| Objective:Based on the previous work,this study aimed to investigate the expression and clinical significance of SERPIND1 in epithelial ovarian cancer,as well as its effect on the malignant biological behavior of ovarian cancer cells and the related regulatory mechanisms.And we also researched the interaction proteins of SERPIND1.Further,we have also investigated the upstream regulatory mechanism of SERPIND1.By elucidating the mechanism underlying the promotion of malignant biological behavior of ovarian cancer by SERPIND1,we demonstrated that SERPIND1 could potentially serve as a novel drug target.Methods : Immunohistochemistry and western blotting methods to detect the expression of SERPIND1 in ovarian epithelial tumor tissues,normal ovarian tissue and human ovarian cancer cell lines.And we analyzed the data of the 113 patients with primary epithelial ovarian cancers and compared the clinicopathological parameters with SERPIND1 tissue expression.At the same time,the proteins in tissues were extracted and the expression level of SERPIND1 protein was detected by western blot.The above results were analyzed the differences in SERPIND1 expression between malignant,borderline,benign,and normal ovarian tissues,respectively.The SERPIND1 gene overexpressing ovarian cancer cell line ES-2-SERPIND1-H and the control cell line ES-2-SERPIND1-H-Mock were constructed separately,and the transient transfected cell lines CAOV3-SERPIND1-L and OVCAR3-SERPIND1-L obtained by small interfering RNA were constructed and its control cell lines CAOV3-SERPIND1-L-Mock and OVCAR3-SERPIND1-L-Mock.Western Blot was used to detect the change of SERPIND1 before and after transfection of SERPIND1 gene.Flow cytometry was used to detect changes in apoptosis before and after the differential expression of SERPIND1.The scratch-wound assay and Transwell experiments were used to study the effects of SERPIND1 on the migration and invasion of ovarian cancer cells.The effect of SERPIND1 on the proliferation of ovarian cancer cells was studied using the MTT assay.PI staining to detect differential expression of cells before and after SERPIND1 Cyclic changes.Annexin-V-APC/PI or Annexin-V-PE/7AAD double staining assay was used to detect changes in apoptosis before and after the differential expression of SERPIND1.The proliferation and peritoneal metastasis changes before and after the differential expression of SERPIND1 in ovarian cancer cells were detected in vivo by establishing the subcutaneous xenograft model and the peritoneal xenograft model in nude mice.Analysis of SERPIND1 interacting protein by protein-mass spectrometry in CAOV3 cell line.The results were verified by co-immunoprecipitation.Western blot was used to detect the PI3K/AKT signaling pathway node proteins PI3 K,p-pi3 k,AKT and p-akt before and after the differential expression of SERPIND1.MMP2,MMP9,e-cadherin,n-cadherin,Vimentin and other proteins that closely related to cell migration and invasion were also detected.The PI3K/AKT pathway inhibitor LY294002 was added to the ES-2 cells with stable SERPIND1 overexpression,and the changes in migration,invasion,proliferation,apoptosis,and cell cycle of ovarian cancer cells were monitored.The binding of NF-?B1 protein to the transcriptional regulatory region of SERPIND1 was detected by ChIP assay.Western blot was used to detect the changes of SERPIND1 before and after NF-B1 gene silencing.Results : Immunohistochemistry results showed that SERPIND1 was primarily located in the cytoplasm,with a small amount in the nucleus.The majority of the staining showed evenly distributed yellow or yellow-brown granules or sheets.Positive and strongly positive rates of SERPIND1 expression were reported for the malignant group(90.40% and 67.20%,respectively),borderline group(62.50% and 37.50%,respectively),benign group(20.83% and 16.67%,respectively),and normal ovarian tissue group(12.50% and 0.00%,respectively).SERPIND1 was highly expressed in the malignant group,compared with the borderline,benign,and normal groups(P < 0.05 in all cases).The strongly positive expression rate of SERPIND1 was significantly higher in FIGO stage III–IV epithelial ovarian cancers than in stage I–II epithelial ovarian cancers(85.1% vs.43.5%,P < 0.01).Positive and strongly positive expression