Integrated Analysis Identifies MicroRNA-195 As A Suppressor Of Hippo-YAP Pathway In Colorectal Cancer

Colorectal cancer(CRC)is the fourth leading cause of cancer death worldwide for several decades.The 5-year survival rate for localized stage CRC is 90%,whereas as CRC spreads to the regional lymph nodes or even to distant parts of the body,the 5-year survival rate plunges from 71%to 13%in the United States.The survival of the patients relies on early detection significantly,and the prognosis mainly depends on tumomode-metastasis(TNM)classification.Most of the patients are in poor prognosis while they are often diagnosed at an advanced stage.Although there are various methods to screen biomarkers in CRC,they are often lack of sensitivity or specificity.MicroRNAs(miRNAs)belong to a class of small noncoding RNAs that can regulate the expression level of target genes at the post-transcription phase.Growing evidences indicate that miRNAs can serve as ideal biomarkers for cancer detection and accurate predictions of prognosis,as well as targets for treatment.The number of miRNAs has increased largely from 2002 to 2014,and the latest miRBase version(Release 21,2014)contains 2588 mature human miRNA entries.There are some studies to search biomarkers by analyzing miRNA expression profiling between CRC tissues and paired neighboring noncancerous colorectal tissues,but the results of identifcation signifcantly expressed miRNAs are inconsistent or discrepant among each study.There are several reasons to be responsible for the inconsistent results,such as different technological detection platforms,various methods for data processing,constant discovery of new miRNAs and samples from different backgrounds.With progressively extensive approachability to incongruous results of colorectal cancer(CRC)miRNAs expression data,it is crucial that a paradigm shifts toward inclusion of a"pre-laboratory" integrated analysis step in biologic studies to expedite precision medicine and translational research.Taking into account of these considerations,we have included seven miRNAs expression data from seven different platforms in CRC using MetaDE method,which provided options for gene merging,matching across studies,and gene filtering.Fisher’s method was performed for statistical analysis of significance:modified t test and permutation method were used to extrapolate the P values.One-sided tests were applied to classify the upregulated or downregulated DE miRNAs.The results of integrated analysis of miRNAs expression datasets were validated in the TCGA datasets.MiR-195 is located on chromosome 17 short arm 13.1 zone,highly conserved.MiR-195 is a key member of the microRNA-15/16/195/424/497 family,which is downregulated in various cancers such as gastric cancer,liver cancer,breast cancer and bladder cancer.MiR-195 has been reported as a tumor suppressor in various types of cancer.It is a member of the miR-15/107 family,which is a member of the evolutionary conserved miRNA,which has been considered to have considerable prognostic potential.MiR-195-5p is a mature miRNA produced by miR-195 and needs further studies of its biological function in colorectal cancer.This topic mainly includes the four parts,as the following:Part 1.Integrated analysis of seven miRNAs expression datasets and common differential expression of colorectal cancerBackground Colorectal cancer is one of the most common malignancies in the world.Some studies have screened biomarkers by analyzing miRNA expression profiles between CRC tissues and adjacent non-cancerous colorectal tissues,but the results of identifying miRNAs that are significantly expressed in each study are inconsistent.To overcome these limitations,we need to integrate the results of miRNA expression profiles of colorectal cancer to eliminate the bias between studies.With the colorectal cancer(CRC)miRNA expression profiles are widely present,inconsistent results are more and more.The process and transformation path of miRNA multi-chip integration analysis were proposed and improved.On the one hand,the meta-analysis of microarray was successfully completed for the quality control of miRNA chip,differential expression gene identification and abundant pathway detection.On the other hand,the differential expression of the comprehensive analysis of the miRNAs expression data set was further validated in the TCGA data set.To further investigate whether the consistent miRNAs were screened for the overall survival of CRC patients,the significance of their diagnostic value and prognostic predictions was explored.Methods A public microarray repositories were curated to search through the PubMed,GEO and Array Express.Only original experimental studies that screened for different miRNAs between CRC and PANT in human were the first choice for inclusion.All datasets were normalized individually on the base-2 logarithm by Robust Multi-Array Average(RMA)and LIMMA package and annotated by converting different probe IDs to gene IDs.All miRNA names were standardized according to miRBase version 17 via miRBase Tracker and consisted