Research On The Mechanism Of Exosome-shuttling MiR-135 In The Livertropic Metastasis Microenvironment Of Colorectal Cancer

Background and Objects:Colorectal Cancer(CRC)is currently the third leading cause of Cancer death in the world.It is one of the leading causes of cancer death.Liver-tropic metastasis determines poor prognosis of CRC patients.CRC is occult and most diagnosed as advanced.The death of patients with colorectal cancer is mainly due to the occurrence of metastasis.Liver metastasis is frequent occurrence patients with colorectal cancer.Nevertheless,the precise mechanism of metastases to liver is unclear.Here,we aim to identify CRC-related miRNAs using microRNA microarray.Furthermore,large clinical samples of anther two independent CRC cohorts are used to verify the expression of the CRC-related miR-135a-5p.Aim to explore the biological effects of CRC-derived exosome-shutting miR-135a-5p and identify the target genes and downstream signaling pathways.We investigate the mechanism of miR-135a-5p and evaluate the diagnosis value of miR-135a-5p for patients with CRC.Methods:1.Identification of Differentially Expressed miRNAs in CRC Tissues1.1 A test set(consisting of 61 CRC cases,61 controls and 61 adenomas)and a validation set(consisting of 131 cases,controls and adenomas)were enrolled from the Jiangsu Tumor Hospital and the Affiliated Hospital of the Xuzhou Medical College,respectively,between2014 and 2015.Six pairs of CRC tissues(3 colon and 3 rectal carcinomas)and the corresponding adjacent noncancerous tissues,as well as six adenomas(all tubulovillous adenomas)were utilized for miRNA microarrays.1.2 The CRC and matched non-tumoral tissues from two independent cohorts were subjected to tissue microarray(TMA)construction.All of the patients were followed-up by a trained clinical specialist through in-person or family contact from the time of diagnosis to death,or last follow-up.The maximum follow-up time was 112.7 months and the median survival time(MST)was 75.0 months.Multivariate Cox regression analysis was used to evaluate the risk of the factors(age,gender,tumor location,tumor grade,TNM stages and miR-135a-5p expression level)for colorectal cancer patients.Furthermore,we investigated the relationship between mir-135a-5p expression and the metastasis of colorectal cancer patients.2.Biological Effects of miR-135a-5p on the Metastasis of CRC2.1 CRISPR-CAS9 was performed to knock out the expression of miR-135a-5p in CRC cells.Next,we detect the effect of miR-135a-5p and hypoxia in CRC cells by RNA-ISH assay.Transwell assay was used to detect the effect of miR-135a-5p for CRC cells invasion and migration ability.2.2 The exogenous miR-135a-5p mimic was transfected into CRC cells.To further evaluate the functional role of miR-135a-5p on the metastases in vivo,luciferase-labeled SW620 cells were subcutaneously implanted into the right flank of nude mouse.Luciferase intensity in tumor,liver,and lung tissues was also analyzed.3.The Regulatory Role of Exosome-Shuttling miR-135a-5p in CRC with Liver Metastasis3.1 Exosomes from the human serum(CRC patients or healthy donors)and cell culture medium were isolated and analyzed by transmission electron microscopy.Levels of exosomes and exosomal miR-135a-5p in the cell culture medium were determined by qRT-PCR.Stratified analysis of colorectal cancer samples was conducted to explore the correlation between the expression level of miR-135a-5p in exosomes of CRC patients and liver metastasis of CRC.Exosomes from healthy donors,CRC patients or CRC cells culture medium were subjected to education mice model.Liver sections of mice were subjected to PKH26(exosome marker)and F4-80(Kupffer cell marker)fluorescent staining.3.2 H&E staining was used to analyze the effects of exosomes and exosomal miR-135a-5p on mouse liver tissue.3.3 RNA-seq was performed to identify the differentially expressed genes between human liver single cells treated with exosomes from normal cultured SW620 miR-135a-5p KO cells and hypoxic