LncRNA RP11-79H23.3 Functions As A Competing Endogenous RNA To Regulate PTEN Expression Through Sponging Hsa-miR-107 In The Development Of Bladder Cancer

ObjectiveStudies have demonstrated for the first time that LncRNA RP11-79H23.3(RP11-79H)might function as a competitive endogenous RNA(ceRNA)for miR-107 to regulate the expression of target gene PTEN,which consequently contributes to migration and invasion of BC.To further highlighted a novel role of RP11-79H23.3 in progression of BC and indicated the molecular mechanism of signaling pathways for BC,It also provided theoretical basis for the diagnosis,treatment and prognosis of new target genes and drugs.MethodsHere,we screened the lncRNA and mRNA expression profiles of BC using the microarray assay.We found that a novel lncRNA,RP11-79H23.3 and drew the corresponding ceRNA network.The expression levels of RP11-79H23.3 and PTEN were detected by RT-qPCRin BC tissues and the matched normal tissues.Functionally,when RP11-79H23.3 was up-regulated or down-regulated in vivo,CCK8 assay was used to detect cell proliferation,wound healing and Transwell assays were used to detect cell migration and invasion.Meanwhile,EJ or T24 living cells were observed for morphological changes under the microscope,and determined for rearranged cytoskeleton by cytoskeleton assays,The effect on cell apoptosis was assessed by Tunel Kit and Hochest33342 assay,The cell cycle progression was analyzed by flow cytometry.To explore RP11-79H23.3 might affect PI3K/AKT signaling pathway protein’s level through regulatung target gene PTEN and the expression of apoptotic proteins(Caspase 3,Bax,Bcl-2)were detected by Western Blot.The xenograft model of nude mice was established in vitro,Immunofluorescence and immunohistochemistry were adopted to detect tumorigenesis,angiogenesis and lung metastasis in vivo.Mechanistically,we performed bioinformatic website to predict the binding sites of miR-107 and RP11-79H23.3 or PTEN,Next,Dual-luciferase reporter assays were performed to detect the miR-107 could directly target RP11-79H23.3 and bind to PTEN in BC cells,Furthermore,we evaluate the subcellular localization and expression of RP11-79H23.3 and miR-107 in bladder cancer cells by FISH assay.RIP experiment were used to prove the binding relationship between RP11-79H23.3 and miR-107 at cellular level,The products were purified and enriched to detect the targetmicroRNAs by RT-qPCR.Finally,To verify the regulated relationship between miR-107,RP11-79H23.3 and PTEN,We explored whether RP11-79H23.3 could regulate target genes PTEN through mi R-107 by RT-qPCR assay.ResultsWe analyzed the lncRNA and mRNA expression profiles of BC and RT-qPCR experiments and found that a novel lncRNA,RP11-79H23.3,was significantly down-regulated in BC tissues and cells as well as the expression corresponded with that of PTEN.Meanwhile,RP11-79H23.3 expression was negatively correlated with clinical stage in BC.Functionally,CCK8,Edu,and Colony formation experiments showed that overexpression of RP11-79H23.3 inhibited cell proliferation,Wound healing,Transwell and cytoskeleton assays demonstrated that up-regulating RP11-79H23.3 significantly suppressed cellmigration,invasion and mobility,whereasoverexpression of RP11-79H23.3 could induce cell apoptosis and represses BC cell cycle progressionin vitro.Moreover,up-regulating RP11-79H23.3 inhibited the tumorigenesis,angiogenesis and lung metastasis in vivo,RP11-79H23.3 knockdown exerted a contrary role.Mechanistically,we used dual luciferase reporter assays confirm that RP11-79H23.3 could directly bind to miR-107 and abolish the suppressive effect on target gene PTEN,which regulate PI3K/AKT signaling pathway.In addition,we investigated the subcellularlocalization of RP11-79H23.3 and mi R-107 in BC cells by FISH.The results indicated that RP11-79H23.3and mi R-107 mainly distributed in cytoplasm.Subsequently,to further confirm the endogenous binding between RP11-79H23.3 and miR-107 at the cellular level,we performed RNA immunoprecipitation(RIP)to pull down microRNAs connected with RP11-79H23.3 and confirmed with qPCR.More importantly,Ed U and Transwell assay showed that ectopic expression of miR-107 abolished the decreased proliferation by RP11-79H23.3 overexpression in EJ cells,whereas miR-107 inhibitor reversed the increased proliferation by RP11-79H23.3 knockdown in T24 cells.Furthermore,RT-qPCR assay detected that the up-regulation of miR-107 decreased the expression of PTEN in BC cells,and the expression of RP11-79H23.3 and PTEN were consisten.Taken together,we demonstrated that RP11-79H23.3 might act as a competitive endogenous RNA(ceRNA)for miR-107 to regulate tumor suppressor PTEN,which could reveal the new molecular mechanism of the development of bladder cancer via PI3K/AKT signaling pathway.Conclusion1.Up-regulation of RP11-79H23.3 expression could inhibit proliferation,invasion,metastasis,angiogenesis,lung metastasis and promote apoptosis of bladder cancer cells in vitro and vivo.2.RP11-79H23.3 was significantly down-regulated in bladder cancertissues and positively correlated with PTEN expression level.3.RP11-79H23.3 might act as a competitive endogenous RNA(ceRNA)for mi R-107 to regulate the expression levels of PTEN,which could contribute to pathogenesis and development of BC via PI3K/AKT signaling pathway.