CAFs Promote The Stemness And Chemoresistance Of Colorectal Cancer By Transferring Exosomal LncRNA H19

Colorectal cancer is a common malignancy of the digestive tract,and its occurrence is thought to be closely related to diet,environment,and genetics.With the rapid changes in eating habits and living environment,the incidence of colorectal cancer in China is also rising.Although the technology for the treatment of cancer continues to develop,more than 40%of patients with colorectal cancer inevitably die from the recurrence and metastasis of the tumor.Chemotherapy is one of the important means to control cancers including colorectal cancer.Multidrug resistance of tumor cells is the main reason for clinical failure of chemotherapy.Therefore,the molecular mechanism underlying the multidrug resistance,invasion and metastasis of colorectal cancer is of vital importance for the clinical treatment of colorectal cancer.Long noncoding RNA(lncRNAs)is a heterogeneous class of transcripts longer than 200 nucleotides and limited protein-coding potential.LncRNAs participate in various cellular or biological processes by interacting with various biological macromolecules such as DNA,chromatin,proteins,and RNAs(including mRNAs,microRNAs,and lncRNAs).Recent studies have shown that IncRNAs are abnormally expressed in a variety of human cancers and can participate in important cellular and molecular activities such as epigenetic modifications,transcriptional regulation,RNA editing,and protein translation and modification,and regulate the occurrence,growth,apoptosis,and invasion of tumors.LncRNAs are differentially expressed in colorectal cancer and are associated with colorectal cancer growth,distant metastasis,and overall survival.They can also be used as molecular markers to predict the prognosis of colorectal cancer patients.LncRNAs not only affect the tumor cells themselves,but also affect other cells in the tumor microenvironment.The tumor microenvironment is a local microenvironment composed of tumor cells infiltrated by immune cells,stromal cells,and secreted active media.The subset of cells with stem cell-like characteristics in tumor cells is called cancer stem cells.This subset of cells plays a key role in the occurrence,recurrence,metastasis and chemotherapy resistance of cancer.The tumor-associated fibroblasts(CAFs)are the major stromal cells in the tumor microenvironment.They play an important role in tumor proliferation,metastasis,angiogenesis and chemotherapy resistance through paracrine processes.However,the relationship between lncRNAs and CAFs and their role in the development of colorectal cancer,maintenance of sternness,and resistance of chemotherapeutic agents have not been known so far.In this study,we first performed whole genome transcriptome profiling by RNA sequencing to analyze the tissue samples of the induced colorectal cancer mouse model,and screened out the differentially expressed IncRNA H19.Meanwhile,we analyzed the expression of lncRNA H19 in different stages of colorectal cancer in the TCGA database,and found the expression of LncRNA H19 correlated with the sternness of colorectal cancer.The effect of LncRNA H19 on colorectal cancer sternness and oxaliplatin resistance was confirmed by in vitro and in vivo experiments.We further explored the upstream regulation mechanism of IncRNA H19 in colorectal cancer,and revealed the specific molecular mechanism of IncRNA H19 downstream regulation of colorectal cancer sternness and drug resistance.This study has deepened our understanding of the occurrence of colorectal cancer,the maintenance of colorectal cancer sternness and the tolerance of chemotherapy drugs,and provided new ideas and targets for the clinical treatment of colorectal cancer.1.LncRNA H19 promotes sternness and oxaliplatin resistance of colorectal cancer.In this study,we first established a mouse model of induced colorectal cancer(CAC)and sequenced the whole transcriptome of the tissue and screened the differentially expressed IncRNA H19.Pathway analysis of the model sample mRNA sequencing results revealed that there are some key signal pathways such as cell adhesion molecules,xenobiotic metabolism,inflammatory bowel disease,and drug metabolism.Meantime,we analyzed sequencing data for different colorectal cancer stages(stage I,stage II,stage III,stage IV and metastatic stage IV)of the lncRNA H19 in the TCGA database.The results showed that lncRNA H19 was significantly higher in the colorectal cancer data set(average TPM = 18.32-72.47)than normal tissue(average TPM = 2.85)(adjusted P-values 1.33E-08-4.76E-03).We found that lncRNA H19 levels were significantly upregulated in