| Objectives To study the effect of IL-17/IL-17 R on the chemo-resistance of hepatocarcinoma and the related mechanism by analyzing IL-17/IL-17R-JAK2/STAT3 signaling pathway,which inhibiting the apoptosis susceptibility of hepatocarcinoma induced by chemotherapy through regulating the autophagy.Methods Serum IL-17 levels of 30 cases of HCC patients were detected by ELISA assay and compared with those of 30 healthy cases.The IL-17 R levels of SMMC-7721 cell and L02 cell were detected by western blot.Then oxaliplatin was chosen and screened an IC50 dose with MTT method for SMMC-7721 cells.Western blot analysis was performed to detect the levels of IL-17 R,apoptosis related proteins and autophage related proteins as well as p-JAK2 and p-STAT3 of SMMC-7721 cells induced by oxaliplatin.The AG490 and LY294002 were used to inhibit the JAK2/STAT3 and the PI3K/AKT signaling pathway respectively,and the autophage related proteins were detected,as well as the regulation effect of PI3K/AKT signaling pathway on JAK2/STAT3 signaling pathway.By means of increasing IL-17 and interfering IL-17 R with the specific antibody or si RNA of IL-17 R,the levels of apoptosis and autophage and the activation of JAK2/STAT3 signaling pathway were detected by western blot.Besides,the autophage inhibitor 3-MA was used prior to adding the IL-17 or blocking the JAK2/STAT3 signaling pathway to observe the apoptosis of SMMC-7721 cells.Results 1 Prior to chemotherapy,the serum IL-17 level of 30 cases of HCC patients was(77.36±22.90)pg/ml,which was higher than that of the control(26.65±4.92)pg/ml but significantly added up to(142.41±33.25)pg/ml after treatment,(P<0.05).Likely,the IL-17 R expressed scarcely in L02 cell but apparently in SMMC-7721 cell,especially after chemotherapy.2 IL-17R-blocking and IL-17-promoting assays were designed to observe the effect of IL-17/IL-17 R on oxaliplatin-induced apoptosis.The result of western blot showed that Bcl-2 protein expression decreased and Bax protein and Cleaved-caspase3 protein expression increased in oxaliplatin-treated SMMC-7721 cells.Compared with the untreated cells,the results above suggested that the apoptosis was induced by oxaliplatin treatment.3 Beclin-1 and LC3 expression in SMMC-7721 cells increased obviously under the stimulus of oxaliplatin.In addition,the expression of Beclin-1 and Lc3 attenuated markedly when the IL-17 R was blocked with the specific neutralizing antibody or interfered by the si RNA,and increased accordingly when the cells were exposed to excessive IL-17.4 The levels of p JAK2 and p STAT3 increased in oxaliplatin-stimulated cells on account of the ascending IL-17/IL-17 R and decreased as soon as the IL-17 R was blocked or interfered,suggesting the JAK2/STAT3 pathway was activated by the IL-17/IL-17 R in chemotherapy.Moreover,both of the PI3K-AKT and JAK2/STAT3 signaling pathways could upregulate the oxaliplatin-induced autophagy,and the latter performed predominantly.5 An obvious apoptosis was observed when the oxaliplatin-treated cells were processed with AG490 and 3-MA separately,revealing that the JAK2/STAT3 deregulated the oxaliplatin-induced apoptosis via up-regulating autophage.Another similar test showed that the apoptosis of SMMC-7721 cells increased markedly when stimulated by 3-MA,even exposed to excessive IL-17,implying that the mechanism of IL-17/IL-17 R deregulating the susceptivity of HCC to apoptosis relied on the autophage activation.Conclusions 1 IL-17/IL-17 R levels increased largely when the HCC cells were treated with chemotherapy.2 IL-17/IL-17 R up-regulated the expression of autophage via the JAK2/STAT3 signaling pathway.3 The IL-17/IL-17R-JAK2/STAT3 signaling pathway might involve in the resistance of HCC to chemotherapy through down-regulating the apoptosis susceptibility. |
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