The Role And Mechanism Of Ovarian Cancer Exosomal SPOCD1-AS Remodeling Peritoneal Mesothelial Cells To Promote Cancer Peritoneal Metastasis

Tumor metastasis is a multi-factor,multi-step cascade concerning tumor cell behavior and target tissue specificity.As the mediators of cell-to-cell communication,exosomes carry active factors such as nucleic acids,proteins and lipids,and participate in all stages of the metastasis cascade.Long non-coding RNA(lncRNA)has been reported to be widely present in exosomes,and plays a complex role in gene regulation,which has become a focused area these years.Ovarian cancer remains the highest fatality rate of gynecologic malignant tumors.Widespread abdominal metastasis is one of the main causes of poor prognosis of patients with advanced ovarian cancer.In the peritoneal environment,the mesothelial layer composed of peritoneal mesothelial cells is the first barrier to prevent the invasion of bacteria and tumors.The mesothelial cells undergo the mesothelial-mesenchymal transition(MMT)process to facilitate tumor peritoneal implantation,however,the mechanism behind remains elusive.To further explore the mechanism of exosomal lncRNA in remodeling peritoneal mesothelial cells and abdominal metastasis of ovarian cancer is of great importance for finding new targeted therapies and improving the prognosis of ovarian cancer patients.In this study,we first isolated and identified exosomes from ovarian cancer cell lines and ovarian cancer ascites,and co-cultured the extracted exosomes with peritoneal mesothelial cells.The MMT process of mesothelial cells was assessed by morphology observation,western blot analysis,and migration assay,the ability of ovarian cancer cell adhesion to mesothelial cells was detected by adhesion assays.We further screened and verified the key lncRNA SPOCD1-AS in the MMT process of mesothelial cells induced by cancer exosomes.We then utilized gene regulation,an orthotopic mouse model to confirm the role of SPOCD1-AS in inducing the MMT process of mesothelial cells and further verify that SPOCD1-AS is packaged by ovarian cancer exosomes and delivered to mesothelial cells to alter the phenotype,thus promoting peritoneal metastasis.We next used RNA pull-down and RNA immunoprecipitation assays to confirm that SPOCD1-AS induces the MMT process of mesothelial cells via binding to G3BP1 protein.In addition,we demonstrated that the two phenylalanine residues(F380/F382)of G3BP1 are the key sites for binding of SPOCD1-AS and G3BP1.We generated peptides which block SPOCD1-AS/G3BP1 interaction,effectively inhibit the MMT phenotype of mesothelial cells,and diminish peritoneal metastasis in orthotopic mouse model.This study aims to clarify the new mechanism of exosomal lncRNA in the peritoneal metastasis of ovarian cancer,and provide new ideas for exploring new targets for ovarian cancer therapeutics.Part Ⅰ Ovarian cancer-secreted exosomes induce the MMT process in mesothelial cellsObjective:To confirm that exosomes derived from ovarian cancer induce the MMT process of peritoneal mesothelial cells and promote ovarian cancer cell adhesion to mesothelial cells.Methods:Ultracentrifugation was used to extract exosomes from ovarian cancer cells(SKOV3,A2780)and immortalised ovarian surface epithelial cells(IOSE-80).Transmission electron microscopy,particle size analysis,and Western blot analysis were utilized to identify the isolated exosomes.The uptake of exosomes by mesothelial cells(MeT-5A)was observed by exosome uptake experiment.Then,exosomes were co-cultured with mesothelial cells.The MMT process of mesothelial cells was assessed by morphology observation,western blot analysis and migration assay,the ability of ovarian cancer cell adhesion to mesothelial cells was detected by adhesion assays.In addition,exosomes of ovarian cancer patient ascites were extracted by precipitation kit and co-cultured with MeT-5A cells to observe the effect of inducing the MMT process of mesothelial cells.Results:1.Typical exosomes can be isolated from ovarian cancer cells and ovarian cancer patient ascites.2.Compared with blank control and immortalised ovarian surface epithelial cell exosomes,ovarian cancer cell exosomes can promote the MMT phenotype of mesothelial cells and enhance the adhesion of ovarian cancer cells to mesothelial cells.3.Compared with blank control and benign tumor peritoneal fluid exosomes,ovarian cancer ascites-derived exosomes can also promote the MMT phenotype of mesothelial cells and enhance the adhesion of ovarian cancer cells to mesothelial cells.Conclusions:Ovarian cancer-derived exosomes promote the MMT phenotype of mesothelial cells and enhance the adhesion of ovarian cancer cells to