| Purpose: In China,the morbidity of esophageal cancer is about 13/10 million and esophageal squamous cell carcinoma is the predominant histologic type,accounting for over 80% in all cases.Given that remarkable accomplishment had been achieved in diagnosis and multimodality in the last few decades,the prognosis of ESCC remains discouraging with a dismal 5-year survival rate less than 20%.Local invasion and metastatic dissemination is commonly thought should be responsible for the poor prognosis of ESCC and various studies have demonstrated that Epithelial-Mesenchymal Transition(EMT)plays a key role in the invasion and migration of tumor cells.EMT converts epithelial cells to a mesenchymal phenotype,including polarity loss,adhesion ability decline and enhancement of cell migration and invasion competence.MicroRNAs are small approximately 22 nucleotide long noncoding single-stranded RNA molecular that could post-transcriptionally regulate gene expression by inhibiting translation of target mRNAs or directly degrade mRNA via binding the 3’ untranslated region of its target gene.In esophageal squamous cell carcinoma,we firstly found that the expression of miR-214 in tumor tissues was significantly downregulated compared to the adjacent normal tissues.miR-214 expression was verified negatively correlated with lymph node metastasis which reminds us that miR-214 could participate in the invasion and metastasis of esophageal squamous cell carcinoma.CrkL(CT10 regulator of kinase like protein)is a member of Crk family and it was found involved in the conduction of multiple signaling pathways.Many studies have shown that Crk L plays an important role in cell proliferation,adhesion,metastasis,response to pathogens,and apoptosis.CrkL was also found upregulated in ovarian cancer,gastric cancer,chronic myeloid leukemia and non-small cell lung cancer.In our previous work,the miR-214 target genes,which may be associated with EMT in esophageal squamous cell carcinoma,were screened by gene expression profiling and bioinformatics analysis.It was found that the 3’-untranslated region(3’-UTR)of the CrkL contained miR-214 potential binding site.Based on this,our study was to investigate the effect of miR-214-CrkL pathway on the proliferation,migration and invasion of esophageal squamous cell carcinoma and to explore its possible mechanism for providing scientific evidence for miR-214-mediated targeted therapy in the future.Methods: The present study is divided into three sections.1.The expression of miR-214 and CrkL in mRNA level in tumor tissues and adjacent normal tissues were detected by qPCR and the expression of CrkL in protein level was examined by Western-blot and immunohistochemical staining.The correlation between of miR-214/CrkL expression and tumor invasion/lymph node metastasis were analyzed.2.The expression of CrkL and EMT markers including E-cadherin and N-cadherin in the mRNA and protein levels of the esophageal squamous cell carcinoma cells were detected by qPCR and Western-blot after transfecting Eca-109 cells with miR-214 mimic / inhibitor and the corresponding negative control.Transwell and Wound-Healing assay were performed to detect the change of invasion and migration ability in transfected Eca-109 cells.3.miR-214 mimic / pcDNA.CrkL were co-transfected into Eca-109 cells and then check if the high expression of Crk L could offset the inhibitory effect of miR-214 to EMT.Dual-Luciferase assay was conducted to verify if CrkL is a target gene of miR-214.Results: The mRNA expression of miR-214 in ESCC tissues was significantly downregulated(P <0.05).The results of PCR and Western Blot confirmed that CrkL was upregulated in tumor tissues and mRNA and protein level(P <0.05).Down-regulation of miR-214 were strongly associated with tumor invasion(p=0.040)and lymph node metastasis(p=0.033)while CrkL was positively correlated with tumor invasion(p=0.013)and lymph node metastasis(p=0.012).CrkL and N-cadherin were downregulated(P <0.05)and E-cadherin was upregulated in cells tranfected with miR-214 mimics(P <0.05)while Crk L and N-cadherin were upregulated(P <0.05)and E-cadherin was downregulated in cells tranfected with miR-214 inhibitor(P <0.05).At the same time,Transwell experiment confirmed that the number of cells invaded to the other side of upper well were significantly larger and smaller in miR-214 inhibitor group and miR-214 mimics group repectively than the control group(P <0.05),Wound-Healing assay also proved the migration speed of cells were faster and slower in cells transfected with miR-214 mimics and inhibitor repectively compared to the control group(P <0.05).After the successful cotransfection with miR-214 mimics / pcDNA.CrkL,the PCR and Western Blot results showed that the expression of E-cadherin was lower than that in the control group(P <0.05)and N-cadherin was significantly higher compared to the control group(P <0.05).Transwell and Wound-Healing results also showed that the number of invaded cells were significantly reduced and the migration speed was significantly decreased after transfected with miR-214 mimics / pcDNA.Crk L.The Dual-luciferase assay confirmed that there is a binding site of miR-214 in the 3 ’untranslated region of CrkL,indicated that miR-214 could inhibit CrkL expression.Conclusion: In our study,miR-214 is lowly expressed in esophageal squamous cell carcinoma and its underlying downstream regulatory protein Crk L is highly expressed.After overexpression of miR-214,CrkL were significantly down-regulated at the mRNA and protein level,and the migration and invasion ability of the miR-214 over-expressed cells were significantly decreased.Upregulated the expression of miR-214 and CrkL simultaneously found that high CrkL can counteract miR-214’s effect in EMT inhibition,and restored cell migration and invasion competence,luciferase gene reporter test also confirmed that miR-214 target inhibition on CrkL. |
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