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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Journal of Hematology & Oncology 1/2017

Integrated analysis identifies microRNA-195 as a suppressor of Hippo-YAP pathway in colorectal cancer

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2017
Autoren:
Min Sun, Haibin Song, Shuyi Wang, Chunxiao Zhang, Liang Zheng, Fangfang Chen, Dongdong Shi, Yuanyuan Chen, Chaogang Yang, Zhenxian Xiang, Qing Liu, Chen Wei, Bin Xiong
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13045-017-0445-8) contains supplementary material, which is available to authorized users.

Abstract

Background

With persistent inconsistencies in colorectal cancer (CRC) miRNAs expression data, it is crucial to shift toward inclusion of a “pre-laboratory” integrated analysis to expedite effective precision medicine and translational research. Aberrant expression of hsa-miRNA-195 (miR-195) which is distinguished as a clinically noteworthy miRNA has previously been observed in multiple cancers, yet its role in CRC remains unclear.

Methods

In this study, we performed an integrated analysis of seven CRC miRNAs expression datasets. The expression of miR-195 was validated in The Cancer Genome Atlas (TCGA) datasets, and an independent validation sample cohort. Colon cancer cells were transfected with miR-195 mimic and inhibitor, after which cell proliferation, colony formation, migration, invasion, and dual luciferase reporter were assayed. Xenograft mouse models were used to determine the role of miR-195 in CRC tumorigenicity in vivo.

Results

Four downregulated miRNAs (hsa-let-7a, hsa-miR-125b, hsa-miR-145, and hsa-miR-195) were demonstrated to be potentially useful diagnostic markers in the clinical setting. CRC patients with a decreased level of miR-195-5p in tumor tissues had significantly shortened survival as revealed by the TCGA colon adenocarcinoma (COAD) dataset and our CRC cohort. Overexpression of miR-195-5p in DLD1 and HCT116 cells repressed cell growth, colony formation, invasion, and migration. Inhibition of miR-195-5p function contributed to aberrant cell proliferation, migration, invasion, and epithelial mesenchymal transition (EMT). We identified miR-195-5p binding sites within the 3’-untranslated region (3′-UTR) of the human yes-associated protein (YAP) mRNA. YAP1 expression was downregulated after miR-195-5p treatment by qRT-PCR analysis and western blot.

Conclusions

Four downregulated miRNAs were shown to be prime candidates for a panel of biomarkers with sufficient diagnostic accuracy for CRC in a clinical setting. Our integrated microRNA profiling approach identified miR-195-5p independently associated with prognosis in CRC. Our results demonstrated that miR-195-5p was a potent suppressor of YAP1, and miR-195-5p-mediated downregulation of YAP1 significantly reduced tumor development in a mouse CRC xenograft model. In the clinic, miR-195-5p can serve as a prognostic marker to predict the outcome of the CRC patients.

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Zusatzmaterial
Additional file 1: Table S1. The full-detailed search strategy and searching terms through PubMed, GEO, and Array Express databases. (XLS 26 kb)
13045_2017_445_MOESM1_ESM.xls
Additional file 2: Supplemental R script. The R code for receiver operating characteristic curve analysis. (DOCX 15 kb)
13045_2017_445_MOESM2_ESM.docx
Additional file 3: Supplemental Experimental Procedures. (DOCX 27 kb)
13045_2017_445_MOESM3_ESM.docx
Additional file 4: Table S2. The 23 full data sets which were potentially relevant studies assessed for eligibility. Contributor, assay type, platform, source accession, and PMID were listed alongside sample size (normal/tumor). (XLS 34 kb)
13045_2017_445_MOESM4_ESM.xls
Additional file 5: Table S3. Datasets used in final quantitative synthesis and integrated analysis. Contributor, assay type, platform, source accession, and PMID were listed alongside sample size (normal/tumor). (XLS 27 kb)
13045_2017_445_MOESM5_ESM.xls
Additional file 6: Table S4. Gene-level results in 10 overexpressed differentiated expressed miRNAs in CRC compared with ANT. Statistical measures of integrated analyses are listed. (XLS 24 kb)
13045_2017_445_MOESM6_ESM.xls
Additional file 7: Table S5. Gene-level results in 11 underexpressed differentiated expressed miRNAs in CRC compared with ANT. Statistical measures of integrated analyses are listed. (XLS 24 kb)
13045_2017_445_MOESM7_ESM.xls
Additional file 8: Table S6. MiRNA-level results in integrated analysis of seven datasets. Statistical measures of integrated analyses are listed. (XLS 32 kb)
13045_2017_445_MOESM8_ESM.xls
Additional file 9 Table S7. 11 underexpressed miRNAs validation results in TCGA-COAD dataset. (XLS 96 kb)
13045_2017_445_MOESM9_ESM.xls
Additional file 10: Figure S1. The four validated downregulated miRNAs may serve as significant prognostic markers in CRC classification. (A) Receiver operating characteristic (ROC) curve analysis were showed a high performance classification accuracy of CRC tissue and normal tissue in TCGA dataset. (B) Kaplan-Meier survival curves of overall survival in TCGA Cohort according to the ratio of miR-195, let-7a, miR-125b or miR-145 miRNAs level in each tumor sample compared to its control, the median value of this ratio in each cohort was chosen as the cut-off point. (PDF 1081 kb)
13045_2017_445_MOESM10_ESM.pdf
Additional file 11: Table S8. Seventy-one consensus target genes were summarized by 4 different target prediction algorithms. (XLS 150 kb)
13045_2017_445_MOESM11_ESM.xls
Additional file 12: Table S9. Enrichment analysis of predicted miR-195 targets in KEGG cell signaling pathway database. (XLS 25 kb)
13045_2017_445_MOESM12_ESM.xls
Additional file 13: Figure S2. KEGG cell signaling pathway was shown for HIPPO pathway. The most significantly enriched by the predicted targets of miR-195 (P = 6.47E-05). Red frame shows the predicted miR-195 targets. (TIF 83 kb)
13045_2017_445_MOESM13_ESM.tif
Additional file 14: Figure S3. Protein-protein interaction network of the consensus target gene of miR-195-5p. (TIF 2196 kb)
13045_2017_445_MOESM14_ESM.tif
Additional file 15: Figure S4. HCT116 cell lines were showed in cell morphology after transfection miR-195-5p inhibitor (10 nM) after 7–10 days. The change in morphology was observed under a light microscope. HCT116 treated with miR-195-5p inhibitor showed mesenchymal features. (TIF 3202 kb)
13045_2017_445_MOESM15_ESM.tif
Additional file 16: Figure S5. Silence of YAP1 expression inhibits colon cancer cell growth, invasion, migration and rescue assay. A. CCK8 assays of DLD1 and HCT116 cells after transfected (un-transfected) with siRNA YAP1. B-C. Shown are representative photomicrographs of colony formation assay after transfected with (without) siRNA YAP1 for eight days. D. Shown are representative photomicrographs of transwell invasion assay after transfected with (without) siRNA YAP1. E. Shown are representative photomicrographs of transwell migration assay after transfected with (without) siRNA YAP1. F. Expression of YAP1 mRNA in siRNA YAP1 treated and blank DLD1 cell. G. Western blot of YAP, TAZ, E-cadherin, Vimentin, ZEB2 protein in siRNA YAP1 treated and blank DLD1 cell. Assays were performed in triplicate. Means ± SD are shown. Statistical analysis was conducted using student’s t-test. *P < 0.05. **P < 0.01. ***P < 0.001. (TIF 3081 kb)
13045_2017_445_MOESM16_ESM.tif
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