| Background and purpose:Ovarian cancer is one of the three malignant tumors of female pelvic cavity reproductive organs,its incidence is second only to cervical cancer,endometrial cancer and the third,the incidence of ovarian malignant tumor in recent years has been a rising trend,growing at a rate of 1 ‰ each year.Because the ovary in the depths of the pelvic cavity,the index of early detection and diagnosis methods of the lack of specificity,thus may lead to the discovery of ovarian cancer,its already late clinical stage or already shift,so the poor treatment effect of ovarian cancer,the prognosis is bad.In recent years,as the tumor cells to destroy the loss and chemotherapy level unceasing enhancement,the mortality of the patients with ovarian cancer also gradually improved,but due to the ovarian cancer patients was found to be more advanced and may not be timely and effective treatment,the 5-year survival rate was 90%in the early days,patients with advanced ovarian cancer patients with a 5-year survival rate of less than 30%,the mortality of ranked the top three of malignant tumor of department of gynaecology.The occurrence and development of ovarian cancer is a complicated process with multiple pathways,stages and mechanisms.Although many studies have been conducted to elucidate the pathogenic factors of ovarian cancer,the molecular mechanism of the biological behavior of ovarian malignant tumors remains unclear.More and more evidence has shown that the loss of cancer genes or the desire to be activated by genes and their interactions with each other is the molecular basis for the development of ovarian cancer.Therefore,research and explore the pathogenesis of ovarian cancer,further defined the malignant behavior of ovarian cancer molecular cell biology,looking for more effective monitoring of ovarian cancer molecular biology index,study of important genes associated with ovarian cancer and its molecular mechanism mediated signal pathway will be facing current ovarian cancer clinical treatment and severe issue to be resolved.Ectopic viral integration site-1(Evil)gene is a reverse transcriptase gene loci and its coding protein with zinc finger transcription factor structure,located in chromosome 3 q26,his gene length is 60000 bp of exon 16,is a member of the SET/PR transcription factor family.Evil gene plays a very important role in the growth and development of mammals,and it is found that Evil gene is a carcinogenic transcription factor associated with malignant myeloid tumor.Evil gene,as a reverse transcription factor,binds to the DNA of the target gene through a specific GACMGATA sequence,thus giving full play to its inhibitory or promoting function to its downstream gene expression.Although in the past a lot of research has confirmed that the Evil gene and blood is closely related to the development of tumor,with the deepening of the researchers all over the world to the Evil gene research,Evil gene in the field of other solid tumors are also constantly being found.An increasing number of studies have found that Evil is also closely related to the biological behavior of solid tumors.Recent studies have shown that its in the invasion and metastasis of malignant tumor also plays an important role,for example,found in breast cancer,colon cancer,pancreatic cancer and other tumors Evil highly expressed,and the prognosis of patients with a negative correlation.At home and abroad,there is no correlation Evil and ovarian cancer,researchers report in the role of Evil in ovarian cancer mechanism is not clear,their expression in ovarian cancer,its expression is involved in the occurrence and development of ovarian cancer invasion,metastasis and its problems have not yet seen in ovarian cancer chemotherapy sensitivity.Therefore,this topic proposed study Evil expression in ovarian cancer,to analyze its expression and ovarian cancer occurrence,development and the correlation of invasion,metastasis and influence on ovarian cancer chemotherapy drug sensitivity,and through experiments in vivo and in vitro regulation of the expression of ovarian cancer cell proliferation,apoptosis,invasion,metastasis and angiogenesis ability,the influence of preliminary discussion Evil and chemotherapy for ovarian