Mechanism Of CircRNA Hsa＿circ＿0011324 Promoting The Progression Of Endometrial Cancer

BackgroundEndometrial cancer(EC)is one of the most common malignant tumor of female reproductive system.Although the prognosis of EC is generally good,the 5-year overall survival(OS)of patients with advanced EC is still poor.Spen paralogue and orthologue c-terminal domain containing 1(SPOCD1)shows oncogenic effects in several solid tumors.circ RNA plays an important regulatory role in tumors.This study intends to identify SPOCD1-related circ RNAs in the database,and to elucidate the biological and clinical roles of some key circ RNAs,as well as the underlying mechanism.Furthermore,the relationship between key circ RNA and host gene SPOCD1,and the expression and regulatory mechanism of host gene SPOCD1 in EC are also explored.This study will find potential diagnostic markers and therapeutic targets,providing new ideas for clinical diagnosis and treatment.Part I,hsa＿circ＿0011324 promotes the progression of endometrial cancer through hsa-miR-497/16-5p /m TOR pathwayObjective: To clarify the expression,clinical significance and mechanism of SPOCD1-related key circ RNA(hsa＿circ＿0011324)in EC.Methods: circ RNAs derived from SPOCD1 gene were searched based on circ Base database,and their expression were detected by q RT-PCR in cancer and adjacent tissues of EC patients.q RT-PCR was used to detect the expression of hsa＿circ＿0011324 in endometrial cancer cell.The stability of hsa＿circ＿0011324 was verified by RNase R.Fluorescence in situ hybridization(FISH)was used to detect the cellular localization of hsa＿circ＿0011324 in endometrial cancer cell(Ishikawa and RL95-2).The relationship between the expression of hsa＿circ＿0011324 and clinicopathology was determined by Chi-square and Fisher’s test.Cox regression analysis was used to analyze the prognostic risk factors.Kaplan-Meier was used to determine the correlation between hsa＿circ＿0011324 and patient prognosis.After overexpressing hsa＿circ＿0011324 in the endometrial cancer cell,the changes of phenotype were detected by CCK-8,clone formation and migration assay.RNA22 and Targetscan predicted that the downstream target genes of hsa＿circ＿0011324 was hsa-miR-497/16-5p.Pearson correlation analyzed the relationship between hsa＿circ＿0011324 and hsa-miR-497/16-5p in EC.Different expression levels of has＿circ＿0011324 and has-miR-497/16-5p on the changes of cancer cell function were clarified.The direct interaction between hsa＿circ＿0011324 and hsa-miR-497/16-5p,hsa-miR-497/16-5p and m TOR were verified by AGO2-RIP and dual luciferase reporter assay.Results:1.Among the 7 circ RNAs derived from SPOCD1,only hsa＿circ＿0011324expressed highly in EC.2.High expression of hsa＿circ＿0011324 was correlated with pathological differentiation,body mass index,grade,lymph node metastasis and stage of EC patients(P<0.05).It was an independent risk factor for EC patients(P<0.05).High expression of hsa＿circ＿0011324 was associated with poor prognosis in EC patients(P<0.05).3.Hsa＿circ＿0011324 was highly expressed in Ishikawa and RL95-2.Hsa＿circ＿0011324 was mainly localized in the cytoplasm of Ishikawa,and a small amount of expression was present in the nucleus.In RL95-2,hsa＿circ＿0011324 was expressed in cytoplasm and nucleus.High expression of hsa＿circ＿0011324 promoted proliferation,clone formation and migration in Ishikawa and RL95-2(P<0.05 or P<0.01).4.The expression of m TOR protein in hsa＿circ＿0011324 high expression group was higher than that in hsa＿circ＿0011324 low expression group(P<0.05).The expression level of hsa-miR-497/16-5p in EC was negatively correlated with hsa＿circ＿0011324(P<0.05).5.Overexpression of has-miR-496/16-5p could rescue hsa＿circ＿0011324’ s regulation on m TOR expression(P<0.05)as well as cancer cell phenotype including proliferation,clone formation