CDIPTOSP Regulates The Proliferation And Migration Of Ovarian Cancer Cells By Promoting KLF17 Degradation Through The SMD Pathway

Objective: Ovarian cancer is the most deadly malignancy in gynecology,most of which are primary tumors and a small proportion of which are metastases from other sites,with poor prognosis and high recurrence rate,and the underlying mechanisms of its progression are complicated and unclear.As an important class of regulatory molecules,long-stranded non-coding RNAs play a crucial role in many biological processes such as epigenetic,transcriptional and post-transcriptional regulations.We identified a conserved cancer-testis-associated long non-coding RNA,CDIPTOSP,and no studies have been reported on the regulation of ovarian carcinogenesis at CDIPTOSP expression level.Long non-coding RNAs can regulate m RNA stability by recruiting proteins to degrade m RNA.Among them,STAU1-mediated m RNA decay pathway is one of the ways of m RNA degradation by long non-coding RNAs in mammalian cells.Abnormal expression of KLF17,an oncogenic factor,has been associated with the development of several cancers.In this study,we investigated the expression and function of CDIPTOSP,STAU1 in ovarian cancer cell lines,and further investigated that CDIPTOSP,STAU1 co-regulate KLF17 to affect the functional phenotype of ovarian cancer cells,thus revealing new molecular targets in ovarian cancer.Methods: 1.Long non-coding RNAs associated with ovarian cancer were mined through the public database GEO/TCGA/GTEx,and characteristic long non-coding RNAs were identified by differential analysis and prognostic analysis.CDIPTOSP silencing plasmids were transfected in ovarian cancer cells SKOV3 and A2780,divided into si-NC group and si-CDIPTOSP group,The transfection efficiency was calculated by RT-q PCR,the cell absorbance was measured by CCK-8 to reflect cell proliferation,the migration ability was measured by Transwell,and the tumorigenic ability was measured by clonogenesis assay.The weight and growth trend of subcutaneous transplanted tumors and lung metastases were detected by nude mice,and the proliferation and cell morphological changes were observed by HE staining and Ki-67 immunofluorescence assay.2.The interacting proteins of CDIPTOSP were identified by RNA pull down,the results of RNA pull down were identified by mass spectrometry,and the binding to the interacting proteins was verified by Western Blot assay.The downstream interacting protein silencing plasmids were transfected in ovarian cancer cells SKOV3 and A2780,and divided into si-NC group and si-STAU1 group.RT-q PCR,CCK-8 assay,Transwell and clone formation assays were performed to detect transfection efficiency,cell proliferation viability and migration ability as well as the ability of cells to become tumour-forming in vivo.3.Ovarian cancer cells SKOV3 were transfected with CDIPTOSP,STAU1 silencing plasmids,sequencing analysis of si-NC group,si-CDIPTOSP group and si-STAU1 group to find co-regulatory proteins,and software to predict the binding sites of CDIPTOSP and downstream target genes.RT-q PCR were performed to detect the effect of CDIPTOSP and STAU1 on the m RNA expression levels of downstream co-regulatory proteins.The enrichment of STAU1 in RNA pull down by CDIPTOSP and KLF17 was examined by RIP-q PCR assay.4.Phenotype rescue assay to detect the regulatory relationship of CDIPTOSP as well as downstream target proteins on ovarian cancer.The CDIPTOSP and KLF17 si RNA were transfected in ovarian cancer cell lines SKOV3 and A2780 and divided into si-NC group,si-CDIPTOSP group,si-CDIPTOSP+si-KLF17 group,CCK-8 viability assay and clone formation assay to detect proliferation of ovarian cancer cells;Transwell assay to detect migration of ovarian cancer cells silencing phenotype rescue assay;in ovarian cancer cell lines SKOV3 and A2780 transfected with CDIPTOSP,KLF17 over-expression vector,divided into EV group,OV-CDIPTOSP group and OV-CDIPTOSP+OV-KLF17 group.CCK-8 viability assay and Transwell assay was performed to detect the proliferation ability and migration ability of ovarian cancer cells;clone formation assay to detect the ability of ovarian cancer cells to become tumor in vivo transfection.Results: 1.Biological analysis reveals high CDIPTOSP expression in ovarian cancer from GEO and TCGA data,and the higher the expression level,the lower the survival rate in the progression-free survival.In vitro cell function assay showed that the proliferation,migration and clone formation ability of si-CDIPTOSP group were weakened compared with si-NC group.In vitro nude mice tumorigenesis assay results showed that It was confirmed that the weight and growth trend of subcutaneous transplanted tumor,HE staining,and Ki-67 fluorescence signal intensity were weaker in si-CDIPTOSP group than in si-NC group.The HE staining of lung metastases showed the same results.2.RNA pull down revealed the CDIPTOSP-interacting protein STAU1.Silencing of STAU1 for cell function assay showed that the proliferation,migration and clonogenic ability of cells in the si-STAU1 group were reduced compared to the si-NC group.3.RNA-seq identified CDIPTOSP and The downstream target gene KLF17,which is co-regulated by STAU1,was predicted by raw signal that CDIPTOSP and KLF17 m RNA can complementarily bind and the 3’UTR of KLF17 contains Alu element.RT-q PCR examination of si-CDIPTOSP and si-STAU1 followed by elevated KLF17 m RNA expression level The results of the RIP-q PCR assay showed that the STAU1 m RNA enriched to the CDIPTOSP-KLF17 complex was significantly reduced in the si-CDIPTOSP group compared to the si-NC group.4.The results of the silencing phenotype rescue assay showed that KLF17 could reverse the inhibitory effects of CDIPTOSP silencing on ovarian cancer cell proliferation,migration and clone formation;The over-expression rescue assay showed that KLF17 could reverse the promotive effects of CDIPTOSP over-expression on ovarian cancer cell proliferation,migration and clone formation.Conclusions: 1.CDIPTOSP is highly expressed in ovarian cancer and reducing its expression inhibits the proliferation and migration of ovarian cancer cell lines.2.STAU1 is an interacting protein of CDIPTOSP and promotes the proliferation and migration of ovarian cancer cell lines.3.KLF17 is a downstream co-regulatory protein of STAU1 and CDIPTOSP.CDIPTOSP is regulated by binding to KLF17 m RNA 3 ’UTR of Alu sequence target binding,recruiting STAU1 and mediating SMD.4.CDIPTOSP plays a role in promoting malignant biological progression of ovarian cancer cells by targeting and regulating changes in KLF17 through the SMD pathway.