MC-LR Pollution And Its Synergistic Effect With HBX Regulated The PP2A Induced Protein Phosphorylation In HepG2 Cells And Effected On Hepatocarcinogenesis

PART(ESTABLISHMENT OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR WATER MC-LR TESTING AND PRELIMINARY INVESTIGATION ON POLLUTION IN HIGH INCIDENCE AREA OF LIVER CANCER IN GUANGXIObjectiveEstablish a high performance liquid chromatography(HPLC)method for the determination of MC-LR in water,and the MC-LR contamination in different water sources in the high incidence area of liver cancer in Guangxi in May and October 2017 was investigated.Methods1.Take 500 mL of water sample,filter with 0.45 μm organic filter,and filter the filtrate through C18 column,elute with 10 mL of ultrapure water,20 mL of 20%methanol,and 15 mL of 80%methanol(add 0.05%trifluoroacetic acid).The content of microcystins in water samples was determined by chromatographic column with mobile phase of 0.1%phosphoric acid-acetonitrile(67:33),flow rate of 1.0 mL/min,column temperature of 45℃ and wavelength of 238 nm.The recovery rate and detection limit of microcystins under different conditions were compared,and the high performance liquid chromatography method of MC-LR in source water was optimized.2.In May and October 2017,collect one bottle(500 mL)of source water,terminal water and pond water in 28 villages of Fusui County,Guangxi Province.After storage and precipitation at 4 C,the water was filtered,extracted,concentrated and volumetrized to 1 mL within 24 hours.The content of microcystins in different water sources was detected and compared by the above-mentioned optimized HPLC method.Results1.Comparing the results of HPLC detection of microcystins treated with different concentrations of eluent,this study found that 20%methanol eluent is suitable for the quantitative detection of water samples.The addition of 0.05%TFA to the 80%methanol solution eluate significantly improved the recovery of toxins.The mobile phase was 0.1%aqueous phosphoric acid-acetonitrile(67:33),MC-LR had good linearity in the range of 0.05-1.0μg/mL(r2=0.9987),and the peak shape was good;the recovery rate was 90%～110%.The relative standard deviation was 5.4%～9.6%;the detection limit of MC-LR method was 0.028μg/L,and the retention time of MC-LR was 15.291 min.2.In May and October of 2017,57 water samples were collected from different sources in the high incidence area of liver cancer in Guangxi,including 15 sources water,the concentration range of TMCs in May and October is 0～169.21 ng/L and 6.14～168.56 ng/L;28 pieces of terminal water,the concentration range of TMCs in May and October is 0～617.02 ng/L and 0～89.97 ng/L;14 parts of pond water respectively,the concentration range of TMCs in May and October is 0～567.32 ng/L and 1.84-897.26 ng/L.The average concentration of TMCs in the three types of water samples in May and October was statistically significant(P<0.05).The average concentration of TMCs in all water samples were lower than the recommended value of WHO(1μg/L).ConclusionThe optimized method of high performance liquid chromatography for MC-LR detection in water source has high accuracy,sensitivity,recovery and good reproducibility,and can be used for MC-LR detection in water samples;In May and October 2017,the detection values of TMCs in different water sources in Fusui County,a high incidence area of liver cancer in Guangxi,were lower than the recommended values of WHO(1μg/L).PART Ⅱ SYNERGISTIC EFFECT OF MC-LR AND C-TERMINAL TRUNCATED HBX ON HEPG2 CELLS AND ITS EFFECT ON PP2A MEDIATED DOWNSTREAM TARGET OF MAPK SIGNALING PATHWAYObjectiveTo clarify the synergistic effect of MC-LR and C-terminus truncated HBX,to explore their effects on downstream targets of PP2A mediated MAPK signaling pathway,and to elucidate the roles and possible mechanisms of MC-LR and HBX in the proliferation,invasion,migration,cycle and apoptosis of HepG2 cells.MethodsThe lentiviral vector was constructed and transfected into HepG2 cells by using HBXA32,HBXΔ4 and wild-type HBX,which are common in HBX tissues.HepG2 cells transfected with HBXΔ32,HBXΔ4,and HBX plus MC-LR were used as experimental group,and HepG2 cells without HBX fragment were added with MC-LR and negative