The Roles Of Long Noncoding RNA LINC00460 In The Growth And Metastasis Of Cervical Cancer And Its Mechanisms

BackgroundCervical cancer is a common gynecological malignancy.With advances in medical technology,the prevalence of cervical cancer has declined globally,but it remains one of the most fatal diseases among middle-aged women,especially in low-and middle-income countries.The 5-year overall survival rates for cervical cancer FIGO stage Ⅱ,Ⅲ and ⅣA were only 65%,40%and 15%,respectively.About 30-40%of patients with cervical cancer will relapse during treatment,especially in advanced patients.Therefore,it is very important to elucidate the pathogenesis of cervical cancer and to identify new biomarkers for diagnosis and treatment of cervical cancer.Long noncoding RNAs(lncRNAs)are a class of non-coding RNAs,which can bind to microRNAs(miRNAs)as competitive endogenous RNAs,thereby inhibiting target gene expression.Accumulating data indicate that lncRNA dysregulation is correlated with various diseases,especially cancer.Until now,a lot of lncRNAs have been shown to be involved in the regulation of cervical cancer progression.LINC00460 is a newly discovered lncRNA located on chromosome 13q33.2,which often plays an oncogenic role in several cancers.However,the functional mechanism of LINC00460 in cervical cancer remains largely unknown.ObjectiveTo elucidate the expression pattern and functional mechanism of LINC00460 in cervical cancer,and to provide theoretical basis for looking for potential therapeutic targets and diagnosis and treatment biomarkers of cervical cancer.Methods(1)qRT-PCR was used to examine the expression of LINC00460 in cervical cancer tissues and cell lines.(2)After down-regulating the expression of LINC00460 by siRNA silencing technology,the effects of LINC00460 on the proliferation,cycle,invasion,migration and EMT of HeLa and Caski were observed by MTT,EDU immunofluorescence,flow analysis,Transwell,Western blotting and other methods.(3)To determine the regulatory relationship between LINC00460 and miR-361-3p,and miR-361-3p and Gli1,bioinformatic analysis,dual-luciferase reporter assay,qRT-PCR and Western blotting were used.(4)The pair correlation between the expression of LINC00460,miR-361-3p and GLI1 in cervical cancer tissues was analyzed;(5)The changes of Gli1 expression,cell growth and invasion after co-transfection of si-LINC00460 and miR-361-3p inhibitor in cervical cancer cells were observed by rescue experiment.(6)The tumor formation model of nude mice was established to determine the effect of LINC00460 knockdown on tumor growth.Results(1)LINC00460 is up-regulated in cervical cancer tissues and cell lines.(2)Suppression of LINC00460 weakened cancer cell growth,invasion and EMT.(3)LINC00460 had a binding site with miR-361-3p.miR-361-3p had a binding site with Glil 3’UTR.Their binding was verified by double luciferase reporter assays.qRT-PCR and Western blotting further confirmed the effect of miR-361-3p on the expression of LINC00460 and Gli1.(4)miR-361-3p was down-regulated in cervical cancer tissues.LINC00460 was negatively correlated with miR-361-3p.miR-361-3p was negatively correlated with Gli1,while LINC00460 was positively correlated with Gli1.(5)The suppression of cell growth and invasion triggered by LINC00460 knockdown was reversed by miR-361-3p inhibitor.The inhibitory effect on Glil expression induced by LINC00460 knockdown was reversed by miR-361-3p inhibition.(6)The tumors formed by LINC00460 knockdown cells were smaller and of lower weight than those formed from control cells.Immunohistochemical analysis revealed that the LINC00460-knockdown tumors exhibited fewer Ki67-positive cells.ConclusionsLINC00460 is abnormally highly expressed in cervical cancer.Knockdown of LINC00460 inhibits cell proliferation and invasion,and inhibits tumor growth in vivo.LINC00460 functions as a cancer-promoting lncRNA in cervical cancer through LINC00460/miR-361-3p/Glil pathway.