| Ovarian cancer(OC)is a common gynecological malignant tumor in the world.The vast majority of these malignancies are epithelial in origin(as opposed to germ cell or stromal tumors),and they are called epithelial ovarian cancers(EOC).Due to the lack of effective screening methods in the early stage,early diagnosis and treatment are extremely difficult.Approximately,more than 70% of patients get the diagnosis at an advanced stage of EOC.With the limited effective treatments and high rates of chemoresistance,patients at a advanced diseases had a poor prognosis.A recent survey showed that the 5-year survival rate among patients with EOC increased from 36%during 1975–1977 to 46% during 2005–2011(i.e.,in the past 30 years)in the United States.Nonetheless,more than 2/3 of patients eventually experience a relapse after the initial treatment.Once metastasis has occurred,treatment of ovarian cancer is difficult,chemotherapy is the only remaining firstline treatment strategy.How to diagnose ovarian cancer at early stage and how to improve the effect of postoperative chemotherapy has always been a hot and difficult point in the clinical research.In-depth study of the pathogenesis of ovarian cancer,and optimization of early detection methods,are of great significance for the diagnosis and treatment of EOC patients.Milk fat globule EGF factor 8(MFG-E8),also known as lactadherin,is a secreted glycoprotein and is universally present in mammals.Research has revealed that MFG-E8 plays an important role in wound healing,arterial remodeling,and angiogenesis.In recent years,more and more researchers focused on the involvement of MFG-E8 in cancers.Studies have shown that MFG-E8 is expressed in a variety of tumors,e.g.,in esophageal cancer,thyroid cancer,prostate cancer,melanoma,breast cancer,colon cancer,and lymphoma.The MFG-E8 overexpression has been correlated to tumor progression via multiple pathways in several types of cancer cells.A number of studies indicate that MFG-E8 contributes to CSC maintenance,induces chemoresistance of CSCs,and increases their tumorigenic potential through Hedgehog and STAT3 signaling.However,the role of MFG-E8 in EOC,the related mechanisms and the correlation between MFG-E8 and CD133 expression are still unknown.Here we focus on the above issues to develop our research,two aspects are involved: 1.To investigate the expression levels and clinical significance of MFG-E8 and CD133 in EOC;2.To explore the effect and related mechanisms of silencing MFG-E8 gene on cell biological characteristics.The results can provide new ideas and strategies for the pathogenesis and treatment of epithelial ovarian cancer.Part one The expression levels and clinical significance of MFG-E8 and CD133 in epithelial ovarian cancerObjective: The aim of our study was to test whether there is an association between high expression of MFG-E8 and CD133 presence or clinical outcomes of patients with EOC.Methods: Eighty-eight patients with epithelial ovarian cancer with complete clinical and pathological data were enrolled as paraffin specimens,and 38 benign epithelial ovarian tumors were selected as control group.Detection of MFG-E8 expression in EOC and benign ovarian tumors by immunohistochemistry,CD133 expression levels was also analyzed by immunohistochemistry in EOC tumor specimens.The relationship between MFG-E8,CD133 and clinical pathological data of EOC patients was retrospectively analyzed,as well as the correlation of MFG-E8 and CD133 expression levels.The data were analyzed using SPSS 22.0 statistical software,P<0.05 was considered statistically significant.Results:1.Clinical pathological characteristics data of EOC specimensA total of 88 patients with epithelial ovarian cancer in this study,Among them,40 patients(45.5%)were premenopausal and 48(54.5%)were post-menopause.According to histological classification,serous ovarian cancer is the most common tissue type,account for 72.7%,followed by endometrioid ovarian cancer in 14 cases(15.9%),mucinous ovarian cancer is the least common,a total of 10 cases(11.4%).There are 44 patients(50%)with lymph node metastasis.According to the degree of tissue differentiation,58 pateints(65.9%)was in a low differentiation.64patients(72.7%)were