SW620 WT cells.We found that CD30(Tumor Necrosis Factor Receptor Superfamily,Member 8,TNFRSF8)was targeted on the 3'UTR by miR-135a-5p based upon the prediction from the miRbase.Luciferase reporter assay was performed to detect the binding effect between CD30 and miR-135a-5p.Furthermore,we verify the regulation of exosomal miR-135a-5p on the expression of TRAF2,p65,TNF-?and IL-2.3.4 We used multiple databases to predict target genes of miR-135a-5p and finally LATS2that were mutual results in the nine databases.Luciferase reporter assay was performed to detect the binding effect between LATS2 and miR-135a-5p.ISH and ChIP assays were performed to detect the effect of miR-135a-5p on YAP1/TEAD/MMP axis.3.5 To further evaluate the functional role of exosomal miR-135a-5p in the clinic PDX models about the pre-metastatic niche progression,we constructed the liver metastasis mice model pre-educated with exosome from PDX CRC cells by artificially constructing the miR-135a-5p.The expression levels of miR-135a-5p,CD30,TNF a,LATS2,YAP1 and MMP7 in nude mouse liver tissues were detected by qRT-PCR assay.The exogenous miR-135a-5p mimic and inhibitor were transfected into PDX CRC cells.Transwell assay was performed to detect the PDX CRC cells migration and invasion abilities.The liver-metastasis model of CRC was constructed.Immunofluorescence assay was performed to confirm the exosomal miR-135a-5p,which contributed to the pre-metastasis niche process in CRC liver metastasis.4.The Diagnostic Value of Serum miR-135a-5p in CRC with Liver metastasisTotal RNA was extracted from serum of colorectal cancer.The expression levels of miR-135a-5p in serum of the same patient with CRC metastasis(2010)and without metastasis(2015)were detected by qRT-PCR assay.To investigate the association between miR-135a-5p and patients with early colorectal cancer(stage I/II),the TMA samples of colorectal cancer patients(stage I/II)were divided into the miR-135a-5p high-expression group and the miR-135a-5p low-expression group.The diagnostic value of serum miR-135a-5p in patients with CRC was determined by ROC curve.Results:1.Identification of Differentially Expressed miRNAs in CRC Tissues1.1 Differential Expression of miRNAs in CRC TissuesThe results showed that miR-135a-5p was the exclusive miRNA which progressively-upregulated from NOR to CRC.The AUC values of miR-135a-5p in the patients with CRC from Testing,Valadation and Combination cohorts were 0.73,0.77 and0.74,respectively(P<0.0001).It indicated that the expression level of miR-135a-5p in tumor tissue samples has a high efficacy in the diagnosis of colorectal cancer.1.2 The Association between the Expression Level of miR-135a-5p and the Risk of CRCConsistent with previous findings,the results showed that miR-135a-5p levels were significantly elevated in CRC tissues compared with matched non-tumor tissues.Additionally,multivariate Cox regression analysis indicated that miR-135a-5p might be an independent indicator of CRC prognosis;high miR-135a-5p levels were correlated with shorter OS time and unfavorable pathological parameters such as metastasis.2.Biological Effects of miR-135a-5p on the Metastasis of CRC2.1 The Functional Study of miR-135a-5p in CRC cellsThe results showed that miR-135a-5p expression levels in CRC cells were markedly upreg?Lated under hypoxic conditions,which no significant differences were detected in miR-135a-5p knockout cells.Hypoxia significantly increased the migration and invasion potency of CRC cells,and these effects were abrogated upon the deletion of miR-135a-5p.2.2 Biological Study of miR-135a-5p and Liver metastasis of CRC in VivoXenografts with higher miR-135a-5p levels(NC)grew faster than miR-135a-5p KO cells.Accordingly,reduced luciferase signals were detected in both xenografts and liver tissues in mice bearing SW620 cells with miR-135a-5p KO.However,no obvious difference in luciferase signaling was found in the lung,which is frequent site of distant metastasis in CRC.3.The Regulatory Role of