cancer cell biology behavior impact resistant molecular mechanism,so as to provide new biology for clinical patients with ovarian cancer gene therapy target.This study is divided into the following three parts:part I:Evil expression in ovarian cancer tissue and its correlation with clinical pathological features of patients;The second part:Evil expression is a preliminary discussion on the influence of the proliferation,apoptosis and invasion ability of ovarian cancer cells and the molecular mechanism.Part iii:the effect of Evil expression on the sensitivity of chemotherapy drugs to ovarian cancer cells.Part I:Evil expression in ovarian cancer and its significancePurpose:The relationship between the expression of Evil and the clinicopathological parameters of ovarian cancer patients was analyzed by examining the expression level of Evil in ovarian cancer tissue and the clinical data of patients.Methods:1.Immunohistochemistry and immunofluorescence were used to detect the expression of Evil protein in epithelial ovarian cancer tissue,benign tumor tissue and normal ovarian tissue.2.In situ hybridization technique was used to detect the expression of Evil mRNA in epithelial ovarian cancer tissue,benign tumor tissue and normal ovarian tissue.3.The expression of Evil protein in different ovarian cells was detected by Western blot.4.Analyze the relationship between Evil expression and clinicopathological parameters of ovarian cancer patients.Results:1.Within normal ovarian epithelial tissue,in the Evil protein expression and its main expression in benign ovarian tumor and epithelial ovarian cancer tissue on the nucleus of immunohistochemical positive Evil gene expression is the result of a yellowish-brown particles,immunofluorescence positive expression is the result of the green fluorescent particles.The positive expression rate of Evil protein in normal ovarian epithelial tissue was 0%(0/5),and the staining intensity was O.The positive expression rate of the benign ovarian tissue was 18%(4/22),and the staining intensity was 1.The positive expression rate in epithelial ovarian cancer was 55%(30/55)and the staining intensity was 3.Compared with benign ovarian tumor and ovarian cancer tissue,the positive expression rate of Evil protein was significantly higher in epithelial ovarian cancer tissue,with significant difference,P<0.05.The Evil protein in Ⅰ～Ⅳstage epithelial ovarian cancer specimens in the organization relative expression positive rate was 21.41%,48.65%,65%and 91.42%,compared statistically difference between groups(chi-square = 6.024,P<0.05).The later the stage of ovarian cancer,the higher the positive rate of Evil protein expression.2.In normal ovarian epithelial tissue,Evil mRNA was not expressed,Evil was mainly present in the nucleus of benign ovarian tumor and epithelial ovarian cancer tissue,and the positive expression was brown-yellow granule.The positive expression rate of Evil mRNA in normal ovarian epithelial tissue was 0%(0/5),and the staining intensity was O.The positive expression rate of the benign ovarian tissue was 15%(3/20),and the intensity level was 1.The positive expression rate in epithelial ovarian cancer was 48%(26/54)and the staining intensity was 2.Compared with the normal ovarian epithelial tissue,benign ovarian tumor and epithelial ovarian cancer tissue Evil mRNA positive expression rate and shading was significantly higher,and a significant difference,P<0.05(figure 4)as the Evil mRNA in the ovarian tissue in situ hybridization results.3.Western blot results show that compared with normal ovarian cell HOSEpiC OVACAR3,ovarian cancer cell SKOV3,A2780,ES2,Caov3 and COC1,Evil protein expression of relative quantity respectively is:0.32±0.01,0.53 ±0.02,0.49±0.01,0.51±0.01,0.31±0.01 and 0.01±0.02,the relationship between the Evil protein expression of ovarian cancer cells and normal ovarian cells,compared with statistical differences.4.There was no significant difference between the abnormal expression of Evil protein and mRNA and the patient’s age and pathological type in epithelial ovarian cancer(P>0.05),but its expression was related to clinical stage,histological differentiation and lymph node metastasis(P<0.05).The later the clinical stage,the worse the tissue