sensitive to platinum and 24(27.3%)were treated with platinum primary resistance.2.Expression of MFG-E8 in benign tumors and EOC tissues and expression of CD133 protein in EOC tissuesMFG-E8 staining was present in the cytoplasm and plasma membrane of cells.34 patients were low expresss of MFG-E8 in benign tumors,and 64 patients were high expressers(72.7%;with MFG-E8 SI >6 in EOC specimens).CD133 staining was also specific to the plasma membrane and cytoplasm and was detected in 54 patients’ tumor specimens(61.4%).Ovarian carcinoma tissues with high MFG-E8 expression and CD133 presence constituted 50cases(56.9%);high expression of MFG-E8 alone was found in 14 cases(15.9%),with CD133 presence alone in four cases(4.5%).Low MFG-E8 expression and CD133 absence were noted in 20 cases(22.7%).3.Correlation between the expression of MFG-E8 and the clinical parameters of EOC patientsAdvanced tumors(FIGO III and IV)had significantly higher MFG-E8expression(P<0.001)than early(FIGO I and II)EOCs.Similarly,high MFG-E8 expression more strongly correlated(P<0.001)with the G3 tumor grade than with G1 or G2.Meanwhile,high expression of MFG-E8 were significantly associated with ascites status(P<0.001)and EOC sensitivity to chemotherapy(P<0.05).In contrast,there was no correlation between the high expression of MFG-E8 and age,menopausal status,histopathological type,or metastatic status of lymph nodes among the EOC patients.4.Correlation between the expression of CD133 and the clinical parameters of EOC patientsThere was no no correlation was found between the CD133 protein expression and the menopausal status,lymphatic metastasis and histopathological type(P>0.05).The CD133 protein expression was significantly associated with FIGO stage,ascites status,pathological grade and platinum sensitivity,and was statistically significant(P <0.05,P <0.01).5.Spearman correlation between immunostaining intensities of MFG-E8 and CD133High expression of MFG-E8 directly and significantly correlated with CD133 presence(R = 0.353,P<0.001).6.The Association between High Expression of MFG-E8 or the presence of CD133 with the Survival of EOC PatientsThe follow-up periods ranged from 10 to 84 months.Kaplan–Meier analysis revealed that high expression of MFG-E8 and the presence of CD133 significantly negatively correlated with overall survival among EOC patients.In addition,the patients with high expression of MFG-E8 and the presence of CD133 in the tumor showed shorter overall survival when compared with patients with low expression of MFG-E8 and absence of CD133(P<0.01,P<0.001).Summary:The results showed that the expression levels of MFG-E8 was higher in EOC compared with benign epithelial ovarian tumors.The high expression of MFG-E8 and the presence of CD133 are closely related to the FIGO stage,tumor grade,sensitivity to chemotherapeutic drugs,and ascites status of EOC patients,and the expression levels of two protein are also associated to each other.High expression of MFG-E8 and the presence of CD133 are significantly associated with overall patient survival and support the notion that both MFG-E8 and CD133 are closely associated with a poor prognosis among patients with ovarian cancer.Part two To sthdy the effect and related mechanisms of silencing MFG-E8 gene on cell biological characteristicsObjective: To explore the effects of MFG-E8 on cell proliferation,cell cycle distribution,adhesion,invasion,migration and response to chemotherapeutic drugs in EOC cells.To study the regulation mechanism involve in these process.Methods: To detect the expression of MFG-E8 protein in SKOV3 and A2780 cells by Western blotting.Cells were transfected with MFG-E8 siRNA and NC siRNA,respectively,and the efficiency of transfection was confirmed by Quantitative Real-Time-PCR and Western blotting.CCK8 and plate cloning assay were used to detected cell proliferation.Cell cycle distribution was examined by Flow Cytometry.The effect of silencing MFG-E8 on the adhesion of SKOV3 cells was examined by adherence of cells to artificial basement membrane components.Scratch healing test and Transwell were used to determine the ability of cell migration and invasion.The effect of MFG-E8 siRNA combined with