| Ovarian tumor is a common gynecological tumor,which can occur at any age.Epithelial ovarian cancer is the most common histological type of ovarian cancer,accounting for 50%-70%of ovarian tumors,including serous,mucinous,endometrial and clear cell subtypes.The incidence rate of epithelial ovarian cancer is third in gynecologic malignancies,but the mortality rate is the first place in gynecologic malignancies.The poor prognosis is mainly due to the lack of early detection methods,the lack of effective treatment methods and high drug resistance rate.Due to the lack of typical clinical symptoms and effective screening methods in the early stage,more than 70%of patients with ovarian cancer were found to have advanced stage,which is characterized by extensive metastasis and ascites accumulation in the pelvic cavity,making the disease progress rapidly.Invasion and metastasis are the main causes of death in patients with ovarian cancer,and they are also the important steps that hinder the treatment.Therefore,to explore the molecular mechanism of invasion and metastasis of ovarian cancer and to seek effective targeted treatment is one of the research focuses of epithelial ovarian cancer.The migration and invasion inhibitory protein(M?P),also called invasion inhibitory protein 45(?p45),which is a tumor suppressor gene that located on chromosome 1p36.22,encoding a highly hydrophilic protein composed 388 amino acids,a high hydrophilic protein,and its molecular weight is predicted to be 45kDa.Recently,M?P can participate in the proliferation and metastasis of tumor cells through PI3K/Akt,NF-kB,Rac-1 and other signaling pathways.It has been reported that M?P has low expression in endometrial cancer,non-small cell lung cancer,prostate cancer,breast cancer,glioma and other malignant tumors.Upregulation of M?P expression can significantly inhibit the proliferation,invasion and metastasis of tumor cells.NF-kB(nuclear factor-?B)is a transcription factor,which is composed of two major subunits p65 and p50,to induce the transcription of related genes.NF-?B signaling pathway can regulate the expression of many genes,such as intercellular adhesion molecule-1(IC AM-1),vascular cell adhesion molecule-1(VC AM-1),tumor necrosis factor-?(TNF-?).These cytokines have been reported to play a significant role in the development of malignant tumors at many levels,including promoting growth,proliferation,invasion,metastasis and angiogenesis.ObjectiveTo detect the expression of M?P in different ovarian tissues,to explore the effect of M?P on the proliferation and invasion of ovarian cancer SKOV3 cell line,and to research whether M?P plays a role through NF-?B signaling pathway.Methodsl.The positive expression rate of M?P protein and the relative expression of M?PmRNA in 45 cases of epithelial ovarian cancer and 25 cases of normal ovarian tissue were detected by immunohistochemistry SP and qRT-PCR respectively,and the correlation between M?P and the clinicopathological characteristics of epithelial ovarian cancer was analyzed.2.SKOV3 cell line was cultured,SKOV3 cells were transfected with pcDNA3-M?P as the experimental group,SKOV3 cells transfected with pcDNA3 as the negative control group,and SKOV3 cells not transfected as the blank control group.qRT-PCR and Western blot were used to detect the relative expression of M?P mRNA and the expression level of M?P protein in the three groups of cells,to verify the validity of the plasmid.3.CCK-8 and Transwell methods were used to detect the effect of M?P on theproliferation and invasion of ovarian cancer SKOV3 cell line.4.Western blot was used to detect the expression of p65,p-p65 and ICAM-1 inSKOV3 cell.Results1.The positive expression rate of M?P protein in epithelial ovarian cancer was significantly lower than that in normal ovarian tissue,and the positive expression rates were 31.1%and 76.0%,respectively.The difference was statistically significant(P<0.001).2.In epithelial ovarian cancer,the positive expression rate of FIGO ?+? M?P protein was significantly higher than that of ?+?,the positive expression rates were 44.0%and 15.0%respectively;in lymph node dissection,the positive expression rates of FIGO ?+? M?P protein were significantly lower than that of no lymph node metastasis group,the positive expression rates were 23.8%and 64.3%respectively.By statistical analysis,the positive expression rate of M?P protein in epithelial ovarian cancer was related to FIGO stage and lymph node metastasis(P<0.05).It was not related to age,histological grade and pathological type(P>0.05).3.The relative expression of M?PmRNA in epithelial ovarian cancer and normal ovarian tissue was 0.969±0.380 and 2.439±0.991,respectively.The relative expression of M?PmRNA in epithelial ovarian cancer was lower than that in normal ovarian tissue.The difference was statistically significant(P<0.01).4.In the cell experiment,the relative expression of M?PmRNA in the experimental group,negative control group and blank control group was 5.856±0.646,1.236±0.116 and 1.077±0.150,respectively;the relative expression of M?P protein was 78.336±5.059,40.338±6.656 and 38.050±1.098.The expression level of M?PmRNA and M?P protein in the experimental group was significantly higher than the negative control group and the blank control group(P<0.001),However,there was no significant difference in M?PmRNA expression and M?P protein expression between the negative control group and the blank control group(P>0.05).It is suggested that the overexpression plasmid pcDNA3-M?P can effectively up regulate the expression of M?P in ovarian cancer SKOV3 cells,which can be used for subsequent experiments.5.Compared with the negative control group and the blank control group,the proliferation of cells in the experimental group was significantly inhibited,and the proliferation rate decreased with time.The difference was statistically significant(P<0.01).6.The number of cell penetrating membrane in 24 hours in experimental group,negative control group and blank control group was 94.200±15.834,149.800±7.949 and 163.600±4.037,respectively.The number of cell penetrating membrane in the experimental group was significantly less than that in the negative control group and the blank control group.That is to say,the cell invasion ability in the experimental group was significantly inhibited.The difference was statistically significant(P<0.001).7.The relative expression of p-65 was 76.027±3.256 and 78.273±3.272 in the experimental group,negative control group and blank control group,respectively,and 80.200±4.409 in the blank control group,with no significant difference(P>0.05);the relative expression of p-p65 was 38.284±4.284,61.768±3.661 and 63.567±3.577,respectively.The expression of p-p65 in the experimental group was significantly lower than that in the negative control group The difference between the control group and the control group was statistically significant(P<0.01).The relative expression of ICAM-1 was 41.577±2.347,65.643±5.142,68.621±4.715.The relative expression of ICAM-1 in the experimental group was significantly lower than that in the control group(P<0.05).Conclusion1.M?P expression was low in epithelial ovarian cancer,and the positive expression rate of M?P protein was related to FIGO stage and lymph node metastasis.It is suggested that the low expression of M?P may promote the development of epithelial ovarian cancer.2.Overexpression of M?P can significantly inhibit the proliferation and invasion of SKOV3 cells.M?P can be regarded as the tumor suppressor gene of ovarian cancer,and may be the potential target of epithelial ovarian cancer treatment.3.M?P may participate in the proliferation and metastasis of epithelial ovarian cancer cells through NF-kB signaling pathway. |
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