mesothelial cells.Part Ⅱ Ovarian cancer exosome-transmitted SPOCD1-AS induces the MMT process of mesothelial cellsObjective:To verify that ovarian cancer exosome-transmitted SPOCD1-AS induces the MMT process of mesothelial cells,thus promoting cancer cell adhesion to mesothelial cells in vitro and facilitating peritoneal metastasis in vivo.Methods:LncRNA sequencing and qRT-PCR were used to verify the key lncRNA SPOCD1-AS in the process of ovarian cancer exosome-induced MMT phenotype of mesothelial cells.The full length of SPOCD1-AS was determined by RACE and Northern blot assy,and its protein coding potential was analyzed by software.qRT-PCR was used to detect the expression of SPOCD1-AS in ovarian cancer cells and exosomes.SPOCD1-AS intracellular distribution was analysed by FISH.SPOCD1-AS was overexpressed or silenced by overexpression lentivirus or shRNA,respectively.Exosomes with SPOCD1-AS high expression and low expression were purified from SPOCD1-AS stable overexpression IOSE-80 cells and SPOCD1-AS stably silenced A2780 cells,respectively,and co-cultured with mesothelial cells.Finally,a mouse ovarian orthotopic model was established.Various exosomes and exosome secretion inhibitor GW4869 were intraperitoneally injected to observe the effects of ovarian cancer exosomes and exosomal SPOCD1-AS on ovarian cancer peritoneal metastasis.Results:1.SPOCD1-AS is significantly upregulated in mesothelial cells treated with exosomes derived from ovarian cancer cells.Compared with IOSE-80 cells,SPOCD1-AS is highly expressed in ovarian cancer cells;compared with IOSE-80 exosomes,SPOCD1-AS is highly expressed in ovarian cancer cell exosomes.2.SPOCD1-AS promotes the MMT process of mesothelial cells.3.SPOCD1-AS knockdown ovarian cancer cell-derived exosomes do not have the ability of inducing the MMT phenotype of mesothelial cells,while SPOCD1-AS overexpression of normal cell-derived exosomes can mediate the MMT phenotype of mesothelial cells.4.In vivo experiments show that the exosomal SPOCD1-AS can promote the peritoneal metastasis of ovarian cancer,while inhibiting the secretion of exosomes can significantly reduce the peritoneal metastasis of ovarian cancer.Conclusions:1.SPOCD1-AS induces the MMT process of mesothelial cells.2.Ovarian cancer exosome-transmitted SPOCD1-AS induces the MMT process of mesothelial cells and facilitates ovarian cancer peritoneal metastasis.Part Ⅲ SPOCD1-AS induces the MMT process via binding with G3BP1Objective:To further explore the molecular mechanism of SPOCD1-AS promoting the MMT process of mesothelial cells.Methods:RNA pull-down followed with mass spectrum and Western blot analysis and RIP assay were conducted to verify the interaction of SPOCD1-AS and G3BP1.Then SPOCD1-AS and G3BP1 co-localization were detected using FISH immunofluorescence analysis.G3BP1 siRNAs were used to verify the role of G3BP1 in inducing MMT phenotype of mesothelial cells.SPOCD1-AS overexpression while G3BP1 downregulation was used to further clarify that SPOCD1-AS induced the MMT process though G3BP1.G3BP1 RRM domain truncated plasmids and F380L/F382L mutant plasmids were constructed and transfected to verify the binding site of SPOCD1-AS and G3BP1.G3BP1 interfering peptides were synthesized to block SPOCD1-AS/G3BP1 interaction.In vivo and in vitro experiments were conducted to verify the interfering peptides’ effect on inhibiting the MMT phenotype of mesothelial cells and reducing the peritoneal metastasis of ovarian cancer.Results:1.There is a binding relationship between SPOCD1-AS and G3BP1 protein.2.Silencing G3BP1 can inhibit the MMT phenotype of mesothelial cells and reduce the adhesion of ovarian cancer cells to mesothelial cells.3.Co-transfection experiments show that SPOCD1-AS induces the MMT process of mesothelial cells through G3BP1.4.The binding of G3BP1 with the RRM domain truncated and F380L/F382L mutation to SPOCD1-AS was significantly diminished.5.G3BP1 interfering peptides can penetrate the cell membrane,competitively bind to SPOCD1-AS to block the interaction of SPOCD1-AS and G3BP1 in cells.6.G3BP1 interfering peptides can inhibit the MMT phenotype of mesothelial cells and reduce the adhesion of ovarian cancer cells,and significantly reduce the peritoneal metastasis of ovarian cancer in mouse models.Conclusions:1.SPOCD1-AS induces the MMT process of mesothelial cells and promotes the adhesion of cancer cells to mesothelial cells through interaction with G3BP1 protein.2.F380/F382 is the key site for SPOCD1-AS interaction with G3BP1.G3BP1 inhibitory peptide can block the interaction of SPOCD1-AS and G3BP1 and inhibit the MMT phenotype of mesothelial cells,thereby diminishing the peritoneal metastasis of ovarian cancer.