and migration(P<0.05).6.Hsa＿circ＿0011324 and m TOR m RNA were enriched in AGO2-RIP,and dual luciferase reporter assay suggested that hsa＿circ＿0011324 could directly interact with hsa-miR-497/16-5p and promoted m TOR expression by sponging hsa-miR-497/16-5p.Conclusion: The high expression of hsa＿circ＿0011324 in EC is associated with poor prognosis of patients.Overexpression hsa＿circ＿0011324 promotes the proliferation and migration of endometrial cancer cell.Hsa＿circ＿0011324 participates in EC progression by targeting m TOR through sponging hsa-miR-497/16-5p.Part II,hsa＿circ＿0011324 is involved in the progression of endometrial cancer by activating SPOCD1/AKT/m TOR pathway via hsa-miR-421Objective: To elucidate the expression of SPOCD1 in EC and its biological function in endometrial cancer cell.To investigate the effect of hsa＿circ＿0011324 on tumorigenesis and SPOCD1/AKT/m TOR pathway in vivo.To clarify whether has＿circ＿0011324 regulates SPOCD1 through hsa-miR-421 and affects cancer cell phenotype.Methods: q RT-PCR and immunohistochemistry were used to detect the expression of SPOCD1 in EC patients(specimens were the same as Part I).After overexpressing or knocking down SPOCD1 in endometrial cancer cell,cell phenotype were detected by CCK8,clone formation and migration assay.Also,the expression of p-AKT and m TOR in cancer cell were detected by Western blotting.Pearson correlation was used to determine the relationship between hsa＿circ＿001132 and SPOCD1.After overexpressing or knocking down of hsa＿circ＿0011324 in cancer cell,the expression of SPOCD1,p-AKT and m TOR were detected by Western blotting.Stably overexpressed hsa＿circ＿0011324 in Ishikawa and RL95-2 and used for tumorigenesis in nude mice.Meanwhile,the expressions of SPOCD1,p-AKT and m TOR were detected by immunohistochemistry.Hsa-miR-421 was predicted to be the downstream target of hsa＿circ＿0011324(https://circinteractome.nia.nih.gov/).AGO2-RIP and dual luciferase reporter were used to verify interaction between hsa＿circ＿0011324 and hsa-miR-421,hsa-miR-421 and SPOCD1.Rescue experiments were performed in endometrial cancer cell,and cancer cell phenotype were detected by CCK8,clone formation and migration assay.Meanwhile,the expressions of SPOCD1,p-AKT and m TOR were detected by Western blotting.Results:1.SPOCD1’s expression was higher in EC.After overexpressing SPOCD1 in endometrial cancer cell,proliferation,clone formation and migration were enhanced,and the expression of p-AKT and m TOR were increased(P<0.05).The expression of hsa＿circ＿0011324 and SPOCD1 in EC patients were positively correlated(P<0.01).2.Hsa＿circ＿0011324 upregulated the expression of SPOCD1,p-AKT and m TOR proteins in endometrial cancer cell(P<0.01).3.Overexpression of hsa＿circ＿0011324 promoted tumor growth in vivo(P<0.05);and the expressions of SPOCD1,p-AKT and m TOR proteins were also increased.4.Hsa＿circ＿0011324 and SPOCD1 were enriched in AGO2-RIP.Dual luciferase reporter assay suggested that hsa＿circ＿0011324 could interact with hsa-miR-421(P<0.05).5.In endometrial cancer cell,overexpressing hsa＿circ＿0011324 promoted proliferation,clone formation and migration(P<0.05),and increased the expression of SPOCD1,p-AKT and m TOR(P<0.05),while supplementation of has-miR-421 weakened the above-mentioned changes generated by hsa＿circ＿0011324overexpression(P<0.05),and the expression of SPOCD1,p-AKT and m TOR were decreased(P< 0.05).Conclusion: SPOCD1 highly expressed in EC,which promotes the proliferation,clone formation and migration of endometrial cancer cell.Hsa＿circ＿0011324 is tumorigenic and activates AKT/m TOR pathway in vivo.The expression of hsa＿circ＿0011324 is positively correlated with SPOCD1.Hsa＿circ＿0011324 sponges hsa-miR-421 to promote the expression of SPOCD1,and thus regulates AKT/m TOR pathway promoting the progression of EC.