| Backgroud:Esophageal cancer is the eighth most common cancer and the sixth leading cause of death from cancer worldwide. Esophageal squamous cell carcinoma (ESCC) is predominant in China. Patients present with dysphagia at a late stage as a result the five-year survival rate is still less than25%. However, the early-stage cancers cofined to the mucosa or submucosa have an overall survival excess of80%. Therefore, early diagnosis is critical for treatment of ESCC.MicroRNAs (miRNAs) are a class of endogenously expressed small noncoding regulatory RNAs, approximately22nucleotides in length, which are known to regulate target gene expression posttranscriptionally by repressing translation or decreasing messenger RNAs (mRNA). By binding to the3’-untranslated region (UTR) of target mRNA, miRNAs show to be of great importance in biological processes including cell proliferation, differentiation and apoptosis. MiRNAs function as oncogene or tumor suppressor in the development of cancers.However, the biological functions of most miRNAs are still unclear so far. The miRNA profile of esophageal squamous cell carcinoma is rarely reported. We aim to screen and identify the miRNAs expression in Chinese patients of ESCC through miRNA microarray. Furthermore, we explore the role of differentially expressed miRNAs to provide a new diagnostic and therapeutic direction for ESCC. Objetives:To screen and identify the differentially expressed miRNAs in ESCC. MiR-139-5p was selected for further validation. To explore the role of miR-139-5p in ESCC in vitro and in vivo.Methods:1. To screen the miRNA profile in ESCC tissues by Agilent miRNA microarray analysis.2. Among the screening results, miR-139-5p was selected for further validation in ESCC and dysplasia tissues and paired normal esophageal tissue specimens by TaqMan qRT-PCR.3. To explore the role of miR-139-5p on proliferation, apoptosis, migration and invasion of ESCC cells, miR-139-5p mimics and miR-139-5p inhibitor were transfected to Eca109and TE-13cells. CCK-8assays were conducted to obeserve the proliferation. To determine whether miR-139-5p could regulate esophageal cancer cell migration and invasion, transwell assays were performed in cell lines. Flow cytometry was executed to determine the effects of miR-139-5p on cell cycle alterations and apoptosis.4. MiR-139-5p was up-regulated in Eca109cells with lentiviral vector, these cells were injected into BALB/c nude mice to oberve the role of miR-139-5p in vivo.5. The target genes of miR-139-5p were predicted by biological information software and confirmed by dual-luciferase reporter assay. The regulating effect of miR-139-5p on the target gene was detected by SYBR Green qRT-PCR and Western blot. RNAi technique was used to down-regulate the target gene expression in ESCC cells. The expression of Cyclin A, Cyclin D1, MMP-2and MMP-9were analyzed by Western blot. Results:1. The results of miRNA microarray analysis showed that there were68miRNAs differently expressed in ESCC tissues (fold change>2). Among them,51miRNAs were up-regulated,17miRNAs were down-regulated. MiR-139-5p was down-regulated both in ESCC and dysplasia tissues (p<0.05). However, there was no relationship between miR-139-5p and clinicopathologic features.2. CCK-8assays showed ectopic expression of miR-139-5p inhibited cells proliferation compared with negative control group, and miR-139-5p inhibitor enhanced the cells proliferation (p<0.05). Colony formation assay showed that transfected with miR-139-5p mimics, cell colony formation was significantly lower than the negative control group, and transfected with miR-139-5p inhibitor, cell colony formation was significantly higher than the inhibitor negative control group (p<0.05). MiR-139-5p mimics induced G1cell cycle arrest and early apoptosis in esophageal cancer cell lines (p<0.05), there were no significant changes when miR-139-5p was knocked down. Ectopic expression of miR-139-5p significantly decreased the migration and invasion capability in esophageal cancer cell lines (p<0.05).3. Up-regulated miR-139-5p in Eca109cells with lentiviral vector, the tumor size was smaller in BALB/c nude mice compared to NC group in vivo (p<0.05).4. Bioinformatics predicted that c-Jun and c-Fos might be targets of miR-139-5p. Dual-luciferase reporter assay showed that miR-139-5p over-expression remarkably repressed the expression luciferase, but had no effect on mutant and negative control vector. qRT-PCR revealed that miR-139-5p mimics didn’t cause degradation of targets mRNA. Western blot analysis showed a down-regulated protein levels. Silencing of c-Jun and c-Fos siRNA in esophageal cancer cells led to decreased expression of Cyclin A, Cyclin D1, MMP-2and MMP-9, which was similar to over-expression of miR-139-5p (p<0.05).ConclusionsThere are68miRNAs differently expressed in ESCC tissues (51miRNAs are up-regulated,17miRNAs are down-regulated). MiR-139-5p is down-regulated in esophageal cancer tissues and cell lines. Ectopic miR-139-5p inhibits cell proliferation, migration, invasion and cell cycle progression in vitro. MiR-139-5p suppresses tumor growth in vivo. C-Jun and c-Fos contain a binding site for miR-139-5p at3’-UTR and are direct targets of it. MiR-139-5p inhibits cell malignant behaviors by negatively regulating c-Jun and c-Fos and its downstream molecules Cyclin A, Cyclin D1, MMP-2and MMP-9. Taken together, miR-139-5p inhibits cell proliferation, migration, invasion and cell cycle progression via or partly through decreasing the AP-1expression and its downstream molecules Cyclin A, Cyclin D1, MMP-2and MMP-9. |
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