| BackgroundWorldwide,ovarian cancer has been one of the most frequent malignant gynecological tumors.More than 52,100 new patients were diagnosed every year in China according to the statistical data in 2015.Epithelial ovarian cancer(EOC)is the most common ovarian cancer.The main stream therapeutic method for the EOC is surgical excision,and more subsequent therapies were replenished to prevent recurrence,such as chemotherapy and radiotherapy.Even though these attempts,the clinical outcome is still not optimistic.Therefore,it is urgently required to unveil the deepgoing molecular mechanism underlying EOC to discover the tumorigenesis.Long noncoding RNAs(lncRNAs)are a vital member in the noncoding RNA family accompanied by microRNA,20-22nt in length,and circular RNA,a covalent closed circular loop.LncRNAs are a series of nucleotide sequence with over 200 nucleotides in length,with their amounting roles in human cancers.For example,lncRNA LINC00460 expression is upregulated in EOC tissues and cells,comparing with its expression in adjacent tissues and cells,and LINC00460 exerts the oncogenic roles in EOC through binding miR-338-3p.LncRNA CASC2 is found to be downregulated in EOC and cells,and it could be able to act as a tumor suppressor and is an independent risk factor for poor prognosis.Thus,these literatures could indicate the emerging role of lncRNA in the EOC.The IncRNA TP73-AS1 is reported to be an oncogene in multiple human cancers,such as osteosarcoma,bladder cancer and clear cell renal cell carcinoma.However,there has been no in-depth study on the concretely mechanism of IncRNA TP73-AS1 in EOC.Objective:We are intended to explore the expression pattern of IncRNA TP73-AS1 in EOC tissues and cells,and to further explore its mechanism on epigenetics.Firstly,we examined the clinical expression levels of IncRNA TP73-AS1 in EOC tissues and EOC cell lines and analyzed the prognostic correlation of TP73-AS1 in TCGA database.Further,the function of TP73-AS1 in EOC cell lines were explored in cell proliferation,apoptosis and invasion by knockdown and overexpression of TP73-AS1.Then the effect of TP73-AS1 on tumor growth in xenograft mice was observed in vivo.Finally,the mechanism of TP73-AS1 in EOC cell lines was detected.The sub-cellular location of TP73-AS1 in SKOV3 cells was determined to analyze the cellular function of TP73-AS1,and then the epigenetic analysis of IncRNA TP73-AS1 on the downstream molecules and histone-modified enzymes were deepgoing studied.Methods1.Expression analysis of IncRNA TP73-AS1 in EOC tissues and cell lines.EOC tumor tissues samples were obtained from 28 patients who underwent surgical resection therapy at the Shandong Provincial Hospital affiliated to Shandong University.The Ethics Committee has approved all these experimental process.Written informed consents were obtained from all patients.Realtime fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of IncRNA TP73-AS1 in EOC tissues and adjacent tissues.Epithelial ovarian cancer cell lines(SKOV3,CAOV3,H08910,OV420)and normal human ovarian epithelial cell line(HOSEPiC)were cultured and detect the expression of IncRNA TP73-AS1 using RT-qPCR.Based on the TCGA database,IncRNA TP73-AS1 expression level was collected,the survival curve was used to compare the survival between TP73-AS1 high expression and low expression.2.Biological function of IncRNA TP73-AS1 in EOC cell lines.3 of TP73-AS1 siRNA duplexes and overexpressed pcDNA-TP73-AS1 plasmid were constructed.The siRNA duplexes and pcDNA-TP73-AS1 plasmid were transfected into SKOV3 and CAOV3 cells using RNAi MAX and lipofectamine 3000,respectively.The siRNA duplex with the maximal inhibitive efficiency was selected by RT-qPCR.The overexpressed effect by pcDNA-TP73-AS1 in EOC cells were verified by RT-qPCR.Cell invasion was detected by Transwell assay,apoptotic cells were detected by propidium iodide(PI)staining and flow cytometry,and cell proliferation was detected by CCK-8 assay.The knockdown lentivirus containing TP73-AS1 siRNA sequence was constructed and infected into SKOV3 cells.After screening by puromycin,the SKOV3 cells with TP73-AS1 knockdown was obtained.Then SKOV3 cells or SKOV3 cells with TP73-AS1 knockdown were injected into nude mice subcutaneously,respectively.The tumor size was detected every 3 days,and mice were sacrificed at 21 days to weigh the tumor.3.The mechanism study of lncRNA TP73-AS1 in EOC cell lines.Total RNA from the nucleus and cytoplasm of SKOV3 cells was extracted,the expression of lncRNA TP73-AS1 were detected by RT-qPCR.The cellular location of lncRNA TP73-AS1 were detected by fluorescence in situ hybridization(FISH).SKOV3 cells were knocked down the TP73-AS1 expression,then the mRNA expressions of component in cyclin dependent kinase inhibitors(CKIs)and cyclin-dependent kinase(CDKs)were