differentiation,and the distant metastasis of lymph nodes,the more abnormal expression of Evil protein in ovarian cancer tissues.Summary:1.Compared with normal ovarian tissue,Evil protein and mRNA are highly expressed in benign ovarian tumors and epithelial ovarian cancer tissues,which are mainly expressed in the cell nucleus and the cell membrane.Its positive expression rate and stain degree index,ovarian cancer tissue is significantly higher than benign ovarian tumor.2.Compared with normal ovarian epithelial cells,Evil protein is highly expressed in ovarian cancer cells.3.The Evil protein expression in 3,epithelial ovarian cancer and ovarian cancer tissue differentiation,clinical stage and the presence of lymph node metastasis related,ovarian tissue differentiation,the worse,the later and associated with distant metastasis lymph node and clinical stage ovarian cancer tissue Evil protein abnormal expressionof the more obvious.Part two:Inhibition of Evil Expression on the Proliferation,Apoptosis,and Invasion of Ovarian Cancer Cells and Its Molecular MechanismPurpose:To explore the effects of Evil expression on proliferation,apoptosis,invasion,metastasis and angiogenesis of ovarian cancer cells,and preliminarily explore the molecular mechanism of Evil in the biological behavior of ovarian cancer cells.Methods:1.GV159 lentivirus vector was used to construct stable cell lines that interfere with Evil expression in OVCAR3 and SKOV3 cells,and qRT-PCR method was used to verify the success of stable cell lines.2.The effect of interference Evil on proliferation ability of ovarian cancer cells was detected by CCK-8 proliferation assay.3.The effects of interference Evil expression on the cloning ability of ovarian cancer cells were detected by using tablet cloning.4.The changes of cyclin D1 and CDK4 were detected by Western blot.5.The effect of interference Evil expression on the migration ability of ovarian cancer cells was detected by Transwell cell migration experiment.6.The influence of Evil expression on the invasion ability of ovarian cancer cells was detected by Matrigel gel invasion experiment.7.The influence of Evil expression on the angiogenesis of ovarian cancer cells was detected by using tubules.8.Western blot assay was used to detect the changes of AKT,GSK3 and Snail.9.Using annexin-v/PI flow cytometry to detect the effects of interference Evil expression on apoptosis of ovarian cancer cells;Westem blot test was used to detect the effects of interference Evil expression on apoptotic proteins in ovarian cancer cells.10.To establish a model of nude mouse transplanted tumor in ovarian cancer,and to detect the influence of Evil expression on the proliferation of nude mouse transplanted tumor;Immunohistochemical method was used to detect the density of MVD in transplanted tumor tissues,and the effect of Evil expression on proliferation and invasion ability of ovarian cancer cells was analyzed.Results:1.In order to further study the Evil impact on the biological behavior of ovarian cancer cell line,according to the expression level of the Evi 1 in ovarian cancer cell line,we present the Evil high expression of OVCAR3 Evil,SKOV3 cells build interference express lentivirus stable cell lines.The expression vector was labeled with green fluorescent protein EGFP,and real-time fluorescence quantitative PCR method was applied to verify the success of the stable plant.The experimental results show that the SKOV3 cells,the interference and transfection Evil empty plasmid expression vector control cells(SKOV3-anti-NC)compared to slow virus Evil expression plasmid vector transfection interference group cells(SKOV3-anti-Evil)in the Evil mRNA level significantly lowered,the difference was statistically significant;OVCAR3 cells,and transfection interference Evil empty plasmid expression vector control cells(OVCAR3-anti-NC)compared to slow virus Evil expression plasmid transfection interference carrier in the experimental group cells(OVCAR3-anti-Evil)Evil mRNA level significantly lowered,the difference was statistically significant.The results of this study showed that interference Evil expression was successful in the establishment of stable cell lines of ovarian cancer.2.We used CCK-8 to detect the effect