| Objective:To elucidate Linc-ROR in the stemness acquisition of Ovarian cancer stem cells(OCSCs),and further investigate the effects of Ovarian cancer derived exosome-mediated Linc-ROR on Ovarian stem cell-like characteristics and tumor progression.Methods:(1)Serum-free suspension culture method was used to induce A2780 cells to produce OCSCs,and sphere-forming experiments,differentiation experiments,and flow cytometry analysis of the proportion of CD133~+cells were used to identify stem cell characteristics;q RT-PCR was used to detect the expression of Linc-ROR m RNA in cells before and after the induction of OCSCs.(2)Cell lines with stable low and high expression of Linc-ROR m RNA were screened by lentivirus transfection,and si-Linc-ROR and p-Linc-ROR were induced to generate si-OCSCs,p-OCSCs and control OCSCs respectively,then evaluates the spheroidizing ability of the three groups of OCSCs by the sphere-forming rate,maximum sphere diameter and sphere-forming period;immunofluorescence staining detected the expression of stem cell marker CD133.Subcutaneous tumorigenesis in nude mice was used to verify the tumorigenicity of OCSCs in vivo.(3)Exosomes from ascites and A2780 cell were extracted by ultracentrifugation,then authenticating them.Under the electron microscope,Nanoparticle tracking analysis(NTA)and Western blot.The ascites and cell-derived exosomes were co-cultured with OCSCs respectively.The groups were:control group(control),ascites-derived exosomes group(ADE+OCSCs),cells-derived exosomes group(CDE+OCSCs).To evaluate the sphere-forming ability by spheres-forming cycle,maximum sphere diameter and spheres-forming rate of each group;the expression of CD133 was detected by immunofluorescence staining under microscope;q RT-PCR was used to detected the expression levels of Oct-4,Nanog and m RNA of the signature genes in the stem cells of each group.(4)The effects of exosomes on the proliferation,migration and invasion of adherent cells after colonization and differentiation of OCSCs were detected by plate cloning assay,scratch assay and transwell assay,and the effect of exosomes on abdominal metastasis of tumors was verified by the establishment of mouse metastatic tumor model.Results:(1)The induced OCSCs growed into spheres in suspension and can differentiate into ovarian cancer adherent cells under specific culture conditions.Flow cytometry analysis showed that the proportion of CD133~+cells was significantly higher than before induction(P<0.05),which proved that the induced spheroids possess the characteristics of stem cells;the results of q RT-PCR showed that the expression level of Linc-ROR m RNA in OCSCs(2.51±0.01)was significantly higher than the expression level of Linc-ROR m RNA in the A2780 adherent cell group(1.02±0.03)before induction(t=22.64,P<0.05).(2)Compared with the negative control group,the OCSCs induced by A2780 cells with low expression of Linc-ROR showed decreased sphere-forming rate,prolonged sphere-forming cycle,and decreased maximum sphere diameter(P<0.05),indicating impaired sphere-forming ability.The CD133~+rate was decreased by immunofluorescence staining.The q RT-PCR results also showed that the m RNA expressions of OCT-4 and Nanog,the signature genes of stem cells,were decreased(P<0.05),while the results were reversed after high expression.After 8 weeks of subcutaneous tumor formation,nude mice were sacrificed and dissected.It was found that the tumor size of si-OCSCs,control-OCSCs and p-OCSCs groups increased in turn,suggesting that knockdown of Linc-ROR can reduce the tumorigenicity of ovarian cancer stem cells in vivo,whereas overexpression of Linc-ROR can enhance the tumorigenicity of ovarian cancer stem cells.(3)Under the electron microscope,the lipid bilayer can be seen clearly,showing the hemispherical or cup-like structure with one side depression.Nanoparticle tracking analysis(NTA)results showed that the exosome average particle size was 67.2nm.Western blot showed that the specific protein molecules HSP70,CD63 and CD9 were positive.After co-culture,OCSCs in the three groups could continue to grow in pellets,among which ADE+OCSCs showed two growth modes.Most cells continued to grow in dry pellets,while a small part differentiated adherently.(3)The spheres-forming rate of OCSCs in the control group,ADE+OCSCs group and CDE+OCSCs group were(1±0.2)%,(4±0.1)%and(10±0.25)%,and the differences were statistically significant(F=1271.015,P<0.05).After co-culture of exosomes with OCSCs,the spheres-forming rate were significantly increased,the spheres-forming rate were shortened and maximum sphere diameters were larger(P<0.05),and the pelleting ability of cells in the group of CDE+OCSCs was enhanced most significantly(P<0.05).Immunofluorescence staining showed that the number of CD133 green fluorescent chromophore cells in OCSCs in the two groups was significantly higher than that in the control group after the addition of exosomes in co-culture,that is,the positive rate of CD133 was higher than that in the control group.The q RT-PCR results showed that the expression level of Oct-4 m RNA in ADE and CDE groups was(3.46±0.24,4.03±0.31),compared with that in control group(1.04±0.12),the differences were statistically significant(F=134.932,P<0.05).The m RNA expression levels of Nanog were(1.57±0.32,2.66±0.15),which were significantly higher than those in the control group(1.00±0.07),and the differences were statistically significant(F=49.329,P<0.05).And the expression of both in CDE+OCSCs group increased more significantly(P<0.05).(4)The results of plate cloning experiment showed that the number of cell clones in control group,ADE group and CDE group was(200±10),(400±15)and(600±17),respectively.The number of clone in ADE group and CDE group was higher than that in control group(F=586.319,P<0.05),and the number of clone in CDE group was more than that in ADE group(t=15.51,P<0.05).The cell migration rate in ADE group and CDE group was(76±4)%and(87±5)%,respectively,which were significantly higher than(60±3)%(F=33.180,P<0.01),and the increase was more obvious in CDE group than in ADE group(t=2.976,P<0.05).Transwell invasion assay in vitro showed that the number of invaded cells in ADE group and CDE group was(160±6)and(250±4),respectively,which were significantly higher than that in negative control group(100±3)(F=840.984,P<0.01),and the increase was more obvious in CDE group than in ADE group(t=21.62,P<0.05).The tumor volume of the ovarian transplantation mice injected with ADE and CDE was significantly larger than that of the control group,and the tumor in situ formed with CDE group was the largest.In addition,anatomy also found that the liver,kidney,intestine and mesentery of nude mice after injection of exosomes had metastatic tumors of varying degrees,but no metastatic lesions were observed in the control group.These results indicate that exosomes from ovarian cancer can promote the growth and peritoneal metastasis of orthotopic xenograft tumor in nude mice.Conclusion:(1)In this study,we confirmed that Linc-ROR plays an important role in the regulation of stem cell-like properties of ovarian cancer(2)Ovarian cancer-derived exosomes can mediate the transfer of Linc-ROR to OCSCs,which is an important regulatory pathway of Linc-ROR.enhance their stem cell-like properties,and promote tumor proliferation,migration,invasion and metastasis.Moreover,CDE were superior to ADE. |
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