| OBJECTIVE:Interleukin 6,a tumor promoting cytokine,plays an important role in the initiation and progression of renal cell carcinoma(RCC).Hepatocyte cell adhesion molecule(hepaCAM),a novel tumor suppressor,is down-regulated or completely lost in an early stage of RCC development upon multiple stimulations.This study aims at exploring the relation between IL-6 and hepa CAM in RCC and the underlying mechanisms METHODS:1)A total of 74 RCC tissue samples and 43 tumor-adjacent nonmalignant tissue samples were collected and detected using immunohistochemistry for the protein expression of IL-6 and hepaCAM.The discrepancies of IL-6 expression(as well as hepa CAM expression)between in RCC tissue and in adjacent tissue were statistically analyzed.The correlation between IL-6 expression and hepa CAM expression in RCC,as well as their relations with certain clinicopathological parameters were also analyzed.2)A total of 30 matched RCC tissue samples,adjacent samples and serum samples from RCC patients were collected,with 13 serum samples from healthy volunteers serving as control.ELISA was used to measure the IL-6 level of serum samples,and q-PCR and western blot were chosen to detect hepaCAM mRNA and protein expression in RCC tissue and adjacent tissue.The differences of IL-6 serum levels between in RCC patients and in healthy volunteers,and the differences of hepa CAM expression between in RCC tissue samples and their paired adjacent samples were statistically analyzed.The correlations between serum IL-6 and RCC tissue hepaCAM expression(mRNA and protein)were also explored.3)In vitro cell culture of human RCC cell lines 786-O,769-P,A498,ACHN and Caki-1 and human proximal tubular cell line HK-2.a)ELISA was used to measure the IL-6 level in supernatants,and q-PCR and western blot were chosen to detect hepa CAM mRNA and protein expression in five RCC cell lines and HK-2.b)769-P and Caki-1 were treated with different exogenous IL-6 concentrations(0、10、20、40ng/ml)or different exogenous IL-6 exposure time(0、6、12、24、48hr),and then subjected to q-PCR and western blot for the analysis of hepaCAM mRNA and protein expression.c)Five cell lines were transfected with siRNA-IL-6,followed by the analysis of hepaCAM mRNA and protein expression using q-PCR and western blot.d)ACHN and 769-P were transfected with hepaCAM-over-expression adenoviruses.ELISA was used to measure the IL-6 level in supernatants,and western blot were chosen to detect the protein expression of hepaCAM,IL-6R and gp130.4)In vitro cell culture of human RCC cell lines 769-P,A498 and ACHN.Cells were divided into five groups: control group,vector adenovirus group,IL-6 treatment group(20ng/ml),hepaCAM adenovirus group and IL-6 treatment + hepa CAM adenovirus group.CCK-8,colony formation assay and cytometry were used to investigate the cell viability,proliferative capacity and cell circle distribution respectively.Western blot was chosen to examine the proliferation-associated molecules: C-myc、PCNA and cyclin D1.5)In vitro cell culture of human RCC cell lines 769-P,A498 and ACHN.a)Cells were transfected with siRNA-IL-6 and then subjected to western blot for the detection of phosphorylation status of STAT3、ERK 和AKT.b)Cells were treated with exogenous IL-6(20ng/ml)alone or in combination with various inhibitors(JAK/STAT3 inhibitor stattic [10uM],MEK/ERK inhibitor U0126[5uM] and PI3K/AKT inhibitor MK-2206[1uM]).Western blot was used to measure the expression of total and phosphorylated STAT3、ERK and AKT.Western blot and q-PCR were used to detect hepaCAM protein and mRNA expression respectively.Cells were pretreated with hepaCAM adenovirus or stattic(10 10uM),alone or combined,followed by exogenous IL-6 addition(20ng/ml).CCK-8 was used to detect the cell viability.d)Western blot was used to investigate the expression of DNMT1,DNMT3 a and DNMT3 b in cells subjected to treatments of siRNA-IL-6 tranfection,exogenous IL-6(20 ng/mL)or Stattic(10 uM),alone or combined.e)Cells