of interfering Evil expression on the proliferation ability of ovarian cancer cells.In SKOV3 and OVCAR3 cells interfering Evil expression and setting Blank control Blank group and negative control NC group,the viability of cells was determined by 1d,2d,3d,4d,5d and 6d after transfection.CCK 8 multiplication experiment results show that the transfection of anti-Evil cell proliferation activity was significantly lower than the start of the 2 days Blank Blank group and negative NC transfection cells(p<0.01),3 d,4 d,5 d and 6 d the active reducing degree more significantly(p<0.001).3.The effect of interference Evil expression on the cloning ability of ovarian cancer cells was detected by using plate cloning.The results showed that there was no statistical difference in the clone formation ability of ovcar3-anti-nc group compared with ovcar3-blank group(141.37±2.449%vs 135.19±1.539%,P<0.05).Compared with the control cells of ovcar3-blank and ovcar3-anti-nc group,the clone formation ability of ovcar3-anti-evil group(32.41±1.579%)was significantly inhibited,and the difference was statistically significant(P<0.001).Compared with skov3-blank group,there was no statistical difference in the cloning ability of skov3-anti-nc group cells(126.45± 1.454%vs.117.09±2.872%,P<0 0.05).Compared with the control cells of skov3-blank and skov3-anti-nc group,the clone formation ability of skov3-anti-evil group(49.21 ±2.953%)was significantly inhibited after interfering with Evil expression,and the difference was statistically significant(P<0.001).4.The changes of cyclin D1 and CDK4 were detected by Western blot.The results showed that in OVCAR3 and SKOV3 cell lines,after knockout Evil expression,the expression of phosphorylated AKT,or P-AKT,was reduced,and the expression level of cyclin D1 and CDK4 protein was decreased.5.The effect of interference Evil expression on the migration ability of ovarian cancer cells was detected by Transwell cell migration experiment.The influence of Evil expression on the invasion ability of ovarian cancer cells was detected by Matrigel gel invasion experiment.Transwell Chambers migration experiment results show that the serum-free culture OVCAR3-Blank after 24 hours,OVCAR3-anti-NC,OVCAR3-anti-Evil,SKOV3-Blank,SKOV3-anti-NC,SKOV3 cells-anti －Evil group composed of ventricular surface wear out to the number of cells were 84.25±0.164、81.91 ±0.334、20.43±0.413、86.95±0.381、81.24±0.328、35.53±0.129.Compared with the cells of ovcar3-anti-nc group,the migration ability of ovcar3-anti-evil group was significantly decreased in Evil expression,and the difference was statistically significant.Compared with the cells of skov3-anti-nc group,the migration ability of skov3-anti-evil group which interfered with Evil expression was significantly reduced,and the difference was statistically significant(P=0.0015).6.Matrigel glue affect the experimental results show that the serum-free culture after 48 hours OVCAR3-Blank,OVCAR3-anti-NC,OVCAR3-anti-Evil,SKOV3-Blank,SKOV3-anti-NC,SKOV3-anti-Evil group composed of ventricular surface wear out to the number of cells were 76.23±0.436、73.36±0.131、23.41 ±0.428、89.04±0.549、84.49±0.249、19.22 ±0.252;compared with OVCAR3-anti-NC group of cells,interfere with the Evil express OVCAR3-anti-Evil group cell invasion ability significantly reduced,difference was statistically significant.Compared with SKOV3-anti-NC cells,the invasion ability of SKOV3 cells-anti-Evil was significantly reduced,and the difference was statistically significant.7.Evil promotes the invasion of ovarian cancer cells through AKT/GSK3.The above experiments have verified that Evil can improve the level of p-akt protein,thereby promoting the activation of AKT.This Western blot experiment confirmed that,after knockout Evil expression in OVCAR3 and SKOV3 cell lines,the level of p-AKT protein decreased,and p-gsk3 expression was decreased in p-AKT downstream,while the total GSK3 was not significantly changed.At the same time,the expression level of Smail protein was also down-regulated,and the expression level of e-cadherin protein related to EMT increased,and the expression of Vimentin protein decreased.8.We used small tubes to form an experiment to detect the influence of Evil expression on the ability