with TCGA for subsequent validation.Any probe that did not map to a gene ID was removed as viral miRNAs or non-miRNA probes.To identify differentially expressed(DE)miRNAs between CRC and PANT,we used MetaOmics software executed as a MetaDE package.For integrated analysis,the mean and standard deviation(SD)filter thresholds were specified as 10%.Considering the different stringencies of the methods,Fisher’s method was performed for statistical analysis of significance:modified t test and permutation method were used to extrapolate the P values.One-sided tests were applied to classify the upregulated or downregulated DE miRNAs.A P value less than 0.05 was considered statistically significant for the DE miRNAs.The results of integrated analysis of miRNAs expression datasets were validated in the TCGA datasets.TCGA-COAD miRNA data and clinical data(level 3)of the corresponding patients(tumor and/or adjacent normal tissue)were downloaded from the TCGA Data portal.Kaplan-Meier method and Cox’s proportional hazards regression model was used to calculate overall survivals,and the differences were analyzed by log-rank test.Receiver operating characteristic(ROC)curve analysis was performed using the code of Mihaly by R Software(version 3.2.3;http://www.r-project.org).Result We located and manually curated 23 published and publicly available CRC datasets.After merging the datasets,32 differential miRNAs were identified.Twenty-five miRNAs were retained after filtering by the mean and SD(10%)to filter small expression intensities and small variation genes.We identified 21 miRNAs showing consistent DE patterns using moderated t-test by adding a fudging parameter,and Fisher’s method by summarizing-log(p-value)across studies and running 300 permutations to eliminate significant influence of the large number of samples.One-sided tests revealed 10 upregulated(highly expressed)miRNAs in CRC compared with PANT and 11 downregulated(lowly expressed)ones in CRC compared with PANT.After combining the effect size,we identified a total of 21 DE miRNAs that showed similar profiles of DE genes to those using Fisher’s method of combining P value(P<1E-19).We further validated the 11 DE miRNAs in TCGA-COAD.Only has-let-7a,has-miR-125b,has-miR-145,and has-miR-195 were significantly downregulated in CRC tumors,and each of these miRNAs could provide a high accuracy on CRC tissue classification as estimated by ROC curve analysis.We could attain a best performance on accuracy by a linear regression model built on a panel of the combined miRNAs(AUC = 1):CRC risk score = 5.091E_miR-125b-57.423E_miR-145-86.136E_let-7a +25.798E_miR-195+1712.826,where E_miR-n = Log 2(expression of microRNA).To further investigate whether the deregulated miRNAs correlate with the survivals of the CRC patients,we performed Kaplan-Meier and Cox’s proportional hazards regression model analysis and found that low miR-195 level was significantly correlated with poor overall survivals of CRC patients,suggesting the prognostic value of miR-195-5p in clinical CRC diagnosis.The downregulation of miR-195 has been consistently observed in the CRC patients with poor overall survivals,indicating that miR-195 may be functionally important in CRC pathogenesis.In contrast,low levels of let-7,miR-125b,or miR-145 in TCGA CRC tissues were correlated with improved overall survivals.Conclusion Compared with a single study of miRNA chip analysis,this study integrates a large sample of multi-center colorectal cancer miRNA chip,while the application of the public TCGA-COAD data were tested.In the CRC,only four miRNAs of has-let-7a,has-miR-125b,has-miR-145 and has-miR-195 were significantly down-regulated,and each miRNA could well distinguish between CRC tissue and Normal tissue,the accuracy of the classification by the receiver operating characteristic(ROC)curve was higher,the area under the curve(AUC)reached 0.887 to 0.989.We can obtain the best classification diagnosis model by establishing a linear regression model combining multiple miRNAs with an accuracy of 100%(AUC = 1).At the same time,it is suggested that there are obvious differences between miRNA and multi-platform,and the expression and quality control of different miRNAs should be emphasized.The combined diagnosis of four miRNAs has high early diagnosis and prognostic value.The expression of miR-195 was consistent with that of seven chip meta-analysis.At the same time,in the test set samples using the cox regression model proved to be independent prognostic factors.Thus,the miR-195 may be a novel candidate marker for the early diagnosis and prognosis of CRC.Part 2.Effects of miR-195-5p on the biological behavior of colorectal cancer cells in vitro and in vivoBackground In previous experiments,we have demonstrated that miR-195-5p has a low expression trend in colorectal cancer tissue and a poor prognosis in colorectal patients with low expression by seven-chip integration analysis and TCGA-COAD microarray,suggesting that miR-195-5p may play a role in the development and progression of colorectal cancer.Thus,we aim to explore the