| Objective:Epithelial ovarian cancer(EOC)is a common gynecological malignant tumor.There are no obvious symptoms in the early stage of ovarian cancer,and the long-term prognosis is poor,which seriously affects women's health.Circular RNA(circ RNA)has a length of several hundred to thousand nucleotides.Due to its covalent closed loop stable structure and rich micro RNA(mi RNA)binding sites,circular RNA can adsorb and release mi RNA's inhibitory effect on target gene,and act as a"mi RNA sponge"to participate in the competitive endogenous RNA network and regulate gene expression.Increasing studies have found that circ RNA can participate in tumorigenesis and progression by regulating the expression of related genes.Exosomes are extracellular vesicles with a diameter of 30 to 100nm,which can carry proteins,DNA,RNA and other functional biomolecules,and transfer them to distant organs or neighboring cells to participate in intercellular communication.Tumor-derived exosomes can build a favorable environment for tumor survival,angiogenesis and invasion,and participate in tumor metastasis.Recent studies have shown that circ RNA can be transferred into exosomes and be significantly enriched in exosomes.However,the expression profile and biological functions of circ RNA in ovarian cancer,and their form in exosomes,remain largely unknown.In this study,we focused on exploring the role and specific mechanisms of circular RNA PUM1 and its exosomal form in ovarian cancer.Methods:1.QRT-PCR was performed to detect the relative expression level of circ PUM1in 15 cases of normal ovaries,11 cases of benign ovarian tumors and 76 cases of epithelial ovarian cancer tissues.2.Cell culture and transfection:The p LCDH-circ PUM1overexpressing plasmid was used to stably transfect ovarian cancer CAOV3 cells,which have lower expression of circ PUM1.The sh-circ PUM1-GFP plasmid targeting the ring-forming sequences was used to break up its circular structure in A2780 cells,which have higher expression of circ PUM1.Puromycin was used to screen for the transfected cells in order to establish circ PUM1 overexpression or silence expression cell lines.3.In the above-mentioned circ PUM1 overexpression or silence expression cell lines and their respective control groups,the MTT experiment was used to determine circular RNA PUM1's effect on cell proliferation ability.4.7AAD/PE-labeled Annexin V was used to stain the above-mentioned circ PUM1 overexpression or silence expression cell lines and their respective control groups to analyze the effect of circular RNA PUM1 on ovarian cancer cell apoptosis with flow cytometry.5.Wound healing assay was performed on the above-mentioned circ PUM1 silence or overexpression cell lines and the respective control groups.By measuring the wound healing rate,the effect of circular RNA PUM1 on the migration ability of ovarian cancer cells was analyzed.6.Cell invasion assay:Transwell assay was used to clarify the effect of circular RNA PUM1 on the invasion ability of ovarian cancer cells.7.In vivo xenograft tumor model:BALB/c nude mice were intraperitoneally injected with 1×10~7 of the above-mentioned circ PUM1 knockout and control ovarian cancer cells.After 4 weeks,the nude mice were sacrificed,intraperitoneal metastasis tumor lesions were excised,measured and photographed.8.Dual-luciferase reporter assay:HEK-293T cells were co-transfected with wild or mutant dual-luciferase plasmid,and mi R-615-5p or mi R-6753-5p mimics with scrambled mimics as control.The relative luciferase signal was presented as firefly luciferase activity normalized to renilla luciferase activity.9.Exploration of mi R-615-5p and mi R-6753-5p's function in ovarian cancer cells:Ovarian cancer cell lines CAOV3 and A2780 were transfected with mi R-615-5p or mi R-6753-5p mimics,respectively.With the above-mentioned in vitro cell functional experiments,namely MTT,apoptosis,wound healing and transwell assays,the role of these two mi RNAs in