The Role And Mechanism Of NF-κB/IL-6/STAT-3 Pathway In Caffeic Acid-inhibited Inflammatory Microenvironment In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the third leading cause of cancer-related mortality worldwide.The cancer-related inflammation has been proposed as a major hallmark of malignancy.In HCC,tumors produce a cancer promoting inflammatory microenvironment via recruiting and/or auto-secreting chemotactic factors,such as interleukin-6(IL-6),IL-8,tumor necrosis factor-alpha(TNFa),signal transducers and activators of transcription-3(STAT-3),and eicosanoids,which promotes cellular proliferation and enhances the malignant properties.Caffeic acid(CaA),a naturally occurring hydroxycinnamic acid derivatives,is an active component in the phenolic propolis extract and also in a wide variety of plants.Caffeic acid has several biological and pharmacological properties,such as antioxidants,anti-inflammatory,immunomodulatory activates,and ffectively anticancer functions.Recent study suggests that CaA reduces the cutaneous TNFa,IL-6,and IL-1β levels in mice.However,the roles of CaA in the regulation of inflammatory microenvironment in HCC,and the molecular mechanisms involved in,remain largely uninvestigated.Methods1.Cell culture and CaA treatmentAfter HepG2,MHCC97H,or RAW 264.7 macrophage cells were conventional cultured for 24 h,they were treated with 20 or 40 μM of CaA for another 24 h.Then such cells were used to determine the levels of IL-6,TNFα,iNOS,and NO.2.Determination of cell viabilityTreated cells were incubated with 20.0 μl of WST-8 solution(Beyotime Co.Ltd)for 4 h.The absorbance at 450 nm was measured with a multimode microplate reader.The values for medium control cells were used to determine the 100%level.3.Plasmid or miRNA transfectionCells were transfected with plasmid,siRNA,miRNA-mimic,or anti-miRNA by using Lipofectamine 2000 reagent for 12 h.Then,such cells were conventional cultured for another 24 h before being used for other experiments.4.Quantitative real-time polymerase chain reaction(qRT-PCR)Total RNA was transcribed into cDNA using AMV Reverse Transcriptase.The qRT-PCR was performed using an ABI 7300 real-time PCR detection system.5.DNA methylation analysisThe genomic DNA was modificated with sodium bisulfite using the EpiTect Kit.DNA methylation was analyzed using a SYBR Green-based quantitative methylation-specific PCR using an ABI 7300 real-time PCR detection system.6.Western blotsProteins were transferred to polyvinylidene fluoride(PVDF)membranes,which were then probed with primary antibody at 4℃ overnight.Then membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h.The immune complexes were detected by the enhanced chemiluminescence.7.Co-ImmunoblotEqual amount of total proteins were separated by 10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis,and transferred to PVDF membranes.Then they were incubated with immunoprecipitates antibody and subsequently with A+G sepharose beads at 4 C overnight.To determine protein-protein interaction,the immunoprecipitates were analyzed by Western blots.8.Enzyme-linked immunosorbent assay(ELISA)Samples or standard protein were added to the wells for 2 h at 37℃.Then biotinylated antibody was added for 2 h,followed by incubating with streptavidin-HRP for another 1 h,follow by adding the substrate,the colorimetric reaction was determined by measuring the absorbance at 450 nm with a multi-well plate reader.9.Statistical analysisData were presented as the means±SD.A Student’s t test,and a one-way analysis of variance(ANOVA)were used to assess significant differences between groups.In our present study,p values<0.05 were considered statistically significant.Results1.CaA inhibited inflammatory response in HCC or macrophageApplication of 20 M CaA treatment of human hepatocellular carcinoma HepG2 and MHCC97H cell lines,or the combined treatment of LPS using 40 M CaA 24 h RAW264.7 cells that CaA can inhibit the expression of two kinds of different cell types in IL-6,TNF,iNOS,NO alpha and/or synthesis level.2.Effects of CaA on NF-κB and STAT-3 pathwayIn HepG2 and MHCC97H cells,CaA inhibited expression and phosphorylation of NF-κB and STAT-3;Interestingly,in RAW264.7 cells,CaA only inhibited the phosphorylation of NF-κB and STAT-3,but had no obvious effects on the expression of these two factors.3.Role of CaA in regulating NF-κB and STAT-3 in HCCIn HepG2 and MHCC97H,CaA elevated miR-124,which bound to the 3’-UTR of RelA and STAT-3 mRNAs,and attenuated the expression of these two factors.We futner found that,CaA elevated the miR-124 via inducing the DNA de-methylation.4.Role of CaA in regulatingf NF-κB and STAT-3 in macrophageIn RAW264.7 cells,CaA blocked the 14-3-3ζ,which inhibited the LPS-induced phosphorylation of NF-κB and STAT-3.In addition,CaA attenuated the 14-3-3ζ via down-regulating the TNFα,but up-regulating the ubiquitination of 14-3-3ζ.5.Role of miR-124 in CaA-inhibited inflammation in HCC cellsKnockdown of miR-124 significantly inhibited the expression and secretion of IL-6 in HepG2 and MHCC97H cells induced by CaA treatment.6.Role of 14-3-3ζin CaA-inhibited inflammation in macrophageHigh expression of 14-3-3ζsignificantly reversed the CaA-inhibited expressions/synthesis levels of IL-6,TNF,iNOS,and NO.Conclusions1.CaA inhibited inflammatory response in HCC or macrophage;2.NF-κB and STAT-3 were involved in CaA inhibited inflammatory response in HCC or macrophage;3.Up-regulation of miR-124 via DNA de-methylation was involved in CaA inhibited NF-κB and STAT-3 and IL-6 secretion in HCC cells;4.Down-regulation of 14-3-3ζ was involved in CaA inhibited NF-κB and STAT-3 and inflammatory response in macrophage.