Protective Effect Of PBX1 In HepG2 Cells Induced By MeHg And Its Related Mechanism

Objective:In this study,HepG2 cells were taken as the research object to explore the effect of PBX1 on the biological behavior of HepG2 cells,and HepG2 cells injury model was established by using MeHg,so as to explore the role and mechanism of PBX1 in MeHg-induced HepG2 cells injury,providing a preliminary working basis for the study of hepatotoxicity of MeHg.Methods:In the first part,PBX1 gene was overexpressed in HepG2 cells by lentivirus packaging method,and divided into groups according to Wild(Wild type)group,Vector(no load)group,and PBX1(overexpression)group to verify the gene transfection efficiency.The effect of PBX1 on the proliferation of HepG2 cells was detected by drawing growth curve.The effect of PBX1 on the clonogenic ability of HepG2 cells was detected by plate cloning assay.Cell scratch assay was used to detect the migration effect of PBX1 on HepG2 cells.Western Blot assay was used to detect the expression levels of proteins related to DNA damage repair in HepG2 cells by PBX1.In the second part,after HepG2 cells were given a certain dose of MeHg,cell survival rate was detected by MTT assay,cell morphological changes were observed by living cell observation system,cell apoptosis rate was detected by Annexin V-FITC/PI assay,and cell DNA damage was detected by comet assay.The expression levels of DNA damage repair and mitochondria-mediated apoptosis-related proteins(γH2AX,PARP-1,PAR,Rad51,Ku70,AIF,Cleaved Caspase 3,CytC)were detected by Western Blot.The third part,through the detection of MeHg after processing each cell survival and apoptosis rate,explore PBX1 of MeHg induced cell damage,the influence of Western Blot detecting DNA damage repair related proteins(γH2AX,PARP-1,Rad51,KU70)and mitochondrial apoptosis of PARP-1/AIF protein involved in pathways,Caspase 3 / CytC(PARP 1,PAR,AIF,Cleaved Caspase 3,CytC)expression level,To investigate the mechanism of PBX1 alleviating MeHg damage to HepG2 cells.Results:1.Biological effects of overexpression of PBX1 on HepG2 cells(1)The HepG2 cell lines of Vector group and PBX1 group were successfully constructed;(2)The results of growth curve showed that the cell number of PBX1 group was significantly higher than that of Wild group and Vector group on the fourth day.(3)The plate cloning experiment showed that the number of clone formation in PBX1 group was significantly higher than that in Wild group and Vector group.(4)The cell scratch experiment showed that the cell migration distance of PBX1 group was significantly higher than that of Wild group and Vector group after24h.(5)Compared with Wild and Vector groups,the expression of γH2AX,PARP-1(116 kDa),Rad51 and Ku70 in PBX1 group were up-regulated by Western Blot.2.The damage effect of methylmercury on HepG2 cells and its mechanism(1)MTT assay was used to detect the survival rate of HepG2 cells treated with different concentrations of MeHg for 24h.The results showed that the survival rate of HepG2 cells decreased gradually with the increase of MeHg concentration.Compared with the control group,the survival rate of HepG2 cells in 0.5,0.75,1.0and 1.25μg/m L groups decreased significantly.(2)Morphological changes of HepG2 cells treated with different concentrations of MeHg for 24h were observed by living cell imaging system.HepG2 cells in the control group were polygonal,with clear boundaries and good refractive properties.With the increase of MeHg concentration,the cell volume became smaller,the cytoplasm was concentrated,the contour was smooth,and the refractive ability became worse.(3)Flow cytometry was used to detect the apoptosis rate of HepG2 cells after treatment with different concentrations of MeHg for 24h.(4)The effect of 1.0μg/m L MeHg on HepG2 cells in different time was detected by basic comet assay.With the increase of time,trailing phenomenon gradually appeared.