Cisplatin treatment on cell proliferation were observed by CCK8 assay.The mRNA expression of ABCB1 and ABCC1 were detected by Quantitative Real-Time PCR.The effect of silencing MFG-E8 on the expression of CDK4,Cyclin D1 and Caspase-3,ETM-related protein,MMP1,MMP2,MMP9 and AKT/m TOR/p70 S6 K signaling pathways were detected by qRT-PCR and Western blotting,respectively.All experiments were repeated three times,and data were analyzed using Graph Pad Prism 5.0statistical software,P<0.05 was considered statistically significant.Results:1.Expression of MFG-E8 in two different ovarian cancer cell lines and the efficiency of siRNA interferenceThe expression of MFG-E8 protein in SKOV3 cells was significantly higher than that in A2780 cells,and SKOV3 was used for following-up experiments.The expressions of MFG-E8 at mRNA and protein level were observed48 h after transfected with MFG-E8 siRNA or negative control siRNA in SKOV3 cells by Quantitative Real-time PCR and Western blotting,respectively.In comparison with vehicle group or control siRNA group,MFG-E8 mRNA expression was decreased by 74.98%,46.22%,88.31% in Msi #1,Msi #2,Msi #3,respectively.Consistant with Quantitative Real-time PCR results,Western blotting analysis showed that the expression of MFG-E8 protein in Msi#3 was significantly lower when compared with the other two groups(P<0.01).The MFG-E8 siRNA#3 sequence can effectively interfere with the expression of MFG-E8,and the subsequent experiments were performed using MFG-E8 siRNA#3.2.Effects of MFG-E8 siRNA transfection on cell proliferation,cell cycle distribution.1)The results of CCK8 assay showed that cell proliferation was significantly inhibited in SKOV3 cells with MFG-E8 siRNA transfection compared with the Csi group both at 48h、72h、96h and 120 h after transfection(P<0.05).We further observed the effects on cell proliferation by colony formation assay in SKOV3 cells.The results indicated that the numbers of the colonies were reduced significantly in cells transfected with MFG-E8 siRNA(P<0.05).The above results revealed that MFG-E8 siRNA transfection could inhibit the proliferation of SKOV3 cells.2)Compared to the Csi group,the proportion of cells in G0/G1 phase was increased significantly(P<0.01)and the proportion of cells in S phase and G2/M phase was significantly decreased in MFG-E8 siRNA transfected group(P <0.01,P<0.05)by FCM assay.The results were confirmed that MFG-8siRNA can lead cells to be arrested at G0/G1 phase.3)The effect of MFG-E8 siRNA transfection on the expression of CDK4,Cyclin D1 and Caspase-3The Western blotting showed that the relative expressions of CDK4,Cyclin D1 and Caspase-3 proteins were lower in Msi group than those in Csi group(P<0.01).The the expression of CDK4,Cyclin D1 and Caspase3 was down-regulated in SKOV3 cells after silencing MFG-E8 gene compared with Csi group.3.Effects of MFG-E8 siRNA transfection on cell adhesion,migration and invasion1)After SKOV3 cells were transfected with MFG-E8 siRNA,the adhesion rate between Csi group and Msi group to Matrigel was statistically significant(P<0.001);the adhesion rate of Msi to FN was also significantly lower than that of Csi group(P<0.05).This indicates that the silencing of the MFG-E8 gene inhibits the adhesion of SKOV3 cells to the artificial basement membrane.2)Transwell assay results showed that the number of cells passing through the chamber of the MFG-E8 siRNA transfection group was significantly lower than that of Csi,both in the invasion and in the migration experiment(P<0.01).3)The scratch test results showed that the speed which cells migrated towards the scratch was lower in MFG-E8 siRNA transfected cells when compared with Csi group.The area recovery rate of the Msi group was significantly lower than that of the Csi group at 24 h or 48h(P <0.01)4)The effect of MFG-E8 siRNA on EMT process in SKOV3 cells qRT-PCR results showed that silencing MFG-E8 gene downregulated the mRNA expression of N-Cadherin,Vimentin and Snail mRNA,and up-regulated the mRNA expression of E-Cadherin.The results were statistically significant(P<0.05,P<0.01,P<0.001).After MFG-E8 siRNA transfection,the relative expression of E-Cadherin protein in Msi group was higher when compared with Csi