| Ovarian cancer(OC)is a malignant tumor of the female reproductive system.A large part of ovarian cancer originates from the epithelial cells of the female reproductive system.Therefore,this type of tumor is named epithelial ovarian cancers(EOC).EOC has the characteristics of late diagnosis,prone to chemotherapy resistance,and low five-year survival rate.A very important reason for the poor prognosis of epithelial ovarian cancer is the lack of early and effective prognostic and diagnostic methods and markers.How to effectively deal with the drug resistance of tumors is a hot and difficult point in the research of ovarian cancer.Better understanding the pathogenesis of ovarian cancer is of great significance for the diagnosis and treatment of EOC patients.Gli(Gli)is a key transcription factor in Hedghog(HH)signaling system.Gli proteins include different subtypes.The activation of this signaling system in adulthood is closely related to the occurrence and development of a variety of tumors.Studies have shown that Gli,as an important transcription factor of the HH signaling system,is also involved in the occurrence and development of a variety of tumors.In recent years,more and more studies have shown that the HH signaling system is closely related to cancer stem cells,and Gli has also been shown to be involved in the formation of cancer stem cells in various of tumors.According to the theory of cancer stem cells,the role of Gli in the occurrence,development,drug resistance,and recurrence of tumors has gradually been paid attention to.In some tumors,NANOG was thought to be a cancer stem cell marker,and NANOG may be a downstream factor of Gli,both of which are related to the malignant behavior of tumors and the formation of cancer stem cells.At present,limited study has been conducted on the role of Gli1 and Gli2,which can activate the HH pathway in EOC,as well as the related mechanisms,and their correlation with the expression of cancer stem cell marker NANOG in EOC.In this study,we focus on the above problems.The research 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 Gli and NANOG in epithelial ovarian cancerObjective: The aim of the first part of the study is to reveal the expression levels of Gli,Gli2 and NANOG in epithelial ovarian cancer,and the correlation between Gli and NANOG expression was analyzed.The relationship between these three protein with clinicopathological characteristics and prognosis of EOC patients was also researched.Methods: Specimens from 61 cases of epithelial ovarian cancer(EOC)and their complete clinicopathological data were collected.Gli1,Gli2 and NANOG protein expression in EOC was detected by immunohistochemistry.The relationship between the expression of Gli1,Gli2 and NANOG and clinicopathological significance and prognosis of EOC patients,as well as the correlation between the expression levels of Gli and NANOG was analyzed.Results:1.The characteristics of clinicopathological data of EOC specimensThis study contain 61 EOC patients with complete clinicopathological data.Among them,20 patients(32.8%)were under 50 years old,and 41patients(67.2%)were 50 years old and above.According to histological classification,serous ovarian cancer is the most common tissue type,with 38cases(62.3%),followed by endometrioid ovarian cancer in 10 cases(16.4%),and mucinous ovarian cancer in 10 cases(16.4%).23 patients(37.7%)had lymph node metastasis,and 40 patients(65.6%)were classified as G2-3.Among 61 patients,44 patients(72.1%)were sensitive to platinum,and 17patients(27.9%)were platinum-resistance.2.Expression of molecules in EOC and their association with clinicopathological features.There were 38 cases(62.3%)of cytoplasmic Gli1+ patients,and its expression was positively correlated with platinum resistance and surgical pathological stage of patients(P<0.05),and its expression also increased with the increase of surgical pathological stage of EOC patients(P<0.05).There were 12 cases(19.7%)with positive expression of nuclear Gli1,and its expression had no correlation with clinicopathological parameters of EOC patients.The cytoplasmic Gli2+ was 34 cases(55.7%),and the cytoplasmic expression of Gli2 had no correlation with the clinicopathological parameters of EOC patients.36 cases(59.0%)were positive for nuclear Gli2 expression.In platinum-resistant cases,the positive rate of nuclear expression of Gli2increased(P<0.001).The expression position of NANOG is mainly located in the cytoplasm,and 36 cases(59.0%)of NANOG+.With the