Research Of IL-8 Gene On The Mechanism Of Invasion And Metastasis In Epithelial Ovarian Cancer

Objective: Being one of department of gynecology tumor, ovarian cancer which is seriously attacking women's health has driven professor's attention which is hard to search out in early stage. There are varieties of tumor of the ovary whose organism construction are complicated and owe different bionomics. The malignant degree of epithelium tissue cancer is higher than interstitial substance cancer. While its therapeutic efficacy can not reach the germinocarcinoma. And this tumor which grows very fast, can't be diagnosed easily in the early stage and its death rate is very high is eager to be researched in the region of woman tumor. So a new therapy should be found. Recently, the research that specialists try to find out the cause and developing relationship of some uncommon genes and ovarian cancer is hitting at the district of boss molecular biology research. Ovarian cancer's cause and developing relationship is a complex and mixed-step process which involves quantities genes and proteins'expression.IL-8 which is found by Yoshimur at 1987 is the first chematropism cell factor belonging to CXC family that plays an important role in the contribution of inflammation and immunologic process. Recently, it has drawn more and more attention of researchers as malignant tumor correlative molecule. Nowadays, it is known that many tumor cell can secrete IL-8, IL-8 overexpression relates to tumorous growth and metastasis though it is not clear that the mechanism of encouraging tumor appearance and development.Recent years, the construction and development of recombination technology can provide new strategy for ovarian cancer in theory and practice——gene therapy can be expected to become new channel of ovarian cancer therapy. Ideal therapeutic gene can inhibit tumor cell's growth effectively without inhibitory action to normal tissue and cell. In this research, to explore the intereffecting mechanism of IL-8 and epithelial ovarian cancer growth, invasion and metastasis , the eukaryotic expression vector of hIL-8 was constructed and expressed in OVCAR-3 cells after transient and stable transfection. Meanwhile pcDNA3.1(+)/IL-8, pcDNA3.1(+) and non-tranfected OVCAR-3 cells were inoculated subcutaneously into every nude mouse respectively according to three divided groups, Mice's general condition and time of tumour growth were observed. The volume of transplanted tumor and pulmonary metastasis were compared with different groups. The foundation was settled for further investigation of tumorigenesis and tumor growth.Method:1 Construct the eukaryotic expression vector of IL-8 gene. Total RNA was isolated from PBMC of healthy volunteers, IL-8 gene encoding fragment was amplified by RT-PCR and cloned into the vector PMD-18T. BamHI/XhoI double digested product of PMD-18T/IL-8 was connected with the vector pcDNA3.1(+) which was digested by BamHI/XhoI .2 Transient and stable transfect recombinant plasmid into OVCAR-3 cells. The recombinant plasmid pcDNA3.1(+)/IL-8 was transfected into OVCAR-3 cells which express IL-8 a little with Lipofectamine2000. The expression of IL-8 was tested in OVCAR-3 cells by RT-PCR ,ELISA and Western blot assay.3 Detect cell proliferation. Detect these cells'reproductive activities by MTT and draw growth curve.4 The cell cycle was detected by flow cytometry (FCM) on three group cells. pcDNA3.1(+)/IL-8 transfected group, pcDNA3.1(+) group and OVCAR-3 none transfected group.5 Measure Bax,Bcl-2,VEGF and MMP-9 expression analysis.The expression of Bax,Bcl-2,VEGF and MMP-9 were detected by FCM on three group cells the same as method 4.6 Using transwell cabin to detect invasive ability of different OVCAR-3 cell lines. Establish vitro invasive model by transwell cabin and matrigel to evaluate the invasive ability of OVCAR-3 cells which stable transfected recombinant plasmid IL-8. Transwell cabin divided into two cabin by polycarbonate fliter membrance(micropore diamerer is 8μm). Micropore allow cell to pass through. Matrigel is composed by typeⅣcollagen,laminin and integrin, similar with basal membrane. The number of cells which pass through the micropore was counted and compared with each other.7 Effect of IL-8cDNA on transplanted tumour growth and lung metastasis. Mice are divided into three group, and OVCAR-3, OVCAR-3/pcDNA3.1(+) and OVCAR-3/pcDNA3.1(+)/IL-8 cells were subcutaneously inoculated into nude mice according to three divided groups. Mice were killed after 9 weeks, the volume of tumour in each group was compared in different time and the expression of CD34 and NF-κB of tumours from different group were detected by