The Effects And Mechanism Of PCB126 On Liver Cancer Epithelial-mesenchymal Transition And Aerobic Glycolysis

Polychlorinated biphenyls(PCBs),the classic persistent organic pollutants(POPs),are halogenated hydrocarbon compounds that have been widely used in industry.Due to the large number of improper handling of PCBs,they enter the environment and accumulate,causing harm to human health.3,3'4,4',5-pentachlorobiphenyl(PCB126)is a dioxin-like PCB.Hepatocellular carcinoma(HCC)is a kind of malignant humantumors.At present,the incidence of liver cancer has been increasing year by year.Epidemiological studies have shown a certain relationship between the occurrence of HCC and PCBs exposure,but the molecular mechanism is not clear.In this study,we explored the effects of PCB126 on epithelial-mesenehymal transition(EMT)and the Warburg effect in HCC cells and its underlying mechanisms,The main research as follow:Part one: After Bel-7402 and SMMC-7721 cells were treated with increased concentrations of PCB126 from 10-11 to 10-7 M for 24 or 48 h,the expressions of EMT markers,and the effect of PCB126 on cell viability were examined.The data showed that PCB126(10-11 to 10-7 M)exposure for 48 h significantly decreased E-cadherin expression and increased N-cadherin and Vimentin mRNA levels.In additon,we found the morphology of both Bel-7402 and SMMC-7721 cells were changed.The MTT assay indicated that PCB126 treatment did not affect HCC cell viability.Cell adhesion analysis showed that PCB126 treatment resulted in a significant decrease in intercellular adhesion.These results indicated that PCB126 promotes EMT of HCC cells.Part two: After SMMC-7721 and Bel-7402 cells were treated with different concentrations of PCB126(10-10~10-8M)for 48 h,the levels of STAT3,p-STAT3 and Snail1 were detected.The results showed that Snail1 mRNA leveand the protein levels of both p-STAT3 and total STAT3 were strikingly up-regulated in PCB126-exposed HCC cells.Meanwhile,we found that both nuclear and cytoplasmic p-STAT3 was obviously increased.These results suggested that PCB126 activated STAT3 signaling.Furthermore,we tested the effects of WP1066,a specific STAT3 inhibitor,on Snail1 and EMT markers.The data showed that PCB126 induced EMT was inhibited after WP1066 treatment.Additionaly,we found that PCB126 exposure significantly increased pyruvate kinase M2(PKM2)expression and nuclear PKM2 levels.To address the role of PKM2 in PCB126-induced activation of STAT3/Snail1 signaling and EMT,HCC cells were transfected with PKM2 sh RNA or control shRNA.The result indicated that PKM2 knockdown alleviated PCB126-induced up-regulation of p-STAT3.At the same time,PCB126-induced EMT was significantly inhibited.These results suggested that PCB126 activates STAT3/Snail1 signaling through PKM2 and thus promotes EMT in HCC cells.Part three: After HCC cells were treated with different concentrations of PCB126(10-10~10-8M)for 48 h,the mRNA expressions of AhR and CYP1A1 were significantly increased.To verify the role of AhR in PCB126-induced EMT,we used AhR shRNA to suppress the AhR signaling.The results showed that AhR shRNA treatment blocked PCB126-induced expression and nuclear translocation of PKM2,as well as STAT3/Snail1 signaling pathway.Meanwhile,PCB126-induced EMT was markedly inhibited after AhR knockdown.In addition,PCB126 expose increased the level of ROS.NAC(ROS inhibitor)treatment prevented PCB126-induced PKM2/STAT3/Snail1 signaling and EMT.Moreover,AhR shRNA treatment reduced the ROS level.These data showed that PCB126 activates the PKM2/STAT3/Snail1 signaling and promotes EMT by AhR.Part four: To investigate whether ER is involved in PCB126-induced EMT,we used the ER inhibitor ICI 182780 to disturb the ER pathways in PCB126-exposed HCC cells.The data showed that PCB126-induced the alteration in EMT markers and PKM2 were markedly reversed by ICI co-treatment.At the same time,PCB126-induced activation of STAT3/Snail1 signaling was inhibited after ICI treatment.Above results showed that ER plays an important role in PCB126-induced EMT of HCC cells.Part five: To explore the impact of PCB126 on the Warburg effect in HCC cell,SMMC-7721 cells were treated with different concentrations of PCB126(10-11~10-7M)for 24 or 48 h and the expression levels of Warburg effect genes were detected by QT-PCR.The results showed that PCB126 exposure significantly reduced the glucose content in the culture medium,improves the production of lactic acid,and increased the mRNA levels of PKM2,glucose transporter GLUT1,lactate dehydrogenase LDHA and pyruvate dehydrogenase kinase PDK.Then we used RNA interference to knock down PKM2.We found that PCB126-induced Warburg effect was suppressed.These results indicated that PCB126-induced the Warburg effect was mediated by PKM2.In conclusion,PCB126 promotes EMT of HCC cells by activating PKM2/STAT3/Snail1,which is mediated by ER and AhR.Moreover,PCB126 promotes the Warburg effect by PKM2 in HCC cells.These findings may be important mechanisms for PCB126 promoting the development of hepatocellular carcinoma.