group(P<0.01),and the relative expression levels of N-Cadherin,Vimentin and snail+slug were lower in Msi group than those in Csi group(P<0.01,P<0.05).This indicates that silencing the MFG-E8 gene can inhibit the EMT process.5)The effect of MFG-E8 siRNA on the expression of MMP1,MMP2,MMP9The results of qRT-PCR and Western blotting indicated that MFG-E8 siRNA can down-regulate the mRNA and protein expression of MMP1,MMP2 and MMP9,and the results are statistically significant(P<0.05,P<0.001).The results showed that silencing MFG-E8 inhibited the expression of MMP1,MMP2 and MMP9 proteins in SKOV3 cells.4.Effects of MFG-E8 siRNA transfection on SKOV3 cells Cisplatin toxicity1)The results of CCK8 assay showed that with the increasing of drug concentration,the proliferation inhibition rate of each group also increased,and the cell proliferation inhibition rate of Msi group increased significantly(P<0.01).The IC50 of cisplatin in Csi group and Msi group was calculated by Graph Pad Prism 5 statistical software.The IC50 of drugs in Msi group was significantly lower when compared with Csi group(P<0.05).CCK8 assay results showed that downregulation of MFG-E8 expression led to decreased SKOV3 cell proliferation at 48 h or 72 h after using Cisplatin treat the cells(P <0.05).The proliferation of SKOV3 cells treated with MFG-E8 siRNA and Cisplatin was not significantly different at 24h(P>0.05).2)qRT-PCR detection of MFG-E8 siRNA on the mRNA expression of ABCB1 and ABCC1The results of qRT-PCR showed that the mRNA expression of ABCB1 and ABCC1 in Msi group was significantly lower than those in Csi group(P<0.05,P<0.001).5.Effects of MFG-E8 siRNA on AKT/m TOR/p70 S6 k signaling pathway in SKOV3 cellsThe results of Western blotting indicated that MFG-E8 siRNA can down-regulate the protein expression of p-AKT,p-m TOR and p-p70 S6 K,the results are statistically significant(P<0.01).It also showed that silencing MFG-E8 inhibited the phosphorylation of AKT,m TOR and p70 S6,and specifically inhibited the activity of AKT/m TOR/p70 S6 K signaling pathway.Summary:1.MFG-E8 contributed to the process of SKOV3 cells proliferation.Silencing MFG-E8 gene can inhibit the proliferation of tumor cells and arrest the cell cycle in G0/G1 phase,and can reduce the expression of Cyclin D1CDK4 and Caspase-3.2.Silencing MFG-E8 gene can inhibit the adhesion,invasion and migration of SKOV3 cells by inhibiting EMT process and decreasing the expression of MMPs.3.Silencing MFG-E8 gene can increase the sensitivity to chemotherapeutic drugs,and down-regulate the expression of multi-drug resistance proteins ABCB1 and ABCC1 mRNA,indicating that MFG-E8 is associated with chemoresistance.4.The effect of MFG-E8 on EOC in adhesion,migration,invasion and chemotherapy resistance partially dependent on the AKT/m TOR/p70 S6 K signaling.Conclusions:1.The expression levels of MFG-E8 and CD133 are related to FIGO stage,tumor grade,ascites status and chemosensitivity,both of which can be used as prognostic indicators for ovarian cancer;MFG-E8 may be a potential cell tumor marker for epithelial ovarian cancer.2.MFG-E8 is involved in the regulation of EOC cell proliferation.Silencing MFG-E8 can arrest cells in G0/G1 phase and down-regulate the expression of CDK4,Cyclin D1 and Caspase3 proteins.3.Silencing the MFG-E8 gene can increase the sensitivity of cells against anti-tumor drugs,and down-regulate mRNA expression of ABCB1 and ABCC1,and inhibitors against MFG-E8 may become a new target for EOC therapy.4.Silencing MFG-E8 gene may down-regulate the expression of MMP1,MMP2 and MMP9 by inhibiting AKT/m TOR/p70 S6 K signaling pathway,and inhibited EMT process,the adhesion,invasion,migration amd Platinum resistance of SKOV3 cells.In summary,MFG-E8 is highly expressed in epithelial ovarian cancer,and MFG-E8 may be a potential cell tumor marker for epithelial ovarian cancer.We usedRNA interference technology to silence the MFG-E8 gene.In a series of in vitro studies,we confirmed that MFG-E8 is involved in the process of proliferation,invasion,metastasis and Cisplatin resistance of tumor cells,thus providing new ideas and strategies for treatment of MFG-E8 positive malignant tumors. |
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