the early stage of colorectal cancer and further increased with the development of colorectal cancer.In addition,the expression of lncRNA H19 in metastatic stage IV colorectal cancer tissue was also increased compared to stage IV.At the same time,we further validated the high expression of lncRNA H19 in colorectal cancer samples and found that there is a correlation between the lncRNA H19 level and the expression of the sternness marker of colorectal cancer.To further explore the effect of IncRNA H19 on the sternness of colorectal cancer cells,we interfered with or overexpressed the lncRNA H19 in colorectal cancer cells.The results showed that overexpression of LncRNA H19 significantly promoted the expression of pluripotent transcription factors and cancer stem cell markers and up-regulated the proportion of ALDH1high cells in colorectal cancer.Further functional analysis revealed that the overexpression of lncRNA H19 significantly promoted the sphere-propagating capacity.Conversely,lncRNA H19 interference significantly inhibited the sphere-propagating capacity.Given the important role of cancer stem cells in drug resistance,we conclude that lncRNAH19 can promote resistance to chemotherapeutic drugs of colorectal cancer.We evaluated the chemosensitivity of colorectal cancer cells through conventional first-line chemotherapy drugs(oxaliplatin)widely used in colorectal cancer.The results showed that overexpression of lncRNA H19 significantly enhanced the oxaliplatin resistance of colorectal cancer cells.Next,we constructed a colorectal cancer cell line that stably interferes with lncRNA H19,and explored the functional changes in sternness of colorectal cancer cells by limiting dilution tumorigenesis assay in vivo.The results showed that knockdown of H19 significantly reduced the frequency of tumor initiation and attenuated the in vivo tumorigenicity of colorectal cancer cells.To explore the effect of lncRNA H19 on oxaliplatin resistance in colorectal cancer in vivo,we established a mouse xenograft model using IncRNA H19 stably low-expressed colorectal cancer cells.The results showed that knockdown of lncRNA H19 significantly inhibited tumor growth under the treatment of oxaliplatin.In addition,knockdown of lncRNA H19 significantly reduced the number of Ki-67 positive cells,increased the expression of cleaved caspase-3 in tissues,and promoted apoptosis in tumor tissues.These results show that IncRNA H19 has the effect of promoting colorectal cancer cell sternness and chemotherapeutic drug resistance.2.CAFs transfer exosomal IncRNA H19 to promote the sternness and oxaliplatin resistance in colorectal cancerTo further explore the role of IncRNA H19 in colorectal cancer,we assessed the localization and expression of lncRNA H19 in cancer nests(pan-CK-positive)and tumor stroma(a-SMA-positive)by RNA-FISH and immunofluorescence assays.Interestingly,both colorectal cancer and tumor stroma of colorectal cancer showed high lncRNA H19 levels,and lncRNA H19 was found to be significantly higher in the tumor stroma than cancer nests.To further confirm the expression of IncRNA H19 in tumor stroma,we isolated CAFs and normal fibroblasts(NFs)from colorectal cancer tissues and adjacent normal colorectal tissues,and identified purity and phenotype.LncRNA H19 in primary CAFs of colorectal cancer was significantly higher than that of NFs.We used CAFs-derived conditioned media(CAF-CM)and NFs-derived conditioned medium(NF-CM)to treat human colorectal cancer cells and to detect colorectal cancer stem cell markers and functions.The results showed that compared with NF-CM treatment,CAF-CM treatment significantly increased the proportion of ALDH1high cells in colorectal cancer cells,enhanced the sphere-propagating capacity of colorectal cancer cells,and reduced the sensitivity of colorectal cancer cells to oxaliplatin.The exosome secretion was blocked by exosome inhibitor(GW4869).It was found that CAFs might enhance stem cell characteristics and drug resistance of colorectal cancer cells by releasing exosomes.In addition,we found that CAFs upregulated IncRNA H19 levels in colorectal cancer cells and demonstrated that exosomes were involved in this process.At the same time,we excluded the involvement of the TGF-P pathway through the use of inhibitors of the TGF-βreceptor I.To further confirm the role of CAFs-derived exosomes,we isolated the exosomes derived from CAFs and NFs,respectively,and identified them.Next,we demonstrated the effective uptake of colorectal cancer cells into CAFs-derived exosomes.Since CAFs are ingested by colorectal cancer cells through secretion of exosomes and increase cellular IncRNA H19 levels,and CAFs