Increased Expression Of LncRNA CASC9 Promotes Tumor Progression By Suppressing Autophagy-mediated Cell Apoptosis Via The AKT/mTOR Pathway In Oral Squamous Cell Carcinoma

Background: Recent studies showed that lncRNA CASC9 was upregulated and acted as an oncogene in a variety of tumors.However,the expression and biological functions of CASC9 in oral squamous cell carcinoma(OSCC)remain unknown.Objection: To study the expression and clinical significance of CASC9 in OSCC and verify the biological function of CASC9 in vitro and in vivo.Method:The expression of CASC9 was detected by RT-qPCR in 35 matched oral squamous cell carcinomas and adjacent tissues,as well as in OSCC cell line and normal oral mucosal cell line.The expression of CASC9 in 84 specimens of oral squamous cell carcinoma with complete clinical data and follow-up data was detected by in situ hybridization.The correlation between CASC9 expression and the clinicopathological parameters,as well as the survival of OSCC patients were analyzed.Theprotein expression of p-AKT and LC3 B in 84 OSCC was detected by immunohistochemistry,and the correlation between the expression of p-AKT and LC3 B and CASC9 expression was analized.After CASC9 was knockdown in OSCC cells by siRNA,we detected cell viability by MTT assay,apoptosis by flow cytometry and Tunel assay,autophagy under transmission electron microscopy,and AKT,p-AKT,mTOR,p-mTOR,LC3 B,P62,BCL2,BAX protein expression by western blotting.rescue experiments were performed with addition of AKT activators and autophagy inhibitors in the CASC9-silenced OSCC cell line.In vivo tumorigenicity assay was performed with shRNA-interfering OSCC cell lines to detect tumor weight,volume and growth rate in vivo,and LC3 B,P62,BCL2,and BAX mRNA expression was detected in tumors.Result : CASC9 expression in OSCC tissues and cell lines was remarkably upregulated compared with that in adjacent normal tissues and normal oral mucosa cells,respectively(p<0.05).Highly expressed CASC9 is strongly associated with tumor size,clinical stage,regional lymph node metastasis and overall survival time in OSCC patients(p<0.05).In vitro,CASC9 knockdown in OSCC cells SCC15 and CAL27 significantly promotes autophagy and apoptosis,while inhibiting proliferation(p<0.05).Moreover,the expression levels of p-AKT,p-mTOR,P62 and BCL-2 were significantly decreased,while the expression levels of BAX and the LC3BII/LC3 BI ratio were increased in CASC9-knockdown SCC15 andCAL27 cells(p<0.05).After the addition of the AKT activator SC79 in CASC9-knockdown SCC15 and CAL27 cells,we found that the increased autophagy and apoptosis were remarkably rescued(p<0.05).Furthermore,the increased apoptosis was remarkably rescued in CASC9-knockdown OSCC cells treated with the autophagy inhibitor Autophinib.In addition,CASC9 depletion suppressed tumor growth in vivo.Conclusion : Our findings demonstrate that lncRNA CASC9 promotes OSCC progression through enhancing cell proliferation and suppressing autophagy-mediated cell apoptosis via the AKT/mTOR pathway.CASC9 could potentially be used as a valuable biomarker for OSCC diagnosis and prognosis.