Effects Of IL-17d On Human Epithelial Ovarian Cancer Cells In Vitro

Objectives: We will build a stable expression and secretion of IL-17D bygene transfection of human epithelial ovarian cancer cells.And we plan toinvestigate the expression of IL-17D in ovarian cancer cells before and afterthe transfection,as well as the effect and significance of exogenous IL-17Dtransfection to the NKG2D ligand MICAon ovarian cancer cells,to explorethe effect of IL-17D on human ovarian cancer cells and innate immunity invitro.Methods:1Culture human ovarian cancer cells SKOV3,empty vector control cellsSKOV3/vector,exogenous human IL-17D gene stably transfected cellsSKOV3/IL17D and cisplatin-resistant cells SKOV3/CDDP,and human ovarianglands cancer cells OVCAR3,empty vector control cells OVCAR3/vector andIL-17D gene stably transfected cells OVCAR3/IL17D,resuscitative andculture,observe cells morphology by inverted microscope.2Test the IC50of the drug-resistant ovarian cancer cell SKOV3/CDDPto SKOV3through MTT,and calculate resistant index.3Test the hyperplasia of seven types of human epithelial ovarian cancercells at different time points (12,24,48,72,96h) through MTT,and draw thegrowth curve of cells.4Isolate the RNA of the seven types of human epithelial ovarian cancercells.Measure the expression of the IL-17D in the seven types of cells at themRNA level by real-time RT-PCR.5Detect the expression of activating NKG2D ligands MICAon ovariancancer cells surface in seven types of human epithelial ovarian cancer cells,Take the fluorescence index (FI) as the relative content of MICAproteinexpression. FI=samples values/control samples values,value=lg (x-mode value)*340.Result:1The IC50for cisplatin of SKOV3and SKOV3/CDDP was7.83and36.09mg/L.The resistance multiple was4.61.2Human ovarian cancer cells SKOV3and OVCAR3were respectivelyin the group comparison,transfection group grow clearly faster than thenon-transfected group and empty vector group (P<0.05), the difference wasstatistically significant; the difference between the non-transfected group andempty vector group was insignificant (P>0.05); resistance groupSKOV3/CDDP cells grow faster than the parental group (P<0.05),thedifference was statistically significant.There was no significant differencebetween resistant group and transfection group (P>0.05).3Expression of IL-17D mRNA in the seven types of human epithelialovarian cancer cells.Take2-ΔΔCTas the relative quantity of IL-17D.Make the SKOV3cells’ expression as contrast,the expression mass ofSKOV3/vector group,SKOV3/IL-17D group,SKOV3/CDDP group as follows:1.13±0.24,113.11±24.45,91.96±18.13;the difference between groups wassignificant(F=45.40,P=0.000<0.05),and the difference between the SKOV3group and SKOV3/vector group,the SKOV3/IL-17D and SKOV3/CDDP wasinsignificant(P>0.05),the difference within other groups was also significant(P<0.05).Similarly,make the OVCAR3cells’ expression as contrast,the expressionmass of OVCAR3/vector,OVCAR3/IL-17D as follows:1.10±0.07,182.78±16.37;the difference between groups was significant(F=369.89,P=0.000<0.05),and the difference between the OVCAR3group and OVCAR3/vectorgroup was insignificant(P>0.05),the difference within other groups was alsosignificant (P<0.05).Our experiment shows:The expression of IL-17D in SKOV3/IL-17D andOVCAR3/IL-17D were higher than that of the non-transfected group andempty vector group; the expression in SKOV3/CDDP was higher than that ofthe parent group. 4Expression of MICA protein on the surface of seven types of humanepithelial ovarian cancer cells by FCM.SKOV3group:the difference between groups was significant(F=598.61,P=0.00<0.05),and the difference between the SKOV3andSKOV3/vector,SKOV3/IL-17D and SKOV3/CDDP was insignificant(P>0.05),the difference within other groups was also significant (P<0.05).Similarly to OVCAR3group:the difference between groups wassignificant(F=1032.37,P=0.00<0.05),and the difference between the OVCAR3and OVCAR3/vector was insignificant(P>0.05),the difference within othergroups was also significant (P<0.05).Our experiment shows:the expression of MICA was decreased aftertransfection, the expression of MICA on SKOV3/CDDP cells’ surface waslower than that of SKOV3cells.Conclusion:1We can achieve the specific expression of IL-17D in ovarian cancercells.2Expression of IL-17D in multidrug resistant cell SKOV3/CDDP wasmuch higher than the parental cells,suggesting that IL-17D may be related tocell drug resistance,the mechanism must need further discussion.3Cell proliferation rate after transfection of IL-17D acceleratedobviously,suggesting that IL-17D may plays a promoting role in tumor cellgrowth.And the expression of MICA on cells with higher IL-17D wasdecreased,which may cause the weakened killing effect of NK cell to cancercells.Therefore this study can provide theoretical basis for investigating thetargeting therapy and immunologic escape of ovarian cancer.