rates of SERPIND1 were reported for the poorly differentiated group(97.1% and 67.6%,respectively),the moderately differentiated group(90.9% and 75.8%,respectively),and the well-differentiated group(83.3% and 50%,respectively).SERPIND1 expression increased with decrease in the degree of differentiation,but the trend was not significantly different.Meanwhile,positive and strongly positive expression rates of SERPIND1 were higher in the lymph node metastasis–positive group than in the lymph node metastasis–negative group,but the differences were not statistically significant(100% and 82.4% vs.94.6% and 76.8%,P > 0.05 in all cases).Cox regression was used to analyze the effects of FIGO stage,SERPIND1 expression,pathological type,lymph node metastasis,degree of differentiation,and other factors on postoperative survival and recurrence.We found that FIGO stage and SERPIND1 expression were independent risk factors of prognosis of epithelial ovarian cancer(P < 0.05).In addition,our data showed that SERPIND1 overexpression was associated with shorter disease-free survival rates and shorter overall survival times in ovarian cancer patients.The ovarian cancer cell line ES-2 that had low SERPIND1 expression was selected as the experimental cell line for the overexpression of the SERPIND1 gene.Compared with the control group,the SERPIND1-overexpressing ES-2 cells showed significantly increased proliferation,migration,and invasion capacities and an increased proportion of S-phase cells.Conversely,the apoptotic rate was significantly decreased.The expression of the SERPIND1 gene was inhibited in the ovarian cancer cell lines CAOV3 and OVCAR3,compared with the controls,and these cells showed a significant reduction in proliferation,migration,and invasion capacities,a reduced proportion of S-phase cells,and a significant increase in apoptotic rate.We established the subcutaneous xenograft model and the peritoneal xenograft model in nude mice.Tumor growth curves showed that the growth rate of tumors that developed from the ES-2-SERPIND1-H cells was significantly higher than that of the ES-2-SERPIND1-H-Mock cells.The average tumor volume and tumor weight in the ES-2-SERPIND1-H cells group were apparently higher than the control group.An autopsy revealed a number of peritoneal metastatic nodules in the SERPIND1 overexpression group,whereas few metastatic nodules were detected in the control group.The interaction protein of SERPIND1 is Vimentin.The results were confirmed by protein-mass spectrometry and co-immunoprecipitation.In this study,we have found for the first time that SERPIND1 overexpression in ovarian cancer cells resulted in increased expression of p-PI3 K and p-AKT,with concurrent significant reduction in E-cadherin expression and increased expressions of N-cadherin,Vimentin,MMP2,and MMP9.In contrast,the inhibition of SERPIND1 expression in ovarian cancer cells reduced the phosphorylation of PI3K/AKT,increased E-cadherin expression,and reduced the expressions of N-cadherin,Vimentin,MMP2,and MMP9.The results showed that after the addition of the PI3K/AKT pathway inhibitor LY294002,the SERPIND1-overexpressing ES-2 cells exhibited significantly reduced migration,invasion,and proliferation capacities,a reduced proportion of S-phase cells,and a significantly increased apoptotic rate.The ChIP assay revealed that NF-?B1 could bind to the promoter region of SERPIND1 at the-258 to-248(GGTGTTTTCCA)and +131 to +141(AGGCCTTCCTC)from the transcription start loci(Fig.5F,G).Additionally,SERPIND1 expression was also reduced following the inhibition of NF-?B1 expression.Conclusion :SERPIND1 was highly expressed in the malignant group,compared with the borderline,benign,and normal groups.In addition,our data showed that SERPIND1 overexpression was associated with shorter disease-free survival rates and shorter overall survival times in ovarian cancer patients.SERPIND1 promoted the proliferation,migration,invasion,G1-to-S phase transition,and EMT of ovarian cancer cells as well as inhibited their apoptosis via the PI3K/AKT pathway.The interaction protein of SERPIND1 is Vimentin.NF-?B1 could bind to the promoter region of SERPIND1.And NF-?B1 exerted a regulatory effect on SERPIND1 expression. |
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