HOXC13,Regulated By MiR-503,Promotes Proliferation Of Esophageal Squamous Cell Carcinoma

Background:Esophageal cancer is one of the most common malignant gastrointestinal tumors,developed from the dysplasia of esophageal squamous epithelium or esophageal glandular epithelium.According to histology,esophageal cancer can be divided into squamous carcinoma and adenocarcinoma,of which esophageal squamous cell carcinoma(ESCC)accounting for more than 90%in China.Although the treatment effect of ESCC has been improved as a series of new technique applied in clinic,the overall 5-year survival rate of ESCC is still less than 25%.Therefore,it's very important to seek for new therapeutic target in ESCC.The development of tumor is related to the activation of oncogenes or the inactivation of tumor suppressor genes.To search for the key genes that play vital roles in ESCC,we analyzed the TCGA database and found that HOXC13 was significantly highly expressed in ESCC and might be a potential oncogene of ESCC.As a member of HOX gene family,HOXC13 has been reported to be correlated with many types of cancer and usually function as transcription factor.However,the role of HOXC13 in ESCC has not been reported.This study proceeded from cell lines and tissues to explore the expression of HOXC13 in ESCC and its relevance with clinical characteristics.We further verified the influence of HOXC13 on proliferation and apoptosis of ESCC and discover the potential molecular mechanism.This research may provide new targets for the diagnosis and treatment of ESCC.Methods and materials1.The TCGA database was used to validate the expression of HOXC13 in ESCC and its correlation with clinical characteristics and overall survival(OS)of patiens.Real-time quantitative PCR(qRT-PCR)was performed to test the expression of HOXC13 in ESCC tissues and its relationship with tumor size,TNM stage,and lymphatic metastasis.QRT-PCR and western blot were than used to detect the expression of HOXC13 in ESCC cell lines and esophageal epithelial cells.2.The interference and overexpression plasmids of HOXC13 were designed to knockdown or overexpress HOXC13 in ESCC.The Cell Counting Kit-8(CCK-8)assay,colony formation assay,xCELLigence System assay and TUNEL assay were used to explore the influence of HOXC13 on proliferation and apoptosis in ESCC.Nude mouse subcutaneous tumor-bearing experiment was performed to inspect the function of HOXC13 in vivo.3.The regulation of HOXC13 on CASP3 was proved by qRT-PCR and western blot.The inhibitor of caspase-3 pathway,Z-DEVD-FMK was used to observe whether the function of HOXC13 was dependent on CASP3.Luciferase reporter assay and chromatin immunoprecipitation(ChIP)were performed to confirm that HOXC13 directly targets the promotor region of CASP3 and suppresses its transcription.4.The biological function of miR-503 was verified by CCK-8 assay,colony formation assay,xCELLigence System assay and TUNEL assay.Luciferase reporter assay was performed to prove whether miR-503 directly targets the 3'-UTR of HOXC13.And the expression of miR-503 and its correlation with HOXC13 were certified in tissue samples.Results1.HOXC13 was highly expressed in ESCC tissues(p<0.0001)and correlates with the overall survival of patients.The overexpression of HOXC 13 indicated more advanced clinical pathological characteristics.And qRT-PCR of 60 pairs of ESCC tissues revealed that HOXC 13 was up-regulated in 86.7%of tumors and had a relevance with TNM stage.In cellular level,HOXC 13 was also highly expressed in ESCC cell lines compared with esophageal epithelial cells.2.Knockdown of HOXC13 promotes proliferation and suppresses apoptosis of ESCC cells(ECA109 and TE13),while overexpression of HOXC13 got the opposite results.And nude mouse subcutaneous tumor-bearing experiment confirmed that the volume and weight of tumor was smaller in HOXC13 knockdown group.3.The key apoptosis gene,CASP3 was upregulated by knockdown of HOXC13.Inhibitor of caspase-3 pathway partially reversed the effect of HOXC 13 on proliferation and apoptosis in ESCC.Luciferase reporter assay and chromatin immunoprecipitation(ChIP)indicated that HOXC 13 directly targets the promotor region of CASP3 to suppress its transcription.4.MiR-503 was lowly expressed in ESCC and had a negative correlation with HOXC13.Overexpression of miR-503 suppressed the proliferation of ESCC and induced apoptosis,meantime inhibiting the expression of HOXC13.Luciferase reporter assay confirmed that miR-503 directly targets the 3'-UTR of HOXC13.Conclusion1.HOXC 13 was highly expressed in ESCC and correlated with the prognosis the clinical pathological characteristics,which might be a potential biomarker for diagnosis and predicting prognosis.2.HOXC 13 promotes proliferation of ESCC via repressing the transcription of CASP3,and meanwhile HOXC 13 is regulated by miR-503.The miR-503-HOXC13-CASP3 axis plays a vital role the development of ESCC.So this study may provide new therapeutic targets and improve the prognosis of ESCC.