| Background:Ovarian cancer is one of the most malignant cancers of the female reproductive system.Because of the lack of specific symptoms at early stage,it is often diagnosed at late stage.Its high incidence,high recurrence rate and low survival rate seriously threaten the life quality of women,and increase the healthy and economic burden of society.Therefore,it is vital to seek effective screening,diagnosis and treatment targets for OC.Lnc RNA have been verified to play a key role in the occurrence and development of a variety of cancers.NRSN2-AS1 is a novel discovered Lnc RNA,but there are few studies in OC.Objective : To clarify the differential expression and biological function of NRSN2-AS1 during the occurrence and development of OC.To explore the potential downstream targets and underlying mechanisms about NRSN2-AS.To provide possible targets for the preliminary diagnosis and clinical treatment of OC,as well as the theoretical basis for this target.Methods:The relevant information of NRSN2-AS1,PTK2 and MG53 in OC samples was obtained from the TCGA database to analyze the differential expression of them in OC and normal tissues.The expression and distribution of NRSN2-AS1 in OC and normal tissues were assessed by RT-q PCR and FISH assay.Via Cell Function assay,including CCK8 assay,Clone Formation assay and Transwell assay,the changes in the viability,proliferation and migration of OC cells after NRSN2-AS1 knockdown were evaluated.By the establishment of subcutaneous tumor model and lung metastasis model in nude mice,the tumorigenesis and metastasis ability of NRSN2-AS1 silenced-cells were analyzed.The proteins interacted with NRSN2-AS1 were identified by RNA-pulldown assay and Mass Spectrometry.The binding sites of NRSN2-AS1 and PTK2 were predicted by HDOCK,and verified by Western blot assay,immunocoprecipitation assay and RIP assay.The biological phenotype of PTK2 in OC was analyzed by Cell Function assay.The signaling pathway of PTK2 and the relationship between NRSN2-AS1 and the signaling pathway that PTK2 involved in were evaluated via Western blot assay and Immunofluorescence assay.Rescue assay was conducted to analyze the changes of biological phenotype and signaling pathway of OC cells in which β-catenin signal was activated/inhibited or PTK2 was knockdown after NRSN2-AS1 down-regulation or up-regulation.The potential mechanism behind NRSN2-AS1 and PTK2 was analyzed by Protein Half-life assay,IP assay,Cell Function assay and Western blot assay.Results:The expression of NRSN2-AS1 was up-regulated in OC clinical specimens and cells,and the over-expression of NRSN2-AS1 is tightly associated with poor prognosis.The NRSN2-AS1 silence inhibited the activity,proliferation,migration,tumorigenesis and metastasis of OC cells.NRSN2-AS1 interacts with PTK2,and the sites 35-82 of NRSN2-AS1 and the 414-670 of PTK2 were predicted to be binding sites.PTK2 was an oncogene and over-expressed in OC.PTK2 knockdown inhibited the viability,proliferation and migration of OC cells,and acted on β-catenin through both direct and indirect pathways.NRSN2-AS1 targeted β-catenin signal via PTK2.Silencing NRSN2-AS1 decreased the expression of PTK2 and inhibited the activation of β-catenin signal.Activation/inhibition of β-catenin after down-/up-regulation of NRSN2-AS1 rescued the phenotype and molecular expression of OC cells,and the knockdown of PTK2 after up-regulation of NRSN2-AS1 suppressed the oncogenic effect,indicating that NRSN2-AS1 depended on PTK2 acting on β-catenin.Knockdown NRSN2-AS1 decreased the expression,shortened the half-life and enhanced the ubiquitination level of PTK2 in OC.PTK2 was degraded by ubiquitin-proteasome pathway induced by E3 ligase MG53.The interaction between NRSN2-AS1 and PTK2 stabilized the expression of PTK2.MG53 was anti-cancer and low-expressed in OC.The down-regulation or up-regulation of MG53 reversed the changes of ubiquitination level and expression level of PTK2 caused by NRSN2-AS1 silence or up-regulation in OC cells.Conclusion:NRSN2-AS1 is up-regulated in OC tissues,and the knock-down can inhibit the viability,proliferation,migration,tumorigenesis and metastasis of OC cells both in vitro and vivo.NRSN2-AS1 interacts PTK2,which is up-regulated in OC and plays a cancer-promoting role,resulting in the β-catenin signal activation through both direct and indirect pathways.NRSN2-AS1 relays on PTK2/β-catenin signaling pathway to regulate OC progression and stabilizes the expression of PTK2 by preventing its ubiquitin-proteasome degradation,which is induced by E3 ligase MG53.This research reveals the biological phenotype and mechanism of NRSN2-AS1/PTK2/β-catenin signaling axis in OC,as well as provides the potential targets and theoretical basis for the preliminary diagnosis and clinical treatment of OC. |
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