highly express IncRNA H19,we suspect whether the exosome secreted by CAFs carries IncRNA H19.Surprisingly,we detected high levels of IncRNA H19 in CAFs-derived exosomes,and the corresponding NFs derived exosome had the lower IncRNA H19 levels.Further experiments found that both CAFs-derived exosomes and colorectal cancer cells can significantly increase intracellular IncRNA H19 levels.These results indicate that IncRNA H19 is enriched in CAFs-derived exosomes and can be transferred from CAFs to colorectal cancer cells to regulate the IncRNA H19 levels.Next we continued to explore the role of CAFs-derived exosomes in colorectal cancer.The colorectal cancer cells were co-incubated with exosomes of CAFs or NFs cells,after which colorectal cancer cells were evaluated for stemness and oxaliplatin resistance.Compared to the exosomes of NFs,CAFs-derived exosomes increased the sphere-propagating capacity,increased the proportion of ALDHhigh cells,and promoted drug resistance to oxaliplatin.In vivo resistance experiments showed that oxaliplatin treatment alone had a significant effect in inhibiting tumor growth,whereas CAFs-derived exosomes treatment attenuated and reversed the effect of oxaliplatin on tumors.Further experiments showed that CAFs-derived exosomes treatment also enhanced Ki-67 expression,reduced oxaliplatin-induced caspase-3 cleavage expression and attenuated oxaliplatin-induced apoptosis in tumor tissues.In addition,CAFs-derived exosomes treatment significantly increased lncRNA H19 levels in tumor tissues compared to NFs-derived exosome-treated groups.The above results indicate that CAFs-derived exosomes promote sternness and oxaliplatin resistance of colorectal cancer by transferring lncRNA H19.3.The mechanism of Wnt/β-catenin pathway activation by CAFs-derived exosomes in colorectal cancerIn view of the important role of Wnt/β-catenin pathway in intestinal stem cell function,maintenance of cancer stem cell and resistance to chemotherapeutic drugs,we speculated that Wnt/β-catenin pathway is regulated by lncRNA H19 in colorectal cancer.First,we found that overexpression of IncRNA H19 up-regulated Wnt luciferase activity(TOPFlash)and significantly increased β-catenin protein levels.We further found that CAFs-derived exosomes significantly promote the expression ofβ-catenin in colorectal cancer cells.We also analyzed the expression of β-catenin in tumor tissue in the two xenograft models.Compared to the NFs-derived exosomes treatment group,CAFs-derived exosomes treatment significantly increased p-catenin protein levels in tumor tissues.Knockdown of lncRNA H19 significantly reducedβ-catenin levels in tumor tissue.These results indicate that exosomal transfer of lncRNA H19 activates the Wnt/β-catenin pathway in colorectal cancer.Increasing data showed that H19 could regulate many biological processes as endogenous sponges for miRNAs.RNA immunoprecipitation assay demonstrated that lncRNA H19 may interact with miRNA in colorectal cancer cells.Sequence analysis and bioinformatics predictive analysis revealed putative binding sites between lncRNA H19 and miR-141,indicating that miR-141 has ceRNA potential for lncRNA H19.Therefore,we next confirmed that IncRNA H19 can directly interact with miR-141 through the predicted binding site through dual luciferase reporter assays.We also confirmed that β-catenin was a target of miR-141 in colorectal cancer cells and demonstrated that IncRNA H19 regulates the expression of β-catenin by interacting with miR-141.In addition,we have found that miR-141 can regulate the function of sternness of colorectal cancer cells.Finally,further analysis of colorectal cancer specimens revealed that the expression of lncRNA H19 was positively correlated with the immunostaining score of(3-catenin.Clinical sample results also confirm our conclusions on colorectal cancer cells and animal models.The above results show that CAFs-derived exosomes transfer IncRNA H19 to colorectal cancer cells.LncRNA H19 activates the Wnt/β-catenin pathway of colorectal cancer through competitive adsorption of miR-141,which promotes the stemness and drug resistance of colorectal cancer cells.Collectively,we characterized the roles of lncRNAs H19 and CAFs in CRC stemness and oxaliplatin resistance and delineated the underlying molecular mechanism by which CAFs-derived exosomes activated the Wnt/p-catenin signaling by transferring exosomal IncRNA H19.In addition,our results suggest that the combination of lncRNA H19 or Wnt/β-catenin targeting therapy with chemotherapeutics drugs may be effective in inhibiting the sternness of cancer stem cells and overcoming the drug resistance of colorectal cancer.