| Objective:Oral squamous cell carcinoma(OSCC)is one of the most common malignant tumors worldwide,with over 300,000 cases annually.Autophagy,as an important non-selective degradation mechanism,could promote tumor initiation and progression by maintaining cellular homeostasis and the cell metabolism as well as cell viability.CircCDRlas has been shown to function as an oncogene in cancer progression,however,it remains largely unknown as to how autophagy is regulated by circCDR1as in oral squamous cell carcinoma(OSCC).This study aimed to investigate the underlying mechanism of autophagy in circCDR1as and OSCC cells.Methods:1.Immunohistochemistry was used to detect the expression of HIF-1α and autophagy-related protein in 57 OSCC tissues and normal tissues.Autophagy levels were detected by western blot,immunofluorescence and Transmission electron microscopy.2.OSCC cells were transfected circCDR1as overexpression vector.After hypoxia exposure and rapamycin or 3-MA treatment,RT-PCR,Western blot and immunofluorescence were used to evaluate the regulation of circCDR1as on hypoxia-mediated autophagy.3.The effect of circCDR1as-mediated autophagy and endoplasmic reticulum stress(ER stress)response on cell proliferation activity and apoptosis was further verified by 3-MA in a hypoxic microenvironment.4.Western blot and reactive oxygen species(ROS)assay were used to investigate the regulation of downstream signaling pathway and autophagy by circCDR1as in hypoxic microenvironment.5.Based on bioinformatics,the relationship between circCDR1as and miR-671-5p was determined.Then we conducted dual-luciferase reporter assay to observe the interaction of circCDR1as and miR-671-5p.6.To further verify whether circCDR1as could regulate tumor growth and autophagy in vivo.Tca-8113 cells transfected with circCDR1as lentivirus or control vector were subcutaneously injected into nude mice.Results:1.Compared with adjacent tissues,HIF-1α and autophagy-related proteins were elevated in OSCC tissues.After exposure to hypoxic condition,autophagy were significantly increased,but apoptosis was reduced in OSCC cells.2.Western blot and immunofluorescence results suggested that circCDR1as promotes hypoxia-mediated autophagy.Interestingly,OSCC cells showed a significant increase in terms of the circCDRl as expression after exposure to hypoxic condition.3.CCK-8 analysis showed that the cells treated with autophagy inhibitor 3-MA reduced cell viability induced by the circCDR1as.Furthermore,apoptosis rate was declined after 3-MA treatment in Tca-8113 cells overexpressed with circCDR1as.In addition,circCDR1as overexpression promoted the ER stress under both nomoxia and hypoxia conditions.4.CircCDR1as overexpression elevated LAMP2 and TFEB levels in hypoxiccells compared to normoxic cells.Moreover,the results showed that hypoxia-activated phosphorylated AKT and ERK1/2 protein,but downregulated the expression of the phosphorylation of mTOR.In addition,circCDRlas overexpression cells showed higher ROS levels in hypoxic cells compared to normoxic cells.5.Through the intersection of three databases(TargetScan,CircInteractome and RegRNA2.0),we obtained three miRNAs including hsa-miR-671-5p,hsa-miR-7,and hsa-miR-1270.Our data demonstrated that the luciferase intensity was attenuated in the cells co-transfected with miR-671-5p and circCDR1as 3’UTR-WT.6.The results demonstrated that overexpression circCDR1as lentivirus increased the tumor volume and weight on Tca-8113 cell tumor xenograft in nude mice.Immunohistochemistry(IHC)staining showed increased HIF-1α and autophagy-related proteins expression in the tumor isolated from the mice with injection of circCDR1as cells.Conclusion:Collectively,these results revealed that high expression of circCDR1as enhanced the viability of OSCC cells under a hypoxic microenvironment by promoting autophagy,suggesting a novel treatment strategy involving circCDR1as and the inhibition of autophagy in OSCC cells. |
