Study Of The Effects And Underlying Mechanisms Of IL-17(A) In Omentum Metastasis Of Ovarian Cancer

Objective:Clinical OVCA specimens,IL-17A-/-mice,human OVCA cell lines and a mouse OVCA cell line were utilized to study the effects and underlying mechanisms of IL-17 A in omentum metastasis of OVCA,which may provide a novel strategy to improve the treatment of OVCA in the clinic.Methods:1.Sixty EOC specimens were collected to study.IHC staining assay was used to analyze the relationship between the expression of IL-17 A and FIGO stage,and MMP2,MMP9,HSL,FABP4 levels in clinical settings.2.Western blotting assay was used to detect the protein level of IL-17 RA in human OVCA cell lines including A2780,OVCAR3,SKOV3 and a mouse OVCA cell line ID8.3.MTT assay was used to detect the effect of recombinant human IL-17A(rh IL-17A)or recombinant mouse IL-17A(rm IL-17A)on proliferation of A2780,OVCAR3,SKOV3 and ID8 cells.4.Wound healing assay was used to detect the effect of rm IL-17 A on migration ability of ID8,and then neutralizing IL-17 RA m Ab(using m Ig as an isotype control)was used to do the blocking test.5.Transwell chamber invasion test(8.0?m)was used to detect the effect of rm IL-17 A and/or mice primary omentum adipocytes on invision ability of ID8,and then neutralizing IL-17 RA m Ab(using m Ig as an isotype control)and FABP4 inhibitor BMS309403 were used to do the blocking test respectively.6.Western blotting assay was used to detect the dose-dependent and time-dependent effects of rh IL-17 A on protein levels of MMP2,MMP9,HSL and FABP4 in A2780 and OVCAR3 cells.7.Western blotting assay was used to detect the protein level of IL-17 RA in human breast cancer cell lines(MDA-MB-231,MCF-7,T47 D and SK-BR-3),human lung cancer cell lines(H1299,H1752,H460 and A549)and human liver cancer cell lines(SK-HEP-1,BEL-7402,Hep G2 and Hep3B).Western blotting assay was used to detect effects of rh IL-17A(1ng/ml,6h)on protein levels of MMP2,MMP9,HSL and FABP4 in them.8.Western blotting assay was used to detect the effects of rh IL-17 A on protein levels of PPAR-? and p-STAT3 in A2780 and OVCAR3 cells.9.MTT assay was used to detect the IC50 of A2780 and OVCAR3 cells to STAT3 inhibitor stattic.10.Western blotting assay was used to detect the protein levels of MMP2,MMP9,HSL,FABP4 and p-STAT3 on rh IL-17 A treated A2780 and OVCAR3 cells,and stattic was used to do the blocking test.11.The ID8-burden mouse model was established with C57BL/6 wild type and IL-17A-/-mice intraperitoneally(i.p.)injected with ID8 cells.Immunohistochemical staining assay was used to detect the expression of MMP2,MMP9,HSL and FABP4 in tumors.Results:1.IHC staining and analysis in the clinical EOC settings displayed that: IL-17 A level in OVCA markedly increased as the disease progresses based on FIGO staging.IL-17 A level significantly positively related with the levels of MMP2,MMP9,HSL and FABP4 in the clinical specimens.2.IL-17 RA was present in human OVCA cell lines including A2780,OVCAR3,SKOV3 and a mouse OVCA cell line ID8.3.Rh IL-17 A or rm IL-17 A had no obvious effect on the proliferation of A2780,OVCAR3,SKOV3 and ID8 cells.4.Comparing with control group,the migration ability of ID8 was promoted by rm IL-17 A.Furthermore,neutralizing IL-17 RA m Ab could block the enhanced effects of rm IL-17 A on the migration ability of ID8 cell.5.Comparing with control group,the invision ability of ID8 could be increased by rm IL-17 A,or co-culturing with primary omentum adipocytes from IL-17-/-mice or wide type mice,or co-culturing with primary omentum adipocytes from wide type mice after treating with rm IL-17 A.Furthermore,neutralizing IL-17 RA m Ab or BMS309403 could respectively block the enhanced effects of rm IL-17 A on the invision ability of ID8 cell.6.The protein levels of MMP2,MMP9,HSL and FABP4 were significantly enhanced by the treatment of 1ng/ml rh IL-17 A for 6h in A2780 and OVCAR3 cells.7.IL-17 RA,MMP2 and MMP9 was present in human breast cancer cell lines(MDA-MB-231,MCF-7,T47 D and SK-BR-3),human lung cancer cell lines(H1299,H1752,H460 and A549)and human liver cancer cell lines(SK-HEP-1,BEL-7402,Hep G2 and Hep3B),and the protein levels of MMP2 and MMP9 were significantly enhanced by the treatment of rh IL-17 A.HSL and FABP4 was alone present in human OVCA cell lines A2780 and OVCAR3,the protein levels of HSL and FABP4 were significantly enhanced by the treatment of rh IL-17 A.8.The protein level of PPAR-? was significantly enhanced by the treatment of rh IL-17 A in A2780,then rh IL-17 A had no obvious effect on the protein level of PPAR-? in OVCAR3.The protein level of p-STAT3 was significantly enhanced by the treatment of rh IL-17 A in A2780 and OVCAR3 cells.9.The IC50 values of stattic from MTT assay were determined to be 3.499 and2.538 ?M in A2780 and OVCAR3 cells,respectively.10.The protein levels of MMP2,MMP9,HSL,FABP4 and p-STAT3 were significantly enhanced by the treatment of 1ng/ml rh IL-17 A for 6h in A2780 and OVCAR3 cells.Furthermore,stattic could block the enhanced effects of rh IL-17 A on the protein level of FABP4 and had no obvious effect on the protein levels of MMP2,MMP9 and HSL in A2780 and OVCAR3 cells.11.The murine ovary carcinoma models were established.Comparing with IL-17-/-mice,the survival time of wide type mice lessened and the tumor counts increased apparently.The expression of MMP2,MMP9,HSL and FABP4 in tumors from wide type mice were increased than those from IL-17-/-mice.Conclusion:From the analysis of clinical specimens,the results of in-vitro experiments and animal experiments,this study reveals for the first time that IL-17 A can increase the protein levels of MMP2,MMP9,HSL,FABP4 and it can exacerbate the protein level of FABP4 through STAT3 signal pathway in ovarian cancer cells.IL-17 A can promote the omentum metastasis by combining with IL-17 RA.IL-17 A and FABP4 may be the new targets for inhibition of ovarian cancer omentum metastasis.These findings may provide a novel strategy to improve the treatment of omentum metastasis of OVCA in the clinic.