(5)Western Blot analysis showed that the expression of γH2AX protein increased first and then decreased with the increase of time;The expression of PARP-1(116 kDa),PAR,KU70,Rad51,AIF(57 kDa),Cleaved Caspase 3,CytC protein increased with time.3.The damage effect of MeHg on HepG2 cells and its mechanism(1)MTT assay was used to detect the cell survival rate of Control group,Vector group,PBX1 group,MeHg group,Vector+MeHg group and PBX1+MeHg group after treatment with 1.0μg/m L MeHg for 24h.Compared with the Control group,the cell survival rate of the Vector group had no significant change.Compared with the Control group,the cell survival rate in the MeHg group and the Vector+ MeHg group was significantly decreased,which was consistent with the results of the second part of the experiment.Compared with the Vector+MeHg group,the cell survival rate in the PBX1+MeHg group was significantly increased.(2)Flow cytometry was used to detect the apoptosis rate of Control group,Vector group,PBX1 group,MeHg group,Vector+MeHg group and PBX1+MeHg group after treatment with 1.0μg/m L MeHg for 3h.Compared with the Control group,the apoptosis rate of Vector group and PBX1 group had no significant change.Compared with the Control group,the apoptosis rate in Mehg group and Vector+MeHg group was significantly increased,and the results were statistically different.Compared with the Vector+MeHg group,the apoptosis rate of PBX1+MeHg group was significantly decreased,and the results were statistically different.(3)Western Blot assay was used to detect the expression of double-stranded DNA damage and repair related proteins in Control group,Vector group,PBX1 group,MeHg group,Vector+MeHg group and PBX1+MeHg group after treatment with 1.0μg/m L MeHg for 3h.Compared with the Control group,the protein expression of PARP-1(116 kDa),γH2AX,KU70 and Rad51 in the Vector group had no significant difference.Compared with the Control group,the protein expressions of PARP-1(116 kDa),γH2AX,Ku70 and Rad51 in the PBX1 group were significantly increased,which was consistent with the results in Part I.Compared with the Control group,the protein expressions of PARP-1(116 kDa),γH2AX,Ku70 and Rad51 in the MeHg group and the Vector+MeHg group were significantly increased,which was consistent with the results in the second part.Compared with the Vector+MeHg group,the expression of PARP-1(116 kDa)and γH2AX protein in PBX1+MeHg group were significantly down-regulated.Compared with the MeHg group,the protein expressions of KU70 and Rad51 in PBX1+MeHg group were significantly increased.(4)Flow cytometry was used to detect the apoptosis rate of Control group,Vector group,PBX1 group,MeHg group,Vector+MeHg group and PBX1+MeHg group after treatment with 1.0μg/m L MeHg for 24 days.Compared with the Control group,the apoptosis rate of Vector group and PBX1 group had no significant change.Compared with the Control group,the apoptosis rate in MeHg group and Vector+MeHg group was significantly increased.Compared with the Vector+MeHg group,the apoptosis rate of PBX1 +MeHg group decreased significantly.(5)The expression of mitochondria-mediated apoptosis-related proteins in Control group,Vector group,PBX1 group,MeHg group,Vector+MeHg group and PBX1+MeHg group was detected by Western Blot after treatment with 1.0μg/m L MeHg for 24h.Compared with Control group,the expression of AIF(57 kDa),Cleaved Caspase 3,and CytC protein in Vector group and PBX1 group were not significantly changed.Compared with the Control group,the expression of PARP-1(116 kDa),PAR,AIF(57 kDa),Cleaved Caspase 3,and CytC protein in the MeHg group and the Vector+MeHg group were significantly increased,which was consistent with partial results in Part II.Compared with the Vector+MeHg group,the expression of PARP-1(116 kDa),PAR,AIF(57 kDa),Cleaved Caspase 3,CytC protein in PBX1+MeHg group was significantly decreased.Conclusion:1.PBX1 can enhance proliferation of HepG2 cells.2.MeHg can induce DNA damage and mitochondria-mediated apoptosis in HepG2 cells.3.PBX1 can reduce DNA damage and mitochondria-mediated apoptosis in HepG2 cells induced by MeHg.