ovarian cancer has been clarified.10.Verify the adsorption of mi R-615-5p and mi R-6753-5p by circular RNA PUM1 affects the phenotype of ovarian cancer cells:The above cell proliferation,apoptosis,wound healing and transwell assays were used to detect the effects of overexpression of mi R-615-5p or mi R-6753-5p in circ PUM1overexpressing cells.11.Western blot:NF-?B2 and MMP2 were detected in CAOV3 and A2780 cells transfected with mi R-615-5p and mi R-6753-5p,as well as in circ PUM1overexpressing cells and control group.The expression of NF-?B2 and MMP2 was also detected in intraperitoneally injected xenograft tumors.After further overexpression of mi R-615-5p or mi R-6753-5p in circ PUM1 overexpressing cells,the expression of NF-?B2and MMP2 were detected.The above experiments have confirmed that the circular RNA PUM1 adsorbs mi R-615-5p and mi R-6753-5p,and regulates the expression of NF-?B2and MMP2.12.Exosomes extraction and identification:Exosomes were extracted from the culture supernatant of circ PUM1 overexpressing and control CAOV3 cells with exosome extraction kits.Western Blot was used to detect the expression levels of exosomal markers.Transmission electron microscope was used to identify the morphology of exosomes.13.Co-cultivation of circ PUM1 exosomes and peritoneal mesothelial cells:The above circ PUM1 exosomes and the control group exosomes were co-cultured with human peritoneal mesothelial cells.The morphology changes of peritoneal mesothelial cells were observed and photographed by an inverted microscope.Western blot was performed to detect protein expression levels of mesenchymal cell markers,as well as circ PUM1downstream targets NF-?B2 and MMP2.14.Intraperitoneal injection of circ PUM1exosomes in nude mice:After intraperitoneal injection of CAOV3 cells forming tumors,the nude mice were given continuous stimulation of exosomes,which were derived from cell culture supernatant of circ PUM1 overexpressing or control CAOV3 cells.After 3weeks,the nude mice were sacrificed.The abdominal cavity was dissected to observe the tumor size and the number of metastases in the abdominal cavity.The peritoneal tissues were excised and HE stained to observe the structural and morphological changes.The above-mentioned mesenchymal cell markers and circ PUM1 downstream targets NF-?B2and MMP2 were detected with Western Blot in peritoneal tissues.Results:1.QRT-PCR results showed that the expression level of circ PUM1 was up-regulated in ovarian cancer tissues(P<0.05),and was positively related with FIGO stage(P<0.05).2.QRT-PCR results showed that after over-expression of circ PUM1,the expression level of circ PUM1 in cells was apparently higher than that of CAOV3 control group(P<0.05).After releasing the ring structure of circ PUM1,the expression level of circ PUM1 in A2780 cells was significantly reduced(P<0.05),which suggested that circ PUM1 overexpression or silence expression cell lines were successfully established.3.MTT assay results showed that the overexpression of circular RNA PUM1 promoted the proliferation of CAOV3 cells(P<0.05).After silencing circular RNA PUM1,the viability of A2780 cells was significantly reduced(P<0.05).4.The results of cell apoptosis experiments showed that overexpression of circular RNA PUM1 inhibited cell apoptosis(P<0.05),and after silencing circular RNA PUM1,cell apoptosis increased evidently(P<0.05).5.Scratch assay showed that,after overexpression of circ PUM1,the migration ability of CAOV3 cells was significantly improved(P<0.05),and it was significantly decreased after circ PUM1 silence in A2780 cells(P<0.05).6.The results of transwell assay showed that the cell invasion ability was significantly improved after circ PUM1overexpression(P<0.05),and it was significantly suppressed after circ PUM1 silence(P<0.05).7.In vivo xenograft tumor model of nude mice showed that compared with the control group,nude mice injected with circ PUM1 silence cells had smaller intraperitoneal