Exosome-Shuttling miR-135a-5p in CRC with Liver Metastasis3.1 Expression Analysis of Exosomal miR-135a-5p in CRCExosomes from the human serum(healthy donors or CRC patients)were isolated and analyzed by TEM.Briefly,exosomes were dramatically increased in CRC patients.Moreover,CRC patients,especially those with liver metastasis,possessed significantly higher serum exosomal miR-135a-5p levels compared to those without liver metastasis.In addition,higher exosomal miR-135a-5p was observed when exposing SW620 cells under hypoxia conditions.Therefore,we confirmed the increase of exosomes and exosomal miR-135a-5p in CRC patients with liver metastasis and speculated that hypoxia may contribute to this process.In order to study whether the exosomes can be uptaken by the liver and the role of hypoxia in this process,we built up an exosomes-education mice model.The results showed that a higher number of PKH26-positive cells were detected in exosomes-education mice model from hypoxia-treated SW620 cells.Moreover,PKH26 was co-localized with F4-80,indicating that exosomes could be absorbed into the liver by KCs.3.2 Biological Effects of miR-135a-5p on the Liver Metastasis of CRCSerum exosomes collected from CRC patients with liver metastasis and cell culture medium were both used for establishing an education mice model;then SW620 cell was injected by sub-spleen or tail vein.The results showed that mice pre-educated with CRC exosomes exhibited more liver metastases and higher hepatic miR-135a-5p expression than those with healthy donors.Besides,no obvious analogous effect was found in lung.Similar results were also found in the mice pre-educated with exosomes from normoxia-and hypoxia-treated cell culture medium.These results suggested that exosomes containing miR-135a-5p plays an important role in the pre-metastasis niche formation in the liver.To further determine whether the expression level of miR-135a-5p affects the liver-tropic metastatic capacity of CRC,exosomes from CRC cells(miR-135a-5p KO and NC)cultured under hypoxic conditions were used to establish an education mice model.Lacking miR-135a-5p CRC cells failed to form liver metastasis after pre-niche with exosomes.3.3 Regulatory Mechanism of Exosome-Shuttling miR-135a-5p in CRC by repressing immunosuppression signaling.The expression of downstream target genes of miR-135a-5p was identified by RNA-seq analysis.A total of 8 genes were significantly downregulated in liver single cells treated with hypoxic WT exosomes.Among these,we found that only CD30 was targeted on the 3'UTR by miR-135a-5p based upon the prediction from the miRBase.We found that miR-135a-5p extremely blunt the CD30.The results of luciferase reporter assay showed that overexpression of miR-135a-5p significantly repressed the expression of CD30.Of note,the protein levels of CD30 were stimulated by miR-135a-5p depletion in liver from SW620-derived exosomes educated mice.CD30 can activate NF-?B signal pathway after interacting with TRAF2.The reduction of CD30 led to a decrease production of TNF-?and IL-2,which were down-regulators of NF-?B.Immunoprecipitation assays showed that exosomes education significantly reduced the binding capacity of CD30 and TRAF2,as well as CD30 and NF-?B since the exosomes education increased the expression levels of miR-135a-5p in mice model.Moreover,the liver metastases appeared after SW620 implantation in the spleen.Simultaneously,no significant changes in the expression of p65 and TRAF2 were found in liver tissues.Besides,the levels of serum TNF-?decreased after education,suggesting that the exosomes education provides a more conducive environment for the occurrence of CRC liver metastasis and renders the tumor cells more easily implanted in the liver.Importantly,21-day education with exosomes of miR-135a-5p deficiency markedly increased the number of CD4~+T cells and IL-2 levels in liver homogenates.3.4 The Regulatory Role of Exosome-Shuttling miR-135a-5p in the Adhesion Capacity of CRC cellsWe used multiple databases