increase of surgical pathological stage,the expression rate of NANOG increased(P<0.001),and in platinum-resistant cases,its positive expression rate increased(P<0.05)3.Correlation of Gli expression with NANOG in EOC tissue.The protein expression of Gli1 in the cytoplasm was significantly positively correlated with that of NANOG in EOC tissues(r=0.796;P<0.001).Cytoplasmic Gli2 expression was also significantly positively correlated with NANOG levels(r=0.264;P=0.039).However,there was no statistically significant correlation between nuclear Gli2 expression and NANOG levels(r=0.187;P=0.145).4.Gli and NANOG expression are negatively associated with EOC patient survival.Kaplan-Meier analysis showed that the expression of cytoplasmic Gli1,nuclear Gli2 and NANOG expression are negatively associated with EOC patient survival.Overall,Gli1,Gli2 and NANOG expression were significantly associated with reduced survival of patients with EOC.The results of Cox regression showed that the expression of nuclear Gli2 and NANOG are independent prognostic factors for the survival time of patients.The survival time of nuclear Gli2+ patients(P=0.001;HR,2.896;95% CI,1.520-5.517)and NANOG+ patients(P=0.002;HR,2.857;95% CI,1.470-5.550)are shorter.Summary: In EOC tissues,the expression levels of Gli1 and NANOG in the cytoplasm are related to FIGO staging and sensitivity to platinum drugs.The expression level of nuclear Gli2 is correlated with platinum drug sensitivity.The expression of Gli1 and Gli2 in the cytoplasm is significantly positively correlated with the expression of NANOG in epithelial ovarian cancer;the positive expression of cytoplasmic Gli1,nuclear Gli2,and NANOG is closely related to the prognosis of epithelial ovarian cancer.The expression of nuclear Gli2 and NANOG is independent prognostic factors of epithelial ovarian cancer.Therefore,Gli1,Gli2,NANOG may be potential tumor markers of epithelial ovarian cancer.Gli and NANOG have important significance for the prognosis of patients with epithelial ovarian cancer.Part two The effect of inhibiting or activating the HH signaling pathway on the biological characteristics of epithelial ovarian cancer cells and its related mechanismsObjective: Treatmen of HH signaling pathway inhibitor GANT61 and HH signaling pathway agonist SHH was performed on epithelial ovarian cancer cells,then we anlyzed cancer cell proliferation,colony-forming ability,migration,invasion,sensitivity to cisplatin,and related molecular expression.The aim of the second part of the study was to explore the effect of Gli on epithelial ovarian cancer cells,and to study its regulatory mechanism.Methods: The Gli inhibitor GANT61 and the HH signaling pathway agonist SHH were added to epithelial ovarian cancer cells to observe changes of the proliferation,clone formation ability,migration,invasion and cisplatin sensitivity of epithelial ovarian cancer SKOV3 cells.qRT-PCR and Western blotting was used to detect the m RNA and protein expression of Gli1,Gli2,NANOG,multi-drug resistance protein MDR1,E-cadherin.All data were analyzed by SPSS 17.0 and Graph Pad Prism5.0 statistical software,and P<0.05 indicated significant difference.Results:1.The effect of HH pathway agonists and inhibitors on the proliferation of epithelial ovarian cancer SKOV3 cells.The results of the CCK-8 assays showed that the proliferation rate of SKOV3 cells in the GANT61 treatment group decreased in a time-and concentration-dependent manner.The OD value were decreased in 100 ?mol/l group of 24 h treatment,the 50 ?mol/l and 100umol/l groups of treatment,as well as the 20,50 and 100 ?mol/l groups of 72 h treatment.Collectively,this suggested that Gli inhibition via GANT61 treatment reduced ovarian cancer cell proliferation.Compared with the control group,the cell proliferation rate of SHH treatment group showed no significant difference at each time point and each concentration.2.The effect of HH pathway agonists and inhibitors on the colony formation ability of epithelial ovarian cancer SKOV3 cells.The results of the plate colony formation assay showed that the colonyforming ability of SKOV3 cells treated with GANT61 was significantly lower compared with the control group(69.6±2.35% vs.25.1±2.27%),indicating that Gli inhibition reduced colony formation of ovarian cancer cells.The colony formation rate of SHH treatment group decreased from 69.6±2.35%to 58.3±3.13%,the difference was statistically significant.3.Effects of HH pathway agonists and inhibitors on the migration and invasion of