were treated with exogenous IL-6(20 ng/mL)alone or in combination with DNA methyltransferase inhibitor 5-Aza(10uM),siRNA-DNMT1 or siRNA-DNMT3 respectively.Western blot,q-PCR and methylmion Specific PCR were chosen to detect the protein expression of DNMT1、DNMT3b and hepaCAM,the mRNA expression of hepa CAM and the methylation status of hepaCAM promoters.RESULTS:1)IL-6 protein expression was significantly up-regulated in RCC tissue compared to that in tumor-adjacent nonmalignant tissue(P<0.001),whereas hepaCAM expression was significantly suppressed in RCC tissue compared to that in adjacent tissue(P<0.001).Moreover,a strong level of agreement between IL-6 increase and the hepaCAM decrease in RCC tissue was observed(kappa =0.748,P<0.001).IL-6 expression in RCC tissue was found to be positively correlated with tumor histological stage(P=0.019),but no significant associations were observed between hepaCAM protein expression in RCC tissue and various clinicopathological parameters.2)IL-6 level in patient serum was markedly higher than in healthy volunteer serum(P=0.019),and it was positively correlated with tumor histological stage(P=0.035).HepaCAM mRNA and protein expression in RCC tissue were remarkably lower than in adjacent tissue(P<0.001).However,we failed to observe any association between IL-6 level in patient serum and hepaCAM mRNA(protein)expression in RCC tissue.(mRNA: R=-0.1985,P=0.2931;protein:R=-0.2050,P=0.2772).3)Hepa CAM protein was completely lost in RCC cell line 786-0,ACHN and A498,down-regulated in Caki-1 and 769-P,but highly expressed in human proximal tubular cell line HK-2.HepaCAM mRNA expression in 769-Pn and Caki-1 was further suppressed by exogenous IL-6 treatment in a time and dose-dependent manner,so was hepaCAM protein expression in 769-P.Hepa CAM mRNA and protein expression was significantly up-regulated in 769-P 、 A498 、 ACHN and Caki-1 after transfected with siRNA-IL-6.Neither IL-6 level in supernatants nor protein expression of IL-6R and gp130 altered in 769-P and ACHN after transfected with hepaCAM-over-expression adenoviruses.4)HepaCAM over-expression effectively inhibited cell viability and proliferative capacity of ACHN、769-P and A498.In addition,it induced G1 arrest and down-regulated protein expression of C-myc、PCNA and cyclin D1.On the contrary,exogenous IL-6 treatment promoted cell viability and proliferative capacity,increased cell number in S phase,and up-regulated the expression of C-myc 、 PCNA and cyclin D1.Cells subjected to combined treatment of hepaCAM adenovirus transfection and exogenous IL-6 showed lower cell viability,lower proliferative capacity and lower expression of proliferation-associated molecules than control group.5)Phosphorylation levels of STAT3 、 ERK and AKT were down-regulated in RCC cells subjected to IL-6 silencing(p-ERK in A498 did not alter),whereas they were up-regulated in RCC cells treated with exogenous IL-6.Among various inhibitors of signal pathways,only JAK/STAT3 inhibitor stattic was able to remarkably promote hepaCAM mRNA and protein expression in RCC cells,and,moreover,abrogated the IL-6-driven suppression of hepaCAM expression,indicating that IL-6 regulates hepaCAM expression via STAT3 pathway.DNMTs plays an important role in IL-6/STAT3 regulation of hepaCAM.In ACHN and 769-P,IL-6 was capable of inducing promoter hypermethylation and subsequent expressive suppression of hepa CAM by up-regulating DNMT1.In A498,IL-6-induced hepaCAM promoter hypermethylation and expressive suppression was largely attributed to DNMT3 b.Though,in A498,DNMT1 was also able to moderately inhibit hepaCAM expression,this did not involve its catalytic activities of methylation.CONCLUSION:In RCC,IL-6 can up-regulate DNMT1 and DNMT3 b through STAT3 pathway,which induces promoter hypermethylation and subsequent suppression of hepaCAM mRNA and protein expression,eventually resulting in RCC proliferation. |
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