts(CAFs)are the most important cellular components of TME,which participate in the occurrence and development of tumors through the secretion of growth factors,inflammatory factors,and other factors.Exosomes are vesicular structures of intracellular origin with a diameter of about 100 nm.Colon cancer related CAFs communicate with colon cancer cells or other interstitial cells through the release of exosomes that deliver proteins,nucleic acids,lipids,and other molecules for material and information exchange.However,the function and mechanism of CAFs in the development of colon cancer have not been fully elucidated.This study aims to investigate the role and molecular mechanism of miR-522-3p derived from CAFs exosomes in the proliferation,metastasis,and angiogenesis of colon cancer.Methods:1.Extraction and identification of CAFs exosomes and their effects on proliferation,metastasis,and angiogenesis of colon cancerFirst,collagenase digestion was used to extract primary CAFs from colon cancer tissue and normal fibroblasts(NFs)from paired normal colon tissue.The mRNA and protein expression levels of α-SMA,FSP-1,FAP and Vimentin in CAFs and NFs were detected by qRT-PCR,immunofluorescence and Western blot to identify CAFs and NFs.Then,CAFs and NFs exosomes were extracted by ultrahigh speed centrifugation,and characterized by transmission electron microscopy,particle size measurement,and detection of exosome-related markers CD63,CD81,and TSG101 protein.CAFs/NFs exosomes were labeled with PKH26 red fluorescent membrane dye.After incubation with colon cancer cells and HUVECs,the intracellular fluorescence expression was detected to investigate whether CAFs and NFs exosomes could be taken up by colon cancer cells and HUVECs.After incubating colon cancer cells and HUVECs with CAFs/NFs exosomes,the effects of CAFs exosomes on the proliferation,migration and invasion of colon cancer cells and HUVECs in vitro were investigated by MTT cell proliferation,scratch healing and transwell chamber invasion experiments.To explore its role in the angiogenesis of HUVECs based on in vitro angiogenesis experiments,and to explore its regulatory role in the proliferation and metastasis of colon cancer in vivo by constructing nude mice models of subcutaneous transplanted tumors and caudal vein lung metastases.2.Expression of miR-522-3p in CAFs exosomes and its effect on proliferation,metastasis,and angiogenesis of colon cancerFirstly,the expression of miR-522-3p in human colon cancer tissues,cell lines and CAFs exosomes was detected by qRT-PCR,and the relationship between miR-522-3p expression and clinicopathologic features of colon cancer was analyzed.CAFs/ NFs exosomes were used to incubate colon cancer cells and HUVECs,and qRT-PCR was used to detect the expression level of miR-522-3p in the above cells at different time points(0,12,24h),to explore whether miR-522-3p could be delivered to colon cancer cells and HUVECs by CAFs exosomes.Mi R-522-3p expression in CAFs exosomes was silenced by miR-522-3p inhibitor,The effects of CAFs exosome derived miR-522-3p on the growth,proliferation,migration and invasion of colon cancer cells and HUVECs in vitro were investigated by MTT cell proliferation,scratch healing and transwell chamber invasion assay.Based on in vitro angiogenesis experiments,we explored its role in the neovascularization of HUVECs,and then explored its regulatory role in the proliferation and metastasis of colon cancer in vivo by constructing nude mice models of subcutaneous transplanted tumors and caudal vein lung metastases.3.To investigate whether miR-522-3p promotes proliferation,metastasis and angiogenesis of colon cancer cells by directly targeting down-regulation of BMP5 expressionFirst,the potential downstream target gene BMP5 of miR-522-3p was predicted through online miRNA target gene database.Then,miR-522-3p mimics and inhibitor were used to overexpress and silence the expression of miR-522-3p in colon cancer cells and HUVECs,respectively.The effects of miR-522-3p on the mRNA and protein expression levels of BMP5 in colon cancer cells and HUVECs were analyzed by qRT-PCR and Western blot.RNA immunoprecipitation and dual luciferase report analysis confirmed that BMP5 is a downstream target gene directly regulated by miR-522-3p.Finally,BMP5 overexpression vector was used to antagonize the negative regulatory effect of miR-522-3p on BMP5 expression.Based