epithelial ovarian cancer SKOV3 cellsThe results of Transwell migration and invasion assay showed that after treatment with GANT61 50umol/L,the number of transmembrane cells of SKOV3 cells was significantly less than that of the negative control group,while after treatment with SHH 1000ng/ml,the number of transmembrane cells of SKOV3 cells was increased compared with that of the negative control group.4.Effects of HH pathway agonists and inhibitors on cisplatin sensitivity of epithelial ovarian cancer SKOV3 cellsThe IC50 of cisplatin against SKOV3 cells in response to different treatments groups was assessed using the CCK-8 assay.The results showed that with the increase of the time and concerntration of cisplatin,the proliferation ability was decreased.The IC50 of cisplatin were decreased after treatment with GANT61 of 24 h,48 h and 72 h.The results indicated that SKOV3 cells were significantly more sensitive to cisplatin after inhibition of Gli1 and Gli2.However,there was no significant change in the IC50 of cisplatin in the SHH treatment group at each time point compared with the untreated group.5.Expression of molecules in ovarian cancer cells after treatment with GANT61 and SHH.The results showed that Gli1 and Gli2 expression significantly decreased as expected,as did the levels of NANOG and MDR1 after treatment with GANT61.EMT marker E-cadherin was increased after treatment of GANT61.Exogenous SHH did not result in an increase in Gli1 and Gli2,and even caused a slight decrease in these two proteins compared with the control group.SHH treatment only resulted in a slight increase in migration and invasion of the cell and the expression of NANOG and MDR1 levels compared with the control group.Summary:1.Gli is involved in the regulation of the proliferation,clonality,migration and invasion capabilities and cisplatin resistance of epithelial ovarian cancer SKOV3 cells.Inhibiting the expression of Gli can weaken the above biological functions.2.HH pathway inhibitors change the malignant behavior of epithelial ovarian cancer cells and reduce the "stemness" of tumor cells,which may affect through Gli-NANOG pathway.3.The HH pathway agonist SHH failed to have a significant effect on the various malignant biological behaviors of epithelial ovarian cancer SKOV3 cells,and even caused some biological behaviors to be weakened,indicating the coexistence of canonical and non-canonical HH pathways.Part three The effect of silencing Gli1 gene on the biological characte-ristics of ovarian cancer cells and its mechanismObjective: To observe the effect of Gli1,an important subtype of Gli,on the occurrence and development of epithelial ovarian cancer cells from the aspects of tumor cell proliferation,colony formation,migration,invasion and response to cisplatin,and to study its inner mechanism.Methods: The SKOV3 cell line was transfected with Gli1 siRNA,and the transfection efficiency was measured by qRT-PCR and Western blotting.Through in vitro assay,it was observed the effects of silencing Gli1 on the proliferation,clone formation,migration,invasion and resistance to cisplatin of epithelial ovarian cancer SKOV3 cells.We also detect the expression of Gli2,stem cell marker NANOG,multi-drug resistance protein MDR1,epithelial marker E-cadherin after treatment of gene silencing.Each experiment was repeated three times.All data were analyzed with SPSS 17.0and Graph Pad Prism5.0 statistical software,and P<0.05 indicated significant difference.Result:1.Transfection efficiency of Gli1 geneThe qRT-PCR results showed that after SKOV3 cells were transfected with Gli1 siRNA,the interference transfection sequence effectively inhibited the expression of Gli1 m RNA,and the m RNA was reduced by about 76.4%compared with the negative control group.Western blotting results showed that the expression of Gli1 protein in the transfection group was significantly lower than that in the negative control group(P<0.05).It shows that the Gli1 siRNA sequence can effectively interfere with the expression of Gli1 at the m RNA and protein levels,and can be used to complete subsequent experiments.2.The effect of Gli1 siRNA transfection on cell proliferation of SKOV3.The results of CCK8 assay showed that after 24 hours of Gli1 siRNA transfection,the cell proliferation ability was not significantly different from that of the NC group.After 48 and 72 hours of transfection,the proliferation ability of the interferenced group was significantly lower than that of the negative control group(P<0.05).3.The effect of Gli1 siRNA transfection on the ability of cell colony formation.The results of colony formation assay showed that the number of colony formation of SKOV3 cell in the Gli1 siRNA transfection group was significantly less than that of the negative control group.The colony formation rate in the Gli1 siRNA transfection group was(13.67±3.50)%,and the negative control group was(27.70±1.31).