| Background:Ovarian cancer is the highest lethal gynecologic malignancy,the most common is epithelial ovarian cancer(EOC).Because the early symptoms of ovarian cancer are hiding and lack of early meaningful detection markers,so most of the patients have been found for late disease associated with distant metastasis and after treatment,epithelial ovarian cancer patients 5-year survival rate is still significantly low.Although in recent years,there are a lot of research on the mechanism of ovarian cancer development,but does not have a very good judgment,so the underlying molecular mechanisms of tumor’s occurrence and development are still not fully elucidated.In recent studies,a closely link has been discovered between negative costimulatory molecule B7-H3 and the development of ovarian cancer.B7-H3 is a new gene first identified in 2001.Its protein structure was identified in 2013,but its receptor remains unclear.It is a transmembrane protein that can be expressed in tumor cells and antigen presenting cells.This molecule not only exists in the form of membrane protein but also in the serum soluble for:m(soluble B7-H3,sB7-H3).It is mainly about the membrane protein cutted by metalloproteinases.A newly found member of B7/CD28 family(immunoglobulin superfamily),and was identified as an accessory co-stimulatory molecule after initial antigen priming in cooperation with a putative counter receptor.Recently,a number of articles have reported that B7-H3 is highly expressed in many cancers,including colorectal cancer,hepatocellular carcinoma,lung cancer,gastric cancer,breast cancer and prostate cancer.Recent studies found that B7-H3 play different roles in different tumors,It can provide the stimulated response signal of enhancing and maintaining the T cell immune reaction,it also can produce the inhibited response signal of estricting and weakening the T cell immune reaction.B7-H3 plays an important role in cancer and inflammatory diseases,organ transplantation and autoimmune disease and becomes the focus in the immunology and other disciplines.In addition,B7-H3 has been shown to be associated with poor prognosis by the Jak2/Stat3 pathway.Although the expression of B7-H3 in ovarian cancer was mentioned in a document,it was verified only at the tissue level that the high expression of B7-H3 in ovarian cancer and B7-H3-positive tumor vasculature is associated with high-grade serous histological subtype,increased recurrence and reduced survival.At present there is no study on the role of B7-H3 in the biological behavior of ovarian cancer cells and its mechanism.Objective:B7-H3,a co-stimulatory molecule,has been found expressed in ovarian cancer,but its role and mechanism is not clear.In this study,we further verified the expression of B7-H3 in ovarian carcinoma and normal epithelial ovarian tissues.We also analyzed The relationship between clinicopathologic factors and B7-H3 expression in patients with ovarian cancer.Our findings discussed that the affect of B7-H3 in ovarian cancer progression and the mechanism of role.We hope that our studies can provide new target and theoretical basis for immunotherapy of ovarian cancer.Methods:1.Immunohistochemistry was performed to detect the expression of B7-H3 in ovarian cancer tissues and normal ovarian tissues.The relationship between clinicopathologic feature and B7-H3 expression in patients with ovarian cancer is analyzed.2.Immunofluorescence was utilized to detect the location of B7-H3 in ovarian cancer cell lines.3.The capacity of proliferation of A2780and SKOV3 cells after silencing or overexpressing B7-H3 were performed by MTT method.4.After being silenced or overexpressed,the influence of B7-H3 on ovarian cancer cell migration and invasion by transwell assays.5.To analyze the effect of B7-H3 on the different phases of the cell cycle and apoptosis,flow cytometry was used.6.After silencing and overexpressing B7-H3,western blotting method were performed to detect the expression of anti-apoptotic protein,pro-apoptotic protein and a variety of related molecules which were associated with cell invasion,migration and proliferation.The protein expression of related molecules in Jak2/Stat3 pathway was also detected by western blotting.7.B7-H3 was knockdown by small interfering RNA(siRNA)in A2780 cells and SKOV3 cells.The B7-H3 lentiviral vector was used to make the stable interference with ovarian cancer cells(A2780-sh-B7-H3,SKOV3-sh-B7-H3)and stable overexpression of ovarian cancer cells(HO8910-B7-H3-EGFP).Results:1.B7-H3 expression in clinical specimens and the location of B7-H3 in ovarian cancer cell lines.The results showed that the expression of B7-H3 in ovarian cancer was significantly higher than that in normal tissues.Immunohistochemistry revealed that B7-H3 was expressed in the cytoplasm of the tissue,and a few were expressed in the interstitial tissue.2.B7-H3 expression in relation to patient clinicopathologic factors.B7-H3 expression is associated with clinical stage of ovarian cancer,whereas it is not correlated with patient age,tumor size,differentiation degree and vascular invasion.3.The effect of Silencing B7-H3 on cell migration and invasion.Silencing B7-H3 reduced the migration and invasion potential of ovarian cancer cells,the expression of MMP2 was weakened.4.The effect of Silencing B7-H3 on cell proliferation and apoptosis ability.After interference of B7-H3,the proliferation of A2780 cells and SKOV3 cells ability weakened accordingly and the corresponding cells staying at the stage of cell division decreased.While cell apoptosis ability increased,especially increase the early apoptosis of the cells.With the silence of B7-H3 the expression of apoptosis regulator proteins of the Bcl-2 family,including the pro-apoptotic proteins Bax,caspase-8 and cleaved caspase-8 increased,while expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl decreased.5.The effect of overexpression of B7-H3 on ovarian cancer cell invasion,migration and proliferation in vitro.Overexpression of B7-H3 enhances ovarian cancer cell HO8910 invasion,migration and proliferation in vitro.The expression of related protein Bax,caspase-8,cleaved caspase-8 reduced while Bcl-2,Bcl-xl and MMP2 added by upregulation of B7-H3 expression.6.The effect of B7-H3 on Jak2-Stat3 pathway.With overexpressing B7-H3,the expression of pStat3 and its upstream molecule pJak2 increased;anti-apoptotic proteins Bcl-2 and Bcl-xl increased while expression of the pro-apoptotic protein Bax was reduced following upregulated expression of B7-H3 and after joining the AG490(the inhibitor of Jak2)for 48h the results were exactly the opposite.Conclusions:1.The expression of B7-H3 in ovarian cancer was significantly higher than that in normal tissues.B7-H3 expression is associated with clinical stage of ovarian cancer,whereas it is not correlated with patient age,tumor size,vascular invasion and other clinicopathologic feature.2.B7-H3 could affect the biological behavior of ovarian cancer cells,for example it increases the proliferation,invasion and migration capabilities of A2780,HO8910 and SKOV3 cells and suppress cell apoptosis.3.B7-H3 plays a role in ovarian cancer cells which is related to the Jak2-Stat3 pathway. |
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