on MTT cell proliferation,scratch healing and transwell invasion experiments,miR-522-3p promoted the proliferation,migration and invasion of colon cancer cells and HUVECs by targeting downregulation of BMP5 expression.Its role in HUVECs neovascularization was confirmed by in vitro angiogenesis experiments.4.To explore whether CAFs exosomes promote colon cancer cell proliferation,metastasis,and angiogenesis by regulating BMP5expressionFirstly,CAFs/ NFs exosomes were used to incubate colon cancer cells and HUVECs respectively.The mRNA and protein expression of BMP5 in the above cells were detected by qRT-PCR and Western blot,and the effects of CAFs exosomes on BMP5 expression in colon cancer cells and HUVECs were investigated.Then CAFs exosomes were used to incubate colon cancer cells and HUVECs overexpressed with BMP5.Based on MTT cell proliferation,scratch healing and transwell chamber invasion experiments,CAFs exosome derived miR-522-3p promoted the proliferation,migration and invasion of colon cancer cells and HUVECs by targeting downregulation of BMP5 expression.Its role in HUVECs neovascularization was confirmed by in vitro angiogenesis experiments.Results:1.CAFs derived exosomes could be ingested by colon cancer cells and HUVECs,increasing the proliferation,migration,and invasion abilities of colon cancer cells and HUVECs,and inducing neovascularizationqRT-PCR results showed that mRNA expression levels of α-SMA,FSP-1,FAP and Vimentin in CAFs extracted from colon cancer tissues were significantly higher than NFs in the paired normal colon tissues.Immunofluorescence and Western blot results showed that α-SMA,FSP-1,FAP and Vimentin were positively expressed in the cytoplasm of CAFs,while almost no positive expression was found in the cytoplasm of NFs.These results confirmed the successful extraction of CAFs and NFs.Transmission electron microscopy and nanosight particle size detection results showed that CAFs/NFs exosomes were bilayer vesicles with a diameter of about 100 nm.Western blot results showed that CD63,CD81 and TSG101 proteins were positively expressed in CAFs/NFs exosomes.However,no obvious expression of the above marker proteins was found in the supernatant of CAFs/NFs after exosomes were removed.The above results confirmed the successful extraction of exosomes from CAFs/NFs.CAFs/NFs exosomes labeled with PKH26 red fluorescent film dye could be taken up by colon cancer cells and HUVECs.The results of MTT cell proliferation,scratch healing and transwell cell invasion showed that CAFs exosomes could promote the proliferation,migration and invasion of colon cancer cells and HUVECs in vitro,respectively.The results of in vitro angiogenesis showed that CAFs exosomes could promote the neovascularization of HUVECs.The results of subcutaneous tumor transplantation in nude mice showed that the volume,weight and growth rate of tumor mass in the CAFs exosome treated group were significantly higher than those in the NFs exosome treated group.Immunohistochemical results showed that the percentage of CD31 and Vimentin positive cells in the CAFs exosome treated group was significantly higher than that in the NFs exosome treated group.The results of caudal vein lung metastatic tumor model showed that the number of nodules on the lung tissue surface of mice treated with CAFs exosomes increased significantly,which was further verified by HE staining.2.CAFs-derived exosomal miR-522-3p promotes the growth,proliferation,invasion and metastasis of colon cancer cells and HUVECs,and induces neovasculationqRT-PCR results showed increased expression of miR-522-3p in colon cancer tissues,cell lines,and CAFs-derived exosomes,and the expression level of miR-522-3p in III/IV colon cancer patients was significantly higher than that in I/II patients.Meanwhile,in colon cancer patients with lymph node metastasis,the expression level of miR-522-3p was significantly higher than that of patients without lymph node metastasis.qRT-PCR results showed that compared with the cells treated with NFs exosomes,the expression level of miR-522-3p in colon cancer cells and HUVECs incubated with CAFs exosomes was significantly increased in a time-dependent manner.With the prolongation of CAFs exosomes,the expression level of miR-522-3p in the above cells was gradually increased.The results of MTT cell proliferation,scratch healing and transwell cell invasion showed