specific biological functions of miR-195-5p and the possible molecular mechanisms.The expression of miR-195-5p in colon cancer cell lines DLD1 and HCT116 was up-regulated and down-regulated by the transfection method.The proliferation,cloning,migration and invasion of miR-195-5p on colon cancer cells were observed in vitro.We demonstrated that miR-195-5p inhibits colorectal cancer cell proliferation,migration,invasion,and plays a role in tumor suppressor gene in the early cell function experiments.However,micro-environment stability is broken a complex biological process.Simple cell level experiments in vitro,not enough to prove that miR-195-5p in vivi can also inhibit the development of colorectal cancer.In order to further validate the in vivo effect of miR-195-5p,we used MiR-195-5p agomir to transfect colorectal cancer cells and observe the effect of miR-195-5p on tumor growth by nude mice.Methods In present work,the DLD1 and HCT116 cell lines with miR-195-5p expression were screened compared with normal intestinal epithelial cells.The colon cancer cell lines were transiently transfected into miR-195-5p simulant group and miR-195-5p Negative control group,miR-195-5p inhibitor group,miR-195-5p inhibitor negative control group,blank control group.The cell viability was observed under inverted phase contrast microscope.Brdu immunofluorescence staining,clonal formation assay,cell proliferation ability and MTT assay.were detected.Cell viability was measured by scratch test.Transwell chamber model was used to detect cell invasion and migration ability.BALB/c athymic nude mice(female,4-6-weeks old and 16-20 g)were purchased from Hubei Research Center of Laboratory Animal(Wuhan,China).All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of Wuhan University.To establish CRC cancer xenograft model,5 ×106 DLD1 cells were suspended in 150 μL PBS and inoculated subcutaneously into the flanks of the nude mice.After 8 days,the transplanted nude mice were randomly divided into two groups(n = 6 each).MiR-195-5p agomir or miR agomir NC(RiboBio Co.,Ltd,Guangzhou,China)was directly injected into the implanted tumor at the dose of 2 nmol/30μL PBS per mouse every 4 days for seven times.Tumor dimension was measured by length(L)and width(W)with a caliper every 4 days,and the volumes were calculated using the formula:(L × W2)/2.Mice were sacrificed by cervical dislocation after anaesthetized with 10%chloral hydrate at day 36,and the tumors were excised and snap-frozen for protein and RNA extraction.Results We started to interrogate the pathophysiological significance of miR-195-5p down-regulation in CRC and its underlying regulatory mechanisms.We transfected DLD1 and HCT116 cells with miR-195-5p mimic or miR mimic NC,and miR-195-5p inhibitor or miR-195-5p inhibitor NC,separately.We first examined the effect of miR-195-5p on proliferation of DLD1 and HCT116 cells.BrdU incorporation assay revealed that miR-195-5p inhibited DNA synthesis in DLD1 and HCT116 cells.In contrast,miR-195-5p inhibitor treatment could lift this inhibition.In addition,clonogenic assay showed that miR-195-5p mimic treatment decreased the clonogenic survivals of DLD1 and HCT116 cells compared with blank controls,while miR-195-5p inhibitor-treated DLD1 and HCT116 cells showed a reversed phenotype,suggesting miR-195-5p negatively regulates cancer cell proliferation.Supporting this notion,a colorimetric based cellular proliferation assay(i.e.MTT assay)showed consistent phenotypes when treating with miR-195-5p mimic or its inhibitor.Invasion and migration through the basement membrane are characteristics of metastatic cancer cells.We assessed the role of miR-195-5p on the migration and invasion of DLD1 and HCT116 cells.In the scratch wound healing assay,cell motility of cells was monitored at different time points after generation of the wound.The miR-195-5p-expressing cells migrated toward the wound at a much slower rate than the control or the cells treated by miR-195 inhibitors.In the transwell invasion and migration assay,we found that invasion and migration of the miR-195-5p-expressing cells was reduced,and this effect could be reversed by the miR-195-5p inhibitor.These results,taken together,clearly demonstrate that miR-195-5p negatively regulates invasion and migration of colon cancer cells.Both the volumes and weights of the tumors treated with miR-195-5p agomir were significantly reduced relative to those treated with miR agomir NC.Therefore,miR-195-5p significantly inhibits the tumorigenicity of DLD1 cells in vivo.Additionally,in consistent with the proposed regulations of miR-195-5p on Hippo pathway and EMT,expression patterns of YAP,Ki67,Vimentin,ZEB2 and E-cadherin in miR-195-5p-agomir-treated tumors were similar to the in vitro results,substantiating the tumor suppressor function of miR-195-5p in CRC tumorigenesis.Conclusions This study shows that overexpression of miR-195-5p in DLD1 and HCT116 cells inhibits cell growth,proliferation,clonal formation,invasion and migration.Inhibition of miR-195-5p function may contribute to abnormal cell proliferation,migration,and