| Objective:Human and murine OVCA cell lines,clinical OVCA specimens,and IL-17A-/-mice were used to investigate the roles of IL-17 A on the omentum metastasis of OVCA,which may provide new targets for controlling peritoneal(including omentum)metastasis of OVCA.Methods:1.Western blotting assay was used to test the effects of rh IL-17 A on the expression of FABP4,CD36,ATGL,MAGL,HSL,CPT1 C,NF-КB,STAT3,p-NF-КB and p-STAT3 of OVCA cells.Meanwhile,western blotting assay was used to test the effects of rh IL-17 A on the expression of FABP4,NF-КB,p-NF-КB,STAT3,p-STAT3 in OVCAR3 and A2780 cells after pretreatment with NF-КB inhibitor PDTC or STAT3 inhibitor STATTIC,respectively.2.MTS assay was used for detecting the effects of rh IL-17 A on OVCA cell proliferation in the presence of fatty acid(such as palmitic acid,PA)alone,rh IL-17 A alone or both PA and rh IL-17 A at the designated concentration for designated treatment time,respectively.Moreover,FABP4 inhibitor BMS309403,STAT3 inhibitor STATTIC and NF-КB inhibitor PDTC were used to do the blocking test respectively.3.Fatty acid(FA)transfer assay and qualitative and quantitative oil red O-staining assay were used to test the effects of rh IL-17 A on FA uptake of OVCA cells in the presence of PA alone,rh IL-17 A alone or both PA and rh IL-17 A,respectively.Moreover,FABP4 inhibitor BMS309403,STAT3 inhibitor STATTIC and NF-КB inhibitor PDTC were used to do the blocking test respectively.4.Clinical specimens of OVCA were collected.immunohistochemical(IHC)assay was used to detect the expression of IL-17 A and FABP4.Based on the result of IHC,the correlation of IL-17 A with FABP4 and with clinical outcome was analyzed,respectively.5.To evaluate the effects of IL-17 A on peritoneal metastasis of OVCA in vivo,the murine models of ovarian peritoneal cavity implantation were established using C57BL/6 WT mice and the syngeneic murine OVCA cell line ID8.IHC was used to test the expression of IL-17 A and FABP4,and western blotting assay was used to test the effects of IL-17 A on the expression of FABP4,STAT3 and p-STAT3 of tumor tissue.6.Enzyme linked immunosorbent assay(ELISA)was used to test the main cell source of IL-17 A in adipocyte-rich metastatic niche of OVCA.Results:1.Results of western blotting assay showed that the expression of FABP4,p-NF-КB and p-STAT3 were remarkably increased with the treatment of 10ng/ml rh IL-17 A for 24 h of OVCA cells(P<0.05).However,rh IL-17 A had no significant effect on the expression of CD36,ATGL,MAGL,HSL and CPT1 C of OVCA cells(P>0.05).Meanwhile,NF-КB inhibitor PDTC could remarkably block the effects of rh IL-17 A on the expression of p-NF-КB(P<0.01),and had no obvious effect on the expression of FABP4(P>0.05).STAT3 inhibitor STATTIC could remarkably block the effects of rh IL-17 A on the expression of p-STAT3 and FABP4(P<0.05).2.Results of MTS showed that rh IL-17 A alone or PA alone had no significant effect on OVCA cell proliferation(P>0.05).rh IL-17A(10ng/ml)could significantly enhance the proliferation of OVCA cells in the presence of PA(25μM)(P=0.006).Moreover,FABP4 inhibitor BMS309403 and STAT3 inhibitor STATTIC could remarkably block the above effect respectively(P<0.05).NF-КB inhibitor PDTC could not block the enhanced effects of IL-17 A on the proliferation of OVCA cells(P>0.05).3.Results of FA transfer assay and qualitative and quantitative oil red O-staining assay showed that rh IL-17 A alone or PA alone had no significant effect on FA uptake of OVCA cells(P>0.05).rh IL-17A(10ng/ml)could enhance the FA uptake of OVCA cells in the presence of PA(25μM)(P<0.05).Moreover,FABP4 inhibitor BMS309403 and STAT3 inhibitor STATTIC could remarkably block the above effect respectively(P<0.05).NF-КB inhibitor PDTC could not block the enhanced effects of IL-17 A on the FA uptake of OVCA cells(P>0.05).4.Results of IHC showed that the expression of IL-17 A and FABP4 was positively correlated with OVCA FIGO staging,lymph node metastasis and ascites metastasis(P<0.01).And the expression of IL-17 A was positively correlated with the protein expression level of FABP4(P<0.01).5.Experiment in mice showed that IL-17 A could increase peritoneal(including omentum)metastasis in murine OVCA models(P=0.021).IHC and western blotting assay showed that endogenous IL-17 A could enhance the expression of FABP4(P=0.001)and p-STAT3(P<0.01).6.Results of ELISA assay showed that murine macrophage cell line RAW264.7 and primary adipocytes could secrete IL-17 A.Conclusion:With human and murine OVCA cell lines,clinical OVCA specimens,and IL-17A-/-mice,we found that IL-17 A could enhance the expression of FABP4 via STAT3 signaling pathway in turn to promote the uptake of fatty acid,which enhanced the proliferation of OVCA cells.This study indicated that IL-17 A and FABP4 might provide new targets for controlling peritoneal(including omentum)metastasis of OVCA. |
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