of angiogenesis in ovarian cancer cells.Collect OVCAR3-Blank,OVCAR3-anti-NC,OVCAR3-anti-Evil,SKOV3-Blank,SKOV3-anti-NC,SKOV3-anti-Evil group of cell culture supernatant,digestion and HUVEC cell,the experimental group cell culture supernatant HUVEC cell suspension,24 hours after observing small tube cavity formation in HUVEC cells.The experimental results showed that there was no statistical difference between the two groups(P>0.05),whether in OVCAR3 cells or SKOV3 cells,and the formation ability of the small tube lumen in the Blank group and the anti-nc group HUVEC cells.Compared with ovcar3-blank(40.86±0.261)and ovcar3-anti-nc(38.57±0.250),the formation ability of the small tube lumen of ovcar3-anti-evil(20.73±0.230)group that interfered with Evil expression was significantly inhibited,and the difference was statistically significant(P<0.05).And SKOV3-Blank(32.02±0.251),SKOV3-anti-NC group(29.12±0.460)compared with SKOV3-anti-interference Evil expression Evil(13.27±0.158)group of HUVEC cells small tube cavity formation ability more significantly suppressed,comparison between the two groups,the difference was statistically significant(P<0.05).9.AnnexinV/PI staining method,according to the results of the experiment OVCAR3-Blank,OVCAR3-anti-NC,OVCAR3-anti-Evil group of cell apoptosis rate respectively(6.18 ±0.022)%,(6.32±0.035)%and(15.59 ± 0.048)%,OVCAR3-Blank and OVCAR3-anti-cells compared with NC group,cell apoptosis rate has no obvious difference(P>0.05).The apoptosis rate of the cells in the ovcar3-anti-evil group was significantly increased compared with that in the two groups,and the difference was statistically significant(P<0.05).The apoptosis rate of skov3-blank,skov3-anti-nc,skov3-anti-evil group was(7.08±0.041)%,(7.30± 0.055)%and(15.58±0.068)%,There was no significant difference in cell apoptosis rate between skov3-blank and skov3-anti-nc group(P>0.05).The apoptosis rate of the skov3-anti-evil group was significantly increased compared with that in the control group,and the difference was statistically significant(P<0.05).Western blot experiment confirmed that after Evil expression was knocked out in OVCAR3 and SKOV3 cell lines,the expression of Bc12 protein was down-regulated,and the expression of cleaved Caspas3 was up-regulated.The effect of Evil expression on apoptosis of ovarian cancer was suggested.The results of this experiment further demonstrate that Evil inhibits apoptosis of tumor cells and induces the proliferation of ovarian cancer cells.10.We used BALB/c-nu female nude mice to establish an experimental model of subcutaneous transplantation of ovarian cancer cells,which was used to interfere with Evil expression in vivo experimental study on the proliferation and invasion of ovarian cancer.Nude mice subcutaneously transplanted tumor experiment results show that the interference OVCAR3 Evil expression-anti-Evil group of nude mice tumor volume was significantly less than OVCAR3-Blank,OVCAR3-anti-NC nude mice in the control group,with the increase of feeding days,nude mice transplanted tumors had differences increase gradually,the experimental group,respectively,compared with between two control groups,differences were statistically significant(P<0.05).Nude mouse transplantation tumor tissue,immunohistochemical detection of tumor microvascular density,according to the results of CD34 endothelial cell markers in nude mice transplantation tumor tissue staining in tan,positioning in the vascular endothelial cell membrane,compared with OVCAR3-anti-NC group of nude mice,OVCAR3-anti-interference Evil expression Evil average MVD in tumor group of nude mice significantly reduced,compared between the two groups,the difference was statistically significant(P<0.05).The results of this study showed that interfering Evil expression could reduce the angiogenesis of tumor tissue,promote apoptotic necrosis,and inhibit tumor proliferation and invasion.Summary:1.In vitro and in vivo experiments confirmed that interfering Evil expression could inhibit the proliferation,invasion and metastasis of ovarian cancer cells and promote apoptosis of cells.2.The biological effects of Evil in ovarian cancer cells are mainly achieved through activation of the P13K-AKT signaling pathway.Part