to predict target genes of miR-135a-5p and finally found SP3and LATS2 that were mutual results in the nine databases.Previou studies have pointed out that LATS2,a key gene for cell adhesion,is closely related to liver cancer and metastasis.The results of luciferase reporter assay indicated that overexpression of miR-135a-5p significantly repressed the expression of LATS2.LATS2 functions as a tumor suppressor kinase that promotes the adhesion ability of cancer cell by inactivating the oncogenic coactivator YAP1,which transcriptionally regulate the MMP family.Therefore,we first investigated whether miR-135a-5p reduced LATS2 and followed activated YAP1/TEAD/MMP complex to provide a microenvironment that CRC cell is easy to adhere to the liver in the process of pre-metastatic niche.We found that education with exosomes containing miR-135a-5p markedly reduced the hepatic expression of LATS2 in a mice model,while increased levels of YAP1.qRT-PCR analysis showed that exosomes education substantially increased the MMP7transcriptional levels.Furthermore,ChIP and re-ChIP assays showed that the YAP1-TEAD complex specifically bound to the promoter region of MMP7;once again,miR-135a-5p KO reversed the binding.Our res?Lts demonstrate that exosomal miR-135a-5p facilitates liver pre-metastatic niche by promoting cell adhesion,which is regulated by the LATS2-YAP1/TEAD-MMP7 pathway3.5 miR-135a-5p Promotes Liver metastasis in PDX mouse model through Exosome EducationTumor masses from CRC patients with different clinical characteristics were used for PDX model construction.Our data demonstrated that exosomes from M liver cells altered the expression levels of miR-135a-5p,CD30,TNF-?,LATS2,YAP1 and MMP7 in the liver,which further demonstrated that exosomal miR-135a-5p,could regulate the pathway of immunosuppression and cell adhesion in CRC patients with liver metastases.No metastasis with high miR-135a-5p level(N high)and liver metastasis(M liver).Exosomes expressed high miR-135a-5p levels(from N high and M liver cells)dramatically increased the cases of mice that developed liver metastasis and improved the tumor formation rate of liver metastasis,but not the mice educated by the exosomes from the N low cell.4.The Diagnostic Value of Serum miR-135a-5p in CRC with Liver metastasisThe expression level of serum miR-135a-5p CRC patients was significantly higher than the healthy control group,and the AUC value of miR-135a-5p in the serum was 0.83,0.71and 0.74 in different cohorts,respectively.The level of miR-135a-5p in serum of CRC patients after liver metastasis was significantly higher than that before liver metastasis.Moreover,patients with low expression of miR-135a-5p hardly developed into liver metastases,and there is no difference in their survival between treating with chemotherapy or not.According to those results,we found that appropriate chemotherapy is necessary for CRC patients with high expression of miR-135a-5p,which can significantly reduce the risk of liver metastasis and improve the prognosis.Conclusions:1.miR-135a-5p was the exclusive miRNA which was progressively-upregulated from NOR to CRC.The expression level of miR-135a-5p was correlated with shorter OS time and unfavorable pathological parameters such as metastasis.2.miR-135a-5p significantly increased the migration and invasion potency of CRC cells.It could promote the liver metastasis of CRC.3.Exosomal miR-135a-5p could be absorbed into the liver by KCs and promoted the liver metastasis of CRC,inducing the formation of pre-niche mechanism before liver metastasis.1)Exosomal miR-135a-5p inhibits the expression of TNF-?in serum and IL-2 in liver tissues of mice,thereby reducing the differentiation of CD4~+and playing an anti-tumor immune role.2)Exosomal miR-135a-5p facilitates liver pre-metastatic niche by promoting cell adhesion,which is regulated by the LATS2-YAP1/TEAD/MMP7 pathway.4.Serum miR-135a-5p has certain guiding significance for the auxiliary diagnosis and prognosis evaluation in CRC patients.