Role Of ARTEMIN Expression In Laryngeal Squamous Cell Carcinoma And Its Mechanism

BackgroundArtemin(ARTN), a neurotrophic factor, has been implicated in development and progression of several human malignancies. However, the clinical and prognostic significance of ARTN and its receptors has not been investigated in human laryngeal squamous cell carcinoma(LSCC). In the other side, recent studies have shown that Micro RNAs(mi RNAs) could cause its target gene degradation and/or translational repression and also play an important role in the development and progression of human cancers. Dicer, one of the most important enzymes of the mi RNA machinery, performs the final step of biogenesis of mi RNAs. This study aimed to investigate clinical impact and prognostic significance of ARTN or its receptor expression in human LSCC and its impact of human LSCC cells proliferation, invasion and migration in vitro. Secondly, we also observed the expression and clinical significance of Dicer in LSCC and scanned and verified the targeting mi RNA of ARTN and explored the mechanism involved.Method(1) We determined the protein expression of ARTN and its receptor, namely GFRα1, inarchival tissue specimens of 76 LSCC and 26 polyp of larynx by immunohistochemistry. Furthermore, the clinicopathological and prognostic significance of ARTN and GFRα1 expression was analyzed in LSCC. Real-time PCR was used to examine the expression of ARTN and GFRα1 in fresh tissues of 16 LSCC and 14 polyp.(2) Human LSCC Hep-2 cells were transfected with ARTN si RNA or negative control by Lipofectamine. Cell proliferation and invasiveness were evaluated by MTS kit and transwell invasion assay. Cell migrating ability of Hep-2 was detected by transwell migration assay.(3) We detected the expression of Dicer in 76 LSCC and 26 polyp of larynx tissue specimens by immunohistochemistry. The clinicopathological and prognostic significance of Dicer expression was investigated in LSCC.(4) The possible targeting mi RNA of ARTN were forecasted by bioinformatics tool and verified as following: Hep-2 cells was transfected by mi RNA mimics, ASO or negative control to detect the changes of m RNA expression level through real-time RT-PCR. And then the 3′UTR region of ARTN gene was cloned into the Psi CHECK-2 vector which containing luciferase reporter gene and co-transfected with mi RNA mimic into Hep-2 cells, and the relationship of this mi RNA and ARTN was validated by Luciferase Reporter Assay.Results(1) The expression of ARTN and GFRα1 was significantly increased in LSCC compared with polyp tissue specimens. And both the expression of ARTN and GFRα1 was positively associated with p TNM stage in LSCC. Kaplan-Meier survival analyses revealed a strong association between the expression of ARTN or GFRα1 and the survival of LSCC patient. Correlation analysis demonstrated that the expression ARTN was significantly correlated to the expression GFRα1. Moreover, real-time PCR confirmed that the expression ARTN and GFRα1 was significantlyhigher in fresh tumors of LSCC than that in fresh polyp.(2) The proliferation, invasion and migration ability was significantly decreased in Hep-2 cells by transfection of ARTN si RNA, compared with control.(3) The expression of Dicer was significantly higher in LSCC than that in polyp tissue specimens. Moreover, the expression level of Dicer was significantly associated the p TNM stage and tumor Lymph node metastasis. Kaplan-Meier survival analyses revealed a strong association between tumor Dicer expression and the survival of LSCC patient.(4) Targetscans program revealed that ARTN might be a target gene of mi R-223. The expression level of ARTN m RNA was significantly decreased in Hep-2 cells transfected with mi R-223 mimics and significantly increased by transfection with mi R-223 ASO. The luciferase reporter assay revealed that mi R-223 was directly bound to ARTN 3′UTR.Conclusion(1) Altered expression of ARTN and its receptor was frequently observed in LSCC and ARTN might play an important role in the development and progression of LSCC.(2) mi RNA might involve the regulation of ARTN expression and ARTN was a direct target gene of mi R-223.