tumors and a lower total number of tumor nodules(P<0.05).8.Compared with the mutant type of circular PUM1 luciferase reporter plasmid,both mi R-615-5p and mi R-6753-5p apparently decreased the relative luciferase activity of the wild type one(P<0.05),which confirmed that circ PUM1 can bind with mi R-615-5p and mi R-6753-5p.9.After overexpression of mi R-615-5p and mi R-6753-5p in ovarian cancer cells CAOV3 and A2780,cell proliferation was inhibited(P<0.05);cell apoptosis increased significantly(P<0.05);the migration ability was inhibited(P<0.05);the invasion ability of CAOV3 and A2780 cells decreased(P<0.05).10.Further overexpressing mi R-615-5p or mi R-6753-5p in the above-mentioned circ PUM1 overexpression cells respectively,reversed the effects of increased cell proliferation(P<0.05),migration(P<0.05),invasion(P<0.05)and decreased apoptosis(P<0.05).11.Dual luciferase reporter assay was used to verify that mi R-615-5p and mi R-6753-5p bind to the 3'UTR of NF-?B2 and MMP2,respectively.Western Blot showed that the expression of NF-?B2 decreased after the ovarian cancer cell lines CAOV3 and A2780 were transfected with mi R-615-5p;after transfection with mi R-6753-5p,the expression of MMP2 decreased.Overexpression of circ PUM1 can up-regulate the expression levels of NF-?B2 and MMP2.In addition,compared with control xenograft,the expression levels of NF-?B2 and MMP2 in circ PUM1 silence expression cells intraperitoneally injected xenograft tumors were decreased.After further overexpressing mi R-615-5p or mi R-6753-5p in the above-mentioned circ PUM1overexpression cells respectively,and the effect of up-regulation of NF-?B2 and MMP2expression was inhibited.12.We extracted exosomes from culture supernatant of circ PUM1 overexpressing and control CAOV3 cells.Western blot confirmed the expression of exosome-specific markers,such as HSP-70 and CD 9.Transmission electron microscopy showed that the diameter of exosomes was 20-100 nm.QRT-PCR results showed that the expression level of circ PUM1 was higher in the exosomes isolated from the supernatant of circ PUM1 overexpressing cells,than that from control group(P<0.05).13.The HMr SV5 cells treated with exosomal circular RNA PUM1 changed to fibroblast-like cells more obviously,with fewer tight junctions between cells.Western blot showed that FAP-1,vimentin,?-SMA,NF-?B2 and MMP2 were up-regulated in HMr SV5 cells treated with circ PUM1 exosomes compared with the control group.14.The number of tumor foci in exosomal circ PUM1 treated nude mice abdominal cavity was significantly higher than that in control group.HE staining showed that the mesothelial cells in peritoneum treated with circ PUM1 exosomes were loosely arranged,infiltrated with tumor cells,and the submesothelial matrix layer was reactively thickened.Western blot showed that the protein expression of FAP-1,vimentin,?-SMA and the downstream targets of circ PUM1,NF-?B2 and MMP2,were up-regulated in peritoneum treated with circ PUM1exosomes.Conclusion:1.Circular RNA PUM1's expression level is evidently upregulated in epithelial ovarian cancer tissues,and it is positively correlated with FIGO stage.2.Circular RNA PUM1 can affect the phenotype of ovarian cancer cells,more specifically,increase cell vitality,migration and invasion,inhibit cell apoptosis,and promote the tumorigenesis and metastasis ability of ovarian cancer cells in vivo.3.By adsorbing mi R-615-5p and mi R-6753-5p,circular RNA PUM1 relieves the inhibitory effects of these micro RNAs on target genes NF-?B2 and MMP2,thereby up-regulating the expression of NF-?B2 and MMP2,and affects ovarian cancer cell phenotype.4.Circular RNA PUM1 can be taken up by peritoneal mesothelial cells in the form of cancer-derived exosomes.Exosomal circular RNA PUM1 induce peritoneal mesothelial cells to undergo"mesothelial-mesenchymal"transition,and promote the dissemination of ovarian cancer in abdominal cavity. |
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