Screening And Targeted Expression Of Differentially Expressed Gene Promoters In Hepatocellular Carcinoma

OBJECTIVEObjective to explore the differential expression of genes related to the occurrence and development of hepatocellular carcinoma(HCC)in HCC cells and the difference of promoter activity between HepG2 cell and normal hepatocyte HL-7702,so as to obtain the differentially expressed genes of HCC and the promoters that can mediate the targeted expression of genes in HCC cells,it will lay a foundation for further analysis and functional research of the key DNA motif(DNA motif)in the promoter of differentially expressed genes in HCC.METHODS1.The differentially expressed genes and their promoter sequences were predicted by literature review and bioinformatics analysis.2.qRT-PCR was used to detect the expression level of differentially expressed genes in HepG2 and HL-7702 cells.3.The genome of HepG2 cells was extracted,and the promoter regions of differentially expressed genes were sequenced and analyzed.4.The sequenced promoter sequence was synthesized and the restriction sites were added at both ends.Then,the promoters were inserted into the promoter activity detection plasmid pGL3-basic containing luciferase expression gene;The recombinant plasmid was transformed into competent DH5αE.coli cells,and the monoclonal antibody was obtained by screening.After plasmid extraction,the plasmid was identified by agarose gel electrophoresis,and the plasmid was further identified by sequencing.5.The luciferase reporter vector plasmids containing the promoters to be tested were cotransfected into HepG2 cells and HL-7702 cells with the luciferase reporter vector plasmid p RL-TK;Then the promoter activity of the target promoter was detected by double luciferase reporter gene assay.6.Objective to explore the possible DNA motif in the promoter region of HCC related genes by bioinformatics analysis.RESULTS1.TCGA database analysis showed that Clusterin(CLU),Dickkopf(DKK1),Golgi protein-73(GP73),Acrosin binding protein(ACRBP),Alpha fetoprotein(AFP)were differentially high expressed in HCC tissues.The promoter regions(-700/+300)of five genes were obtained by bioinformatics analysis.2.The results of qRT-PCR showed that:the relative mRNA expression levels of CLU,DKK1,GP73,ACRBP and AFP in HepG2 were higher than those in HL-7702.The relative mRNA expression levels of CLU,DKK1,GP73,ACRBP and AFP in HepG2 were 9.38,37.33,17.88,2.69 and 2.39×10~6times higher than those in HL-7702,respectively,and the difference was statistically significant(P<0.05).3.The results of promoter sequencing showed that the promoter sequences of AFP,GP73 and ACRBP in HepG2 cells were compared with those in Genebank,and the mutation sites were found;the promoter sequences of CLU and DKK1were consistent with those in Genebank.4.The sequenced promoter sequence was synthesized and constructed into pGL3-basic plasmid,and the expression plasmids were obtained:pGL3-CLUP,pGL3-DKK1P,pGL3-GP73P reference group,pGL3-GP73P mutation group,pGL3-ACRBPP reference group,pGL3-ACRBPP mutation group,pGL3-AFPP reference group and pGL3-AFPP mutation group.The extracted expression plasmids were subjected to restriction enzyme digestion and electrophoresis:the expression plasmids pGL3-CLUP,pGL3-DKK1P,pGL3-GP73P reference group,pGL3-GP73P mutation group,pGL3-ACRBPP reference group,pGL3-ACRBPP mutation group,pGL3-AFPP reference group and pGL3-AFPP mutation group were digested by single enzyme to form one electrophoresis band with a length of about 6000bp;the plasmids digested by double enzyme to form two electrophoresis bands with a length of about 5000bp and the other length of about1000bp,the length of which was consistent with that of plasmid and promoter.Sequencing results also showed that the target promoter sequence had been inserted into pGL3-basic plasmid promoter region.5.Double luciferase reporter gene assay showed that the promoter activity of expression plasmid pGL3-CLUP,pGL3-DKK1P,pGL3-GP73P reference group,pGL3-GP73P mutation group,pGL3-ACRBPP reference group and pGL3-ACRBPP mutation group in HepG2 was higher than that in HL-7702.The promoter activities of pGL3-GP73P reference group and pGL3-GP73P mutations group in HepG2 cells were 18.757 and 10.822 times higher than those in HL-7702cells,respectively,and the difference was statistically significant.The promoter activity of pGL3-ACRBPP reference group and pGL3-ACRBPP mutation group in HepG2 cells was 13.159 and 10.286 times higher than that in HL-7702 cells,respectively,and the difference was statistically significant.It was also found that the mutation sites of GP73 and ACRBPP promoters could reduce the promoter activity,and the specific mechanism needs to be further studied.After pGL3-AFPP reference group and pGL3-AFPP mutation group were transfected into HepG2 cells and HL-7702 cells respectively,it was found that the promoter activity was poor and unstable,which could not be statistically analyzed.6.The expression plasmids:pGL3-CLUP,pGL3-DKK1P,pGL3-GP73P reference group,pGL3-GP73P mutation group,pGL3-ACRBP reference group and pGL3-ACRBP mutation group were preliminarily explored by MEME in anr mode,nine motifs were obtained,among which two common motifs were found.CONCLUSION:Genes CLU,DKK1,GP73,ACRBP and AFP were differentially expressed in HepG2 cells;The promoter of five genes was screened;GP73 and ACRBP promoter can mediate the gene expression in HepG2 cells and have good promoter activity.It is found that the mutation sites in promoter can affect promoter activity.