immunohistochemical method. The tissue slice of lung was observed.RESULTS:1 After PCR amplification, BamHI/XhoI digestion and DNA sequencing confirmation, the eukaryotic expression vector pcDNA3.1(+)/IL-8 was constructed successfully.2 The IL-8's expressions in quantity were founded in the cells of transient transfection when detected by RT-PCR. The gray scale value of pcDNA3.1(+)/ IL-8 transfected group which was 226.88±5.47 correspondingly againstβ-actin was 1.55, while OVCAR-3 none transfected group which was 120.38±6.72 correspondingly againstβ-actin was 0.82. The growth rate was 189.02%;The IL-8's expressions in cell sap and the cells after transfected pcDNA3.1(+)/ IL-8 6h and 48h were 3.69±0.37, 19.67±0.24, 11.10±1.14 respectively by ELISA, while the others were a little, P<0.05; the specific protein strap was detected only in the former by Western-blot.3 The results by MTT indicated that the light absorption value of OVCAR-3 cells transfected IL-8/pcDNA3.1 were 0.56±0.08, 0.63±0.06, 0.93±0.09, 1.20±0.06, 1.32±0.12, 1.39±0.14 respectively, while OVCAR-3 none transfected group were 0.62±0.07, 0.70±0.10, 0.84±0.12, 0.92±0.10, 0.96±0.14, 1.01±0.10 respectively. There was no statistical significance compared pcDNA3.1(+)/ IL-8 transfected group with the ones not transfected within 3days(P>0.05), while pcDNA3.1(+)/ IL-8 transfected group was obviously more active after 3 days(P<0.05), and the former's increasing rate presents an evident tendency of going up along with the time's extension.4 The cellular S stage in pcDNA3.1(+)/IL-8, pcDNA3.1(+) and OVCAR-3 cells were 55.18±2.98, 39.72±4.16, 31.36±1.46 respectively by FCM. The former was much higher than the others, P<0.05. These proliferation index were 58.83±2.85, 44.32±4.20, 35.31±1.56.The former PI was much higher and the comparison between them has statistical significance(P<0.05).5 The expression of VEGF and MMP-9 in pcDNA3.1(+)/IL-8, pcDNA3.1(+) and OVCAR-3 cells were 430.44±21.70, 358.70±17.77, 335.52±19.68 and 356.15±22.70, 279.37±27.48, 283.56±26.67 respectively; while Bcl-2 and Bax in these cells were 380.86±15.27, 319.15±17.89, 305.04±28.90 and 343.12±26.10, 416.27±25.45, 420.13±23.65 respectively. There was statistical significance against pcDNA3.1(+) transfected group and OVCAR-3 none transfected group (p<0.05), while the comparison between them has no statistical significance.6 Cell number that pass through micropore of OVCAR-3 group, empty plasmid group and IL-8 group is 101.47±13.52, 98.07±13.97, 163.73±12.98. There was statistical significance against OVCAR-3 group, empty plasmid group and IL-8 group. This confirm that IL-8 maybe increase invasive ability of OVCAR-3.7 Effect of transplanted tumor growth in nude mice by IL-8. Days of tumour growth of OVCAR-3 group, empty plasmid group and IL-8 group is 19.70±1.25, 19.90±1.28, 16.00±1.53. Rate of tumor growth is 100%. Tumor volume of IL-8 group is much bigger than other two groups in 7, 8, 9 week . There have no difference in other times. Cancer cells couldn't found in lung in each group.8 Immunohistochemical result of MVD and NF-κB. The MVD and NF-κB in mice treated with IL-8 was much higher than other two groups. Light absorption value of CD34 of OVCAR-3 group, empty plasmid group and IL-8 group are 0.1330±0.0134, 0.1330±0.0134, 0.1942±0.0175. There was statistical significance. Light absorption value of NF-κB of OVCAR-3 group, empty plasmid group and IL-8 group are 0.0794±0.0098, 0.0812±0.0100, 0.1072±0.0049. There was statistical significance.Conclusions: The IL-8 gene is transfected into OVCAR-3 cells effectively by liposome. Moreover, IL-8 also promotes the proliferation of cell by increasing the number of cell S stage, that the mechanism is connected with angiogenesis factor and correlation factor about apoptosis, which provides a foundation for researching the mechanism of IL-8 on ovary cancer. Result of ELISA showed that cell secretes much more IL-8 after empty plasmid transfection, we consider that transfection maybe stimulate the secretion of IL-8, but we could not find any report about this respect, we should study it more deeply in the future. Invasive experiment showed that IL-8 could increases the invasive ability of OVCAR-3. IL-8 enhances the express of VEGF/MMP-9/Bcl-2 and inhibits the espress of Bax. This mechanism is intimate correlated with angiogenesis factors and apoptosis factors. The animal experiment showed that IL-8 couses the growth, invasion and metastasis of tumor by induce the express of NF-κB and increase the value of MVD. All of this provide the theoretical and experimental basis for us to study the mechanism of growth, invasion ,metastasis and prognosis in epithelial ovarian cancer.