%,the results were statistically significant(P<0.05).4.The effect of Gli1 siRNA on the migration and invasion ability of SKOV3 cellsThe results of the Transwell assay showed that in the migration and invasion assay,the number of cells passing through the chamber in the Gli1 siRNA transfection group was significantly less than that in the negative control group,and the difference was statistically significant(P<0.05).5.The effect of Gli1 siRNA transfection on the cisplatin sensitivity of SKOV3 cellsThe results of CCK-8 assay showed that with the increase of drug concentration and time,the proliferation inhibition rate of the two groups of cells increased.(P<0.05).After the treatment of cisplatin for 24 hours,48 hours and 72 hours,the IC50 of the cells in the Gli1 siRNA transfection group was significantly lower than that of the control group,and the difference was statistically significant(P<0.05).It shows that silencing Gli1 can increase the sensitivity of cisplatin.6.The effect of Gli1 siRNA on the expression of related molecules in SKOV3 cells.The qRT-PCR results showed that compared with the negative control group,no significant changes was observed of the gene expression of Gli2 in the transfection group,and the expression of NANOG,MDR1 were(75.93±6.06)% and(77.20±7.81)% of the negative control group.The difference was statistically significant(P<0.05).The expression of E-cadherin was significantly increased in the si Gli1 group.(P<0.05)?Western blotting results showed that the relative expression of Gli2 protein in the negative control group and the Gli1 siRNA transfection group was(0.27±0.02)and(0.26±0.01),no difference was observed between the two groups(P>0.05);the relative expression of NANOG protein between the two groups were(0.91±0.03)and(0.077±0.015)respectively,and the difference was statistically significant(P<0.05).The relative expression levels of the two groups of MDR1 protein were(0.67±0.03)and(0.35±0.02).The relative expression of E-Cadherin protein in the negative control group and Gli1 siRNA transfection group was(0.023±0.006)and(0.437±0.015),the difference was statistically significant(P<0.05).The above results indicate that silencing Gli1 gene will reduce the stemness and chemoresistance of EOC,and inhibit the EMT process.Summary:1.Gli1 siRNA can effectively interfere with the expression of Gli1 at the m RNA and protein level.2.Silencing the Gli1 gene can inhibit the expression of NANOG,a tumor stem cell marker,and reduce the "stemness" of tumor cells through the Gli1-NANOG pathway,reduce cell proliferation,clone formation and metastasis,and increase cisplatin sensitivity.3.Silencing the Gli1 gene can inhibit the expression of epithelial marker E-cadherin and inhibit the process of EMT.4.Gli,as an oncogene,promotes the occurrence and development of tumors,and may cause ovarian cancer to be resistant to cisplatin.Conclusions:1.The expression of Gli is correlated with NANOG,and is negatively correlated with the survival and prognosis of EOC patients.Therefore,Gli1,Gli2,NANOG may be potential tumor markers of epithelial ovarian cancer.Gli and NANOG have important significance for the prognosis of patients with epithelial ovarian cancer.2.Gli is involved in regulating the biological functions of EOC cells.Targeting Gli can inhibit the malignant biological functions of the EOC cells.The results indicating that the regulation of the Gli gene may change the "stemness" of EOC cells through the Gli-NANOG pathway.3.There may be coexistence of canonical and non-canonical HH signaling pathways in EOC.In summary,the expression of Gli in epithelial ovarian cancer is related to clinicopathological characteristics and prognosis.Gli can be used as a potential tumor marker for epithelial ovarian cancer.By regulating the Gli gene,In a series of in vitro studies,we have confirmed that Gli is involved in the proliferation,colony formation,invasion,migration and platinum resistance of EOC cells and the relative mechenisms.Therefore,provide us new ideas and strategies for the diagnosis and treatment of EOC. |
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