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MiR-382 functions as a tumor suppressor against esophageal squamous cell carcinoma.

ABSTRACT: To explore the effect of miR-382 on esophageal squamous cell carcinoma (ESCC) in vitro and its possible molecular mechanism.Eca109 cells derived from human ESCC and Het-1A cells derived from human normal esophageal epithelium were used. Lentivirus-mediated miR-382 was overexpressed in Eca109 cells. The effect of miR-382 on cell proliferation was evaluated by MTT and colony formation assay. For cell cycle analysis, cells were fixed and stained for 30 min with propidium iodide (PI) staining buffer containing 10 mg/mL PI and 100 mg/mL RNase A, and analyzed by BD FACSCalibur™ flow cytometer. For cell apoptosis assay, cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions and analyzed by a dual-laser flow cytometer. Cell invasion and migration abilities were determined through use of transwell chambers, non-coated or pre-coated with matrigel. Levels of proteins related to cell growth and migration were examined by western blotting.Endogenous miR-382 was down-regulated in Eca109 cells compared with Het-1A. Introduction of miR-382 not only significantly inhibited proliferation and colony formation, but also arrested cell cycle at the G2/M phase, as well as promoted apoptosis and autophagy in Eca109 cells. Migration, invasion and epithelial-mesenchymal transition of Eca109 cells were suppressed by overexpressing miR-382. Western blotting results showed that miR-382 inhibited the phosphorylation of mTOR and 4E-BP1.miR-382 functions as a tumor suppressor against ESCC development and metastasis, and could be considered as a potential drug source for the treatment of ESCC patients.

SUBMITTER: Feng J

PROVIDER: S-EPMC5483498 | biostudies-other | 2017 Jun

REPOSITORIES: biostudies-other

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Project description:BackgroundTo study miR-30b-5p expression in esophageal squamous cell carcinoma (ESCC) by comparisons between tumor tissues and matched adjacent non-cancerous tissues to elucidate the correlation between miR-30b-5p expression and ESCC clinical parameters, and to explore the signaling pathways associated with miR-30b-5p and key target genes.MethodsClinical data, cancer tissues, and adjacent non-cancerous tissues of 32 patients diagnosed with ESCC were collected from Taizhou Hospital of Zhejiang Province. The expression levels of miR-30b-5p were determined by real-time polymerase chain reaction (RT-PCR). mRNA data for ESCC tissues and normal tissues, and clinical materials of patients with ESCC were obtained from the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA). Associations between miR-30b-5p expression and clinical features of patients with ESCC and overall survival were explored. A bioinformatics analysis was performed to determine the pathways and key miR-30b-5p targets associated with ESCC. Additionally, a cytological experiment was performed to evaluate the biological functions of miR-30b-5p. Finally, correlations between miR-30b-5p and key targets involved in PI3K/Akt signaling pathways were validated by western blotting.ResultsThe expression level of miR-30b-5p in the 32 ESCC tissues was significantly lower than that in adjacent normal tissues (P<0.01) and was significantly disparate in the T stage, with higher expression in T1 than in T2 (P<0.05). Among the patients with higher expression levels of miR-30b-5p in ESCC tissues than in adjacent normal tissues, patients with higher expression of miR-30b-5p had a better prognosis (P<0.05). An analysis of gene chip data from the GEO database showed similar results. A gene enrichment analysis indicated a series of pathways that may be associated with the downregulation of miR-30b-5p, including focal adhesion, ECM-receptor interaction, and PI3K/Akt signaling pathways. Seven key target genes (PDGFRB, VIM, ITGA5, ACTN1, THBS2, SERPINE1, and RUNX2) were identified; these were found to be upregulated in ESCC tissues and were negatively correlated with miR-30b-5p. Functional experiments showed that miR-30b-5p attenuated migration (P<0.01) and invasion (P<0.05) in the Eca109 cell line. Moreover, the levels of ITGA5, PDGFRB, p-PI3K, and p-AKT, which are involved in the PI3K/Akt signaling pathway, were decreased in the miR-30b-5p-overexpressing Eca109 cell line.ConclusionsUpregulated miR-30b-5p may inhibit migration and invasion in ESCC by targeting ITGA5, PDGFRB, and signaling pathways, such as PI3K/Akt, involved in ESCC regulation. Our results indicate that miR-30b-5p plays an important role in the occurrence and progression of ESCC and is a potential therapeutic target.

Project description:BackgroundMicroRNAs have an important role in diverse biological processes including tumorigenesis. MiR-223 has been reported to be deregulated in several human cancer types. However, its biological role has not been functionally characterized in lung squamous cell carcinoma (LSCC). The following study investigates the role of miR-223-3p in LSCC growth and metastasis and its underlying mechanism.MethodsMicroRNA profiling analyses were conducted to determine differential miRNAs expression levels in LSCC tumor tissues that successfully formed xenografts in immunocompromised mice (XG) and failed tumor tissues (no-XG). RT-PCR and in situ hybridization (ISH) was performed to evaluate the expression of miR-223-3p in 12 paired adjacent normal tissues and LSCC specimens. Cell proliferation and migration were assessed by CCK-8, colony formation and Transwell assay, respectively. The role of miR-223-3p in LSCC tumorigenesis was examined using xenograft nude models. Bioinformatics analysis, Dual-luciferase reporter assays, Chromatin immunoprecipitation (ChIP) assay and Western blot analysis were used to identify the direct target of miR-223-3p and its interactions.ResultsMiR-223-3p was downregulated in LSCC tissues that successfully formed xenografts (XG) compared with tumor tissues that failed (no-XG), which was also significantly reduced in LSCC tissues compared with the adjacent normal tissues. Gain- and loss-of function experiments showed that miR-223-3p inhibited proliferation and migration in vitro. More importantly, miR-223-3p overexpression greatly suppressed tumor growth in vivo. Mechanistically, we found that mutant p53 bound to the promoter region of miR-223 and reduced its transcription. Meanwhile, p53 is a direct target of miR-223-3p. Thus, miR-223-3p regulated mutant p53 expression in a feedback loop that inhibited cell proliferation and migration.ConclusionsOur study identified miR-223-3p, as a tumor suppressor gene, markedly inhibited cell proliferation and migration via miR-223-3p-mutant p53 feedback loop, which suggested miR-223-3p might be a new therapeutic target in LSCC bearing p53 mutations.

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MiR-382 functions as a tumor suppressor against esophageal squamous cell carcinoma.

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