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KRAS Mutation-Responsive miR-139-5p inhibits Colorectal Cancer Progression and is repressed by Wnt Signaling.

ABSTRACT: Introduction: Colorectal cancer (CRC) frequently harbors KRAS mutations that result in chemoresistance and metastasis. MicroRNAs (miRNAs) are usually dysregulated and play important regulatory roles in tumor progression. However, the KRAS mutation-responsive miRNA profile in CRC remains uninvestigated.Methods: miR-139-5p was identified and evaluated by small RNA sequencing, qRT-PCR and in situ hybridization. The roles of miR-139-5p in CRC cells with and without KRAS mutation were determined by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry and transwell assays in vitro and by tumorigenesis and metastasis assays in vivo. Microarrays followed by bioinformatic analyses, luciferase reporter assays and Western blotting were applied for mechanistic studies.Results: miR-139-5p was significantly downregulated in KRAS-mutated CRC cells and tissues compared with their wild-type counterparts. Low miR-139-5p expression was associated with aggressive phenotypes and poor prognosis in CRC patients. miR-139-5p overexpression inhibited CRC cell proliferation, migration and invasion in vitro, sensitized tumors to chemotherapy, and impaired tumor growth and metastasis in vivo. Transcriptomic profiling identified multiple modulators in the Ras (JUN and FOS) and Wnt (CTNNB1 and DVL1) signaling pathways and the epithelial-to-mesenchymal transition (EMT) process (ZEB1) as direct targets of miR-139-5p, and inverse correlations were confirmed in CRC clinical tissues. Aberrantly activated Wnt signaling in KRAS-mutant cells was demonstrated to transcriptionally repress miR-139-5p through TCF4, forming a miR-139-5p/Wnt signaling double-negative feedback loop.Conclusions: We identified miR-139-5p as a KRAS-responsive miRNA and demonstrated its involvement in CRC progression. KRAS mutation disrupted the miR-139-5p/Wnt signaling reciprocal negative feedback mechanism, which might cause miR-139-5p downregulation and derepression of oncogenic signaling pathways and EMT. These results reveal a transcriptional regulatory mode of KRAS-driven malignant transformation and highlight miR-139-5p as a novel regulator of crosstalk between the Ras and Wnt signaling pathways in CRC.

SUBMITTER: Du F

PROVIDER: S-EPMC7330859 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Project description:Glioma is an extremely aggressive malignant neoplasm of the central nervous system. MicroRNA (miRNA) are known to bind to specific target mRNA to regulate post-transcriptional gene expression and are, therefore, currently regarded as promising biomarkers for glioma diagnosis and prognosis. The aim of the present study was to examine the pathogenesis and potential molecular markers of glioma by comparing the differential expression of miRNA and mRNA between glioma tissue and peritumor brain tissue. We explored the impact of screened core miRNA and mRNA on cell proliferation, invasion, and migration of glioma. An miRNA expression profile dataset (GSE90603) and a transcriptome profile dataset (GSE90598) were downloaded from combined miRNA-mRNA microarray chips in the Gene Expression Omnibus (GEO) database. Overall, 59 differentially expressed miRNAs (DEMs) and 419 differentially expressed genes (DEGs) were identified using the R limma software package. FunRich software was used to predict DEM target genes and miRNA-gene pairs, and Perl software was used to find overlapping genes between DEGs and DEM target genes. There were 129 overlapping genes regulated by nine miRNAs between target genes of the DEMs and DEGs. The Chinese Glioma Genome Atlas(CGGA) was analyzed in order to identify miRNAs with diagnostic and prognostic significance. MiR-139-5p, miR-137, and miR-338-3p were validated to be significantly linked to prognosis in glioma patients. Finally, we validated that miR-139-5p affected glioma malignant biological behavior via targeting gamma-aminobutyric acid A receptor alpha 1(GABRA1) through rescue experiments. Low miR-139-5p expression was correlated with survival probability and World Health Organization (WHO) grade. MiR-139-5p overexpression inhibited cell proliferation, migration, and invasion of glioma in vitro. GABRA1 was identified as a functional downstream target of miR-139-5p. Decreased GABRA1 expression was related to similar biological roles as miR-139-5p overexpression while upregulation of GABRA1 effectively reversed the inhibition effects of miR-139-5p. These results demonstrate a novel axis for miR-139-5p/GABRA1 in glioma progression and provide potential prognostic predictors and therapeutic target for glioma patients.

Project description:BackgroundGlioma metastasis, invasion, epithelial-mesenchymal transition (EMT) and chemoresistance indicate poor prognosis. Accumulating evidence reveals that Notch1 is an important factor in tumour progression. However, the role of Notch1 in glioma EMT and associated microRNAs (miRNAs) with the Notch pathway remain controversial.MethodsUtilizing cBioPortal database to examine the gene signature of NOTCH1 (encoding Notch1), CDH2 (encoding N-cadherin) and SNAI1 (encoding Snail-1) in disease-free survival (DFS) and overall survival (OS). We analyzed the Notch1 expression from Oncomine. We used Western blot (WB), immunohistochemistry (IHC) and immunofluorescence to determine protein levels. Transcription was evaluated by quantitative real-time (qRT)-PCR. siRNA and lentivirus were used to knock down Notch1 and overexpress miR-139-5p, respectively. The migration and invasion of glioma cells were assessed by wound healing and transwell assays. Luciferase reporter assays were utilized to verify the relationship between Notch1 and miR-139-5p. A U87-implanted intracranial model was used to study the effect of miR-139-5p on tumour growth and Notch1 suppression efficacy or EMT reversion.ResultsIt revealed the association of NOTCH1, CDH2, SNAI1 genomic alterations with decreases in DFS and OS. Notch1 was upregulated in classical and proneural subtypes of GBM, and associated with tumour grade. Notch1 inhibition suppressed the biological behaviours of metastasis, invasion and EMT. Notch1 was identified as a novel direct target of miR-139-5p. MiR-139-5p overexpression partially phenocopied Notch1 siRNA, whereas the forced expression of Notch1 reversed the effects of miR-139-5p on the invasion of glioma. Moreover, intracranial tumourigenicity and EMT behaviours were reduced by the introduction of miR-139-5p and partially mediated by the decreased Notch1 expression.ConclusionsmiR-139-5p was identified as a tumour suppressor by negatively targeting Notch1, and this work suggests a possible molecular mechanism of the miR-139/Notch1/EMT axis for glioma treatment.

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KRAS Mutation-Responsive miR-139-5p inhibits Colorectal Cancer Progression and is repressed by Wnt Signaling.

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