invasion.MiR-195-5p may exhibit different functions in different tissues,in different cell lines or in different micro-environments.miR-195-5p can inhibit the growth of colon cancer cells in vivo.It is confirmed that miR-195-5p is a tumor suppressor in colorectal cancer.Part 3.Prediction and verification of miR-195-5p target geneBackground MiR-195-5p was down-regulated in colorectal cancer tissues and colon cancer cell lines.Functional experiments showed that upregulation of miR-195-5p expression in colon cancer cell lines DLD1 and HCT116 significantly inhibited cell proliferation,inhibited cell invasion and migration,and inhibited the expression of miR-195-5p,and the opposite results were obtained in vitro.Therefore,miR-195-5p may play a role in tumor suppressor in the carcinogenesis of colorectal cancer.In this part of the study,we used GO,KEGG,PPI and other bioinformatics methods to predict miR-195-5p possible target genes,and double-luciferase reporter gene experiments to verify the depth mechanism and the combinated target.Methods The integrated gene was identified by four different target prediction algorithms.GO(Gene Ontology)analysis explains the main function of the miR-195-5p target gene according to the GO database.The Fisher exact test was used to calculate the significance level(P value)of each GO item to screen out the significant GO term of the common target gene enrichment of miR-195-5p.At the same time,the molecular function,biological process and cell location were summarized respectively.We used the R software for KEGG analysis.We used protein-protein interaction network(PPI)to further screen for possible target proteins and signaling pathways.QRT-PCR and western blot,luciferase reported that miR-195-5p binds directly to predicted binding sites in YAP1 3’-UTR.Results we performed KEGG pathway analysis to elucidate the potential biological functions of miR-195 integrated-signature.Interestingly,the top KEGG pathways enriched for the miR-195 targets were mainly associated with cancer-specific pathways,including Hippo signaling pathway,proteoglycans in cancer,viral carcinogenesis,pathways in cancer and prostate cancer.Meanwhile,protein-protein interaction network(PPI)suggested that YAP1 was the hub gene of the miR-195 targets.We also examined YAP1 expression in this sample cohort comprising the 60 human CRC and PANT.Both qRT-PCR and western blot results showed that YAP 1 was significantly upregulated in CRC compared with PANT.Significantly,miR-195-5p level was inversely correlated with the YAP1 level in the tumors as calculated by Pearson’s correlation(R2 = 0.531,P = 4.02E-11),suggesting that miR-195-5p may negatively regulate YAP1 in CRC.Of note,Kaplan-Meier and Cox’s proportional hazards regression model also revealed that patients with high levels of YAP 1 had shorter overall survivals.To test if YAP1 expression is regulated by miR-195-5p,we cloned the YAP1 3’-UTR into a luciferase reporter plasmid,and quantified expression of the adjacent hRluc coding region.For this purpose,we searched different databases for the potential targets of miR-195-5p that exhibited oncogenic properties.YAP1,which harbors two conserved miR-195-5p cognate sites,namely,162-168 and 1857-1862 of YAP1 3’-UTR,is a predicted target of miR-195-5p.The luciferase reporter plasmid pmiR-RB-REPORTTM-YAP1-3’-UTR or a mutant reporter plasmids carrying point mutations in the putative miR-195-5p binding sites was co-transfected with miR-195-5p mimics or miR mimic NC and inhibitors,separately.The results showed that miR-195-5p suppressed luciferase activity whereas miR-195-5p inhibitor could promote luciferase activity for reporter plasmid carrying wild type YAP1 3’-UTR(P<0.05),but no significant effects were observed for reporter plasmid carrying mutant YAP13’-UTR(i.e.,pmiR-RB-REPORTTM-mut-YAP1-3’-UTR).These results suggest that miR-195-5p binds directly to the predicted binding site(s)in the YAP1 3’-UTR and negatively regulates YAP1 expression.To confirm the direct regulation of miR-195-5p on YAP1 expression,we transfected DLD1 and HCT116 cells with miR-195-5p mimic,miR mimic NC,miR-195-5p inhibitor,and miR-195-5p inhibitor NC.Both qRT-PCR and Western blotting revealed that YAP1 level was reduced in miR-195-5p-expressing cells,while its level was restored in miR-195-5p inhibitor-treated cells.The conversion from epithelial cells to mesenchymal cells,characterized by spindle-type cell morphology in HCT116 cells which treated with miR-195-5p inhibitor.So far,the results suggest that miR-195-5p negatively regulates YAP1 levels in CRC cells.Additionally,expressions of TAZ,Vimentin,ZEB2 and SMAD3 protein were also negatively regulated by miR-195-5p,while E-cadherin was positively regulated by miR-195-5p,suggesting the negative regulations of miR-195-5p on the Hippo pathway and EMT in CRC cells.Conclusions miR-195-5p can directly regulate the expression of YAP 1 by targeting its YAP1 mRNA 3’-UTR.The ectopic expression of miR-195-5p can reduce cell migration and invasion and promote the expression of E-cadherin in EMT markers.