control(NC)as control group.qRT-PCR and Western blotting(WB)were used to detect the expression of HBX gene and protein in each group after transfection,and to determine whether the transfection was successful.In the experimental group and the control group,the activity of PP2A was detected by enzyme-linked immunosorbent assay;proliferation,migration and invasion of HepG2 cells were detected by cell scratch test,Transwell chamber test and plate cloning test;cell cycle and apoptosis of HepG2 cells were detected by flow cytometry;MEK1/2,ERK1/2,p38 and JNK protein expression of downstream MAPK signaling pathway mediated by PP2A was detected by WB.The levels of cell cycle regulatory proteins p53,cdc25 and cdc2 were further detected,and the changes of these cell cycle regulatory proteins were observed after intervention with PP2A protein phosphatase agonist.Results1.This study successfully constructed HepG2 cells expressing C-terminally truncated HBX gene and protein.The green fluorescent signal was observed under microscope.The expression of HBX mRNA and protein was verified by qRT-PCR and WB.2.By comparing the PP2A activity at different MC-LR concentration gradients and different intervention time points,it was found that PP2A activity was the lowest at 24 hours after 10 μM MC-LR intervention.Two MC-LR concentrations of 0 and 10 μM were selected for subsequent cell scratching,migration and invasion,cell cloning and flow cytometry to detect apoptosis and cycle,and the synergistic effects of the two on HepG2 cells were observed at two time points of 0 h and 24 h.MC-LR(0,10 μM)and HBXΔ32 were selected to detect the expression of downstream proteins related to PP2A mediated MAPK pathway at the two time points(12 h,24 h)where PP2A activity was most significantly inhibited.3.The results showed that wound healing distance,cell proliferation,migration and invasive ability of HepG2 cells transfected with HBXΔ32,HBXΔ4 and HBX were significantly higher than those of the control group(P<0.05).4.The distribution ratio of S phase of HBXΔ 4 and HBXA 32 cells treated with 10 μM MC-LR was significantly higher than that of the control group(P<0.05),and the apoptotic rate of HepG2 cells treated with MC-LR and C-terminal truncated HBX was not significantly different between the experimental group and the control group(P>0.05).5.The phosphorylation levels of MEK1/2,ERK1/2,p38 and JNK proteins related to MAPK signaling pathway were compared.The results showed that the phosphorylation levels of MEK1/2,ERK1/2,p38 and JNK proteins in the experimental group were higher than those in the control group(P<0.05),and the expression levels increased with the prolongation of MC-LR exposure time,and there was a linear correlation between them.6.Compared with the control group,the phosphorylation of cyclin p53 and cdc2 protein in the experimental group decreased significantly,and showed a dependence with the prolongation of MC-LR exposure time(P<0.05);The expression of phosphorylated cdc25 protein increased significantly compared with the control group,and showed a dependence with the prolongation of MC-LR exposure time(P<0.05).7.After pretreatment of HepG2 cells with 10 μM PP2A protein phosphatase agonist DES for 12 h,the expression of p53,cdc25 and cdc2 in MC-LR+DES treated group was lower than that in MC-LR treated group,the difference was statistically significant(P<0.05).ConclusionMC-LR and C-terminus truncated HBXΔ32,HBXΔ14 and HBX have certain effects on the proliferation,invasion,migration and cell cycle of human hepatocellular carcinoma HepG2 cells;MC-LR and C-terminus truncated HBXA32 can synergistically increase the expression levels of key proteins in p38,ERK and JNK of three downstream pathways of MAPK;MC-LR and C-terminally truncated HBXA32 can induce changes in phosphorylated protein levels of cell cycle-associated proteins p53,cdc25 and cdc2 through PP2A.These results suggest that after MC-LR and C-terminus truncated exposure to HepG2 cells,MAPK kinase may be the downstream target after PP2A activity is inhibited,and affect the proliferation,invasion and migration of HepG2 cells by regulating the protein phosphorylation level of corresponding downstream target of MAPK pathway.