that CAFs exosome derived miR-522-3p could promote the proliferation,migration and invasion of colon cancer cells and HUVECs in vitro.In vitro angiogenesis experiments showed that CAFs exosome derived miR-522-3p could promote neovascularization in HUVECs.The results of subcutaneous tumor transplantation experiment in nude mice showed that the volume,weight and growth rate of tumor mass in CAFs exosome miR-522-3p silencing group were significantly lower than those in the control group.Immunohistochemical results showed that compared with negative control group,the proportion of CD31 and Vimentin positive expression cells in CAFs exosome miR-522-3p silencing group was reduced.The results of caudal vein lung metastatic tumor model showed that the number of nodules on the lung tissue surface of mice in CAFs exosome miR-522-3p silencing group was significantly reduced,which was further verified by HE staining.3.BMP5 is a downstream target gene directly regulated by miR-522-3p.Mi R-522-3p promotes the proliferation,migration and invasion of colon cancer cells and HUVECs,and induces neovascularization by down-regulating the expression of BMP5The prediction results of miRNA target gene online databases Target Scan,RNAhybrid and miRanda showed that BMP5 was a potential downstream target gene of miR-522-3p.qRT-PCR results showed that up-regulation of miR-522-3p expression could inhibit the mRNA level of BMP5 in colon cancer cells and HUVECs,while down-regulation of miR-522-3p expression could increase the mRNA level of BMP5.Western blot results showed that overexpression of miR-522-3p could down-regulate the protein expression of BMP5 in colon cancer cells and HUVECs,while silencing the expression of miR-522-3p could increase the protein expression of BMP5.The results of double luciferase assay showed that overexpression of miR-522-3p in colon cancer cells and HUVECs could decrease the luciferase activity intensity of BMP5 3’-UTR vector,but had no significant effect on the luciferase activity intensity of mutant vector.RNA immunoprecipitation results showed that compared with the negative control group,the enrichment degree of BMP5 mRNA by Ago2 protein in the miR-522-3p overexpression group was significantly increased.There was no significant difference in the enrichment degree of BMP5 mRNA and miR-522-3p in the miR-522-3p overexpression group and the negative control group.The results of MTT cell proliferation,scratch healing and transwell cell invasion experiments showed that miR-522-3p promoted the proliferation,migration and invasion of colon cancer cells and HUVECs by down-regulating the expression of BMP5.In vitro angiogenesis experiments showed that miR-522-3p promoted neovascularization in HUVECs by downregulating the expression of BMP5.4.CAFs exosomes promote the proliferation,migration and invasion of colon cancer cells and HUVECs,and induce neovascularization by down-regulating the expression of BMP5.qRT-PCR results showed that the mRNA level of BMP5 in colon cancer cells and HUVECs in CAFs exosome treatment group was lower than that in NFs exosome treatment group.Western blot results also showed that the protein expression of BMP5 in CAFs exosome treatment group was significantly lower than that in NFs exosome treatment group.The results of MTT cell proliferation,scratch healing and transwell cell invasion showed that overexpression of BMP5 could partially reverse the CAFs exosome-mediated proliferation,migration and invasion of colon cancer cells and HUVECs.In vitro angiogenesis experiments showed that restoring BMP5 expression could partially reverse CAFs exosomemediated neovascularization in HUVECs.Conclusions:1.Cancer associated fibroblasts exosomal miR-522-3p could promote the proliferation,migration and invasion of colon cancer cells and HUVECs,and induce angiogenesis.2.miR-522-3p is increased in colon cancer tissues,cell lines and tumor associated fibroblasts exosomes,and its expression level is closely related to clinical staging and lymph node metastasis of colon cancer.3.In colon cancer and HUVECs,BMP5 is the downstream target gene directly regulated by miR-522-3p.4.Cancer associated fibroblasts exosomal miR-522-3p increases the proliferation,migration,and invasion ability of colon cancer cells and HUVECs,and induces angiogenesis by down-regulating the expression of BMP5. |
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