three:Inhibition of Evil expression on the sensitivity of chemotherapy drugs to ovarian cancer cellsPurpose:To explore the effect of interfering Evil expression on the sensitivity of chemotherapy drugs in ovarian cancer and its preliminary discussion on drug resistance mechanism.Methods:1.To construct stable cell lines that interfere with Evil expression in OVCAR3 and SKOV3 cells using GV159 lentivirus vector.2.The growth inhibition rate of the cell lines of OVCAR3-blank,OVCAR3-anti-NC,OVCAR3-anti-Evil,and SKOV3-anti-NC and SKOV3-anti-Evil were detected by CCK-8 method,and IC50 was calculated.3.The cloning inhibition rate of ovarian cancer cells in DDP was detected by soft AGAR culture.4.The expression of multidrug resistance protein P-gp and MRP1 in different cell lines were detected by Western blot.5.Western blot was used to detect the changes of MGMT and MMR in different cell lines after Evil expression was suppressedResults:1.The growth inhibition rates of tumor cell lines were determined by CCK-8 assay and IC50 was calculated.We found that the drug resistance index of OVCAR3 and SKOV3 cell lines alone was 28.73 ± 0.75μg/ml and 27.92 ± 1.07μg/ml,while OVCAR3 and SKOV3 The drug resistance index of the combination of empty vector group and DDP were 27.49 ± 0.52μg/ml and 26.32 ± 0.63μg/ml respectively,however,the resistance index of the combination of OVCAR3 and SKOV3 cells interfering Evil expression group with DDP were 13.67 ± 1.08 μg/ml and 15.86 ±1.31μg/ml respectively,which indicated that the sensitivity of ovarian cancer cells to DDP was significantly increased and the drug resistance was significantly decreased after the inhibition of Evil expression in ovarian cancer cell lines OVCAR3 and SKOV3(P<0.05).2.The cell lines of ovcar3-blank,ovcar3-anti-nc,ovcar3-anti-evil,ovcar3-anti-evil,and skov3-anti-nc and skov3-anti-evil,respectively,were cultured and cloned.Experimental results,as shown in the figure,compared with Blank control group and Blank group NC Evil interference group of SKOV3 and OVCAR3 cell clones in DDP inhibition rate increased significantly(P<0.05),the interference Evil expression plus DDP can obviously reduce the ability to clone of ovarian cancer cells,further confirmed the Evil ovarian cancer cells express downgrade for DDP drug sensitivity significantly higher.3.In order to further explore the mechanism of Evil expression on the influence of the drug resistance of ovarian cancer,we used Western blot to detect the expression changes of multidrug resistance protein p-gp and MRP 1 in different cell lines after the down-regulation of Evil.Results as shown in figure,after we found that inhibit Evil,SKOV3 and OVCAR3 cell lines of P-gp and the expression of MRP 1 significantly decreased(P<0.05),which we consider the Evil may be by adjusting the multi-resistant protein to affect ovarian cancer cisplatin resistance.4.It was found that DDP Evil expression in ovarian cancer cells sensitivity is not by the influence of multi-resistant to regulate protein,in this part we continue to use Western blot detection inhibit Evil express different cell lines after DNA damage repair enzyme MGMT,the change of the MMR,to explore possible regulation mechanism of another.As shown as a result,we found that the Evil expression after cut,SKOV3 and OVCAR3 MGMT in cell lines,the MMR protein were decreased significantly,we speculate that the Evil may be through the influence the change of DNA damage repair enzyme DDP resistance to regulate ovarian cancer cells.Summary:1.Evil expression reduction can reduce the resistance of ovarian cancer cells to DDP and increase the sensitivity of chemotherapy drugs in cells.2.Evil may be the mechanism of multidrug resistance protein and DNA damage repair enzyme to jointly influence the drug resistance of tumor cells.Conclusion:1.Evil presents high expression in ovarian cancer and plays the role of proto-oncogene.2.Evil inhibits cell apoptosis through the activation of pi3k-akt signaling pathway to promote proliferation,invasion and metastasis of ovarian cancer cells.3.Evil increased the sensitivity of ovarian cancer cells to DDP through the mechanism of multidrug resistance protein and DNA damage repair enzyme. |
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