detected by RT-qPCR.The Western Blot was used to detect the protein level which correlated TP73-AS1.By RNA-binding protein immunoprecipitation(RIP)assay,the histone-modifying enzymes binding to TP73-AS1 were detected.By chromatin immunoprecipitation(ChIP)assay,the histone-modifying enzymes and the histone binding to p21 gene promoter sequence were detected.After TP73-AS1 siRNA and p21 siRNA co-transfection into SKOV3 cells,cell invasion was detected by Transwell assay,apoptotic cells were detected by PI staining and flow cytometry,and cell proliferation was detected by CCK-8 assay.Expression levels of TP73-AS1,p21 and EZH2 in TCGA database were collected,survival curves of p21 and EZH2 were analyzed,and the expression correlations of TP73-AS1 and EZH2 were compared.Results1.The level of TP73-AS1 in EOC tissue specimens illustrated that TP73-AS1 was drastically over-expressed in the EOC tissue compared to adjacent normal tissue specimens.In accordance with the tissue results,the level of TP73-AS1 in EOC cells(SKOV3,CAOV3,HO8910,OV420)was also markedly increased in relation to normal ovarian epithelial cell line(HOSEPiC).The EOC patients with higher TP73-AS1 level were significantly correlated with the poorer prognosis by Kaplan-Meier analysis.Therefore,this data suggests that IncRNA TP73-AS1 is over-expressed in the EOC tissue and cells,besides,the overexpression was correlated with the poor clinical outcome.2.To explore the biological functions of TP73-AS1 in the EOC cells,the silenced expression siRNA and overexpression plasmid for TP73-AS1 were transfected into EOC cells(SKOV3,CAOV3)to knockdown or increase its expression,respectively.Transwell invasion assay showed that the silenced TP73-AS1 expression inhibited the invaded cell quantity,while the enforced TP73-AS1 expression promoted the invaded cell quantity.Flow cytometryanalysis revealed that the silenced TP73-AS1 expression stimulated the apoptosis of EOC cells,while the enforced TP73-AS 1 expression receded the apoptosis.Proliferative CCK-8 assay showed that TP73-AS1 knockdown decreased the proliferation,while the TP73-AS1 enforced level promoted the proliferation.In vivo xenograft assay illustrated that TP73-AS1 knockdown repressed the tumor weight and growth compared to the negative control.Thus,this data suggests that TP73-AS1 facilitates the EOC cells' proliferation in vitro and in vivo.3.The subcellular location of TP73-AS1 was found to be much more in the nuclear than the cytoplasm fraction,suggesting the transcriptional regulation for TP73-AS1 in the EOC cells.In the SKOV3 cells transfected with siRNA TP73-AS1,CKIs and CDKs were measured using RT-PCR,including p15,p16,p21,p27,p57,CDK2,CDK4,CDK6.The level of p21 was increased in the TP73-AS1 silencing.Western Blot revealed that p21 protein was enforced in the TP73-AS1 silencing transfection.RIP assay showed that TP73-AS1 could bind with EZH2 and H3K27me3.ChIP revealed that EZH2 and H3K27me3 directly bound with p21 promoter region,however,TP73-AS1 silence decreased the binding ability of EZH2 and H3K27me3 with p21 promoter.Kaplan-Meier survival curve analysis based on the TCGA database suggested the poor prognosis of EOC patients with higher EZH2 than the lower group,as well as with lower p21 than the higher group.The interaction within TP73-AS1 and EZH2 was analyzed using the data based on the TCGA,showing the positive interaction of TP73-AS1 and EZH2.Therefore,the data shows that TP73-AS1 epigenetically inhibits p21 transcription through binding with EZH2 and H3K27me3.4.SKOV3 cells invasion and proliferation ability decreased and the apoptosis increased as TP73-AS1 silence.As knockdown TP73-AS1 and p21 simultaneously,the levels of p21 mRNA and protein decreased in the SKOV3 cells.The invasion,proliferation and apoptosis by TP73-AS1 silence were rescued by p21 knockdown.Transwell invasion and flow cytometry assay illustrated that p21 siRNA up-regulated the invasive cells,and reduced the apoptotic cells in the SKOV3 cells.Meanwhile,the CCK-8 assay revealed that p21 siRNA increased the proliferation in the SKOV3 cells.Therefore,data shows that TP73-AS1 regulates the EOC progression through targeting p21.Conclusion1.In this research,we discovered that TP73-AS1 is markedly up-regulated in the EOC tissues and cells.Moreover,this aberrant high level of TP73-AS1 is responsible for the poor clinical outcome for the EOC patients.TP73-AS1 is found to be an oncogene in EOC.2.The mechanical research revealed that TP73-AS1 epigenetically silenced the p21 transcription through binding the EZH2 and H3K27me3 with p21 promoter.3.The work reveals that the TP73-AS1 exerts a critical function in the EOC as an oncogene in the proliferation,invasion and tumor growth,through epigenetically silence p21 protein. |
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