Effects Of Phenobarbital On The Expression Of UGT1A1 Wild Type And Q239X Mutant In Human Hepatocytes In Vitro

Objective Construct a human hepatocyte model with Q239X(c.715 c →T)mutation of uridine diphosphate glucuronosyl transferase 1A1(UGT1A1)gene by CRISPR/cas9 technology.Investigate the effect of Q239 X mutation of UGT1A1 gene on its expression and the effect of phenobarbital on the expression of wild-type,Q239 X heterozygous and homozygous variants of UGT1A1 gene in vitro.Methods1.The model of Q239 X mutation of UGT1A1 gene in human hepatocytes was constructed in vitro.According to CRISPR/cas9 principle,UGT1A1 sgrna1 oligo F/R,identification primer UGT1A1 F/R and homologous recombination single strand donor were designed and synthesized.UGT1A1 sg RNA oligo was introduced into eukaryotic expression plasmid px458(px458-UGT1A1 sg RNA)carrying cas9,and px458-UGT1A1 was transformed into lipo2000 Sg RNA and homologous recombination single strand donor were introduced into HL-7702 cells at the same time.The GFP positive cells were cultured into monoclonal,and then expanded gradually.The genomic DNA of each clone was extracted for PCR amplification,and the PCR products were sequenced.The positive clones with point mutation of UGT1A1 gene were screened out.2.UGT1A1 gene wild-type,Q239 X heterozygous mutant and homozygous mutant hepatocytes were cultured respectively.The PCR amplification products of DNA were agarose gel electrophoresis to detect the expression of DNA gene of UGT1A1 in three groups of hepatocytes.3.The expression of UGT1A1 gene m RNA and protein was detected by real-time fluorescent quantitative PCR(RT-PCR)and Western blotting.4.Different concentrations of phenobarbital were used to induce wild-type hepatocytes,and CCK-8 method was used to detect the cell survival rate of each group.5.Three kinds of hepatocytes with UGT1A1 gene wild type,Q239 X heterozygous mutant and Q239 X homozygous mutant were randomly divided into experimental group and control group.The experimental group was treated with 1640 medium containing 1 mmol/L and 2 mmol/L phenobarbital,and the control group was treated with 1640 medium without phenobarbital,and cultured for 24 h and 48 h.The m RNA and protein expression of UGT1A1 gene were detected by RT-PCR and western blotting.Results1.PCR amplification product identification electrophoresis showed that there were bands at 351 bp,and DNA sequencing showed that the heterozygous and homozygous mutant hepatocyte model of UGT1A1 gene Q239 X was constructed successfully.2.Agarose gel electrophoresis showed that UGT1A1 gene expression was observed at DNA level in wild-type UGT1A1,Q239 X heterozygous mutant and Q239 X homozygous mutant hepatocytes.3.The results of RT-PCR showed that the expression of UGT1A1 m RNA in Q239 X heterozygous mutant and Q239 X homozygous mutant cells(0.30±0.09 and 0.03±0.01)was significantly lower than that in wild-type hepatocytes(1.00±0.00)(P<0.05),30% and 3% of that in wild-type hepatocytes,respectively.Western blotting showed that the expression of UGT1A1 protein in Q239 X heterozygous mutant and Q239 X homozygous mutant cells(1.07±0.03,0.86±0.15)was significantly lower than that in wild-type hepatocytes(1.44±0.15)(P<0.05),74% and 60% of that in wild-type hepatocytes,respectively.4.The results of RT-PCR showed that the expression of UGT1A1 m RNA in the wild-type 1 mmol/L 24 h and 48 h,2 mmol/L 24 h and 48 h experimental groups was significantly higher than that in the wild-type control group(P<0.05),and the expression was 1.59,2.06,1.81 and 2.00 times higher than that in the wild-type control group,respectively;The heterozygous mutation of Q239 X in the UGT1A1 gene 1 mmol/L 24 h and 48 h,2 mmol/L 24 h and 48 h experimental groups were significantly higher than that in the wild-type control group.The expression of UGT1A1 m RNA was 1.41,1.36,1.43 and 1.38 times as much as that of Q239 X heterozygous control group(P<0.05).5.Western blotting results showed that the expression of UGT1A1 protein(2.43±0.41,2.40±0.67,2.46±0.38,2.41±0.33)in the 1 mmol/L 24 h and 48 h wild type group was significantly higher than that in the wild type group(P<0.05),which was 1.69,1.69,1.71,1.70 times higher than that in the corresponding control group(P<0.05).The expression of UGT1A1 protein(1.49±0.08,1.49±0.13,1.45±0.10,1.47±0.17)in experimental group was significantly higher than that in heterozygous mutant control group(P<0.05),which was 1.39,1.46,1.36,1.44 times higher than that in Q239 X homozygous control group(P<0.05).The expression of UGT1A1 gene protein(0.83±0.22,0.91±0.21,0.91±0.21,0.84±0.14)in the homozygous 1 mmol/L24 h and 48 h,2 mmol/L24 h and 48 h groups had no significant change(P>0.05).Conclusion1.In this study,CRISPR/cas9 technology was used to successfully construct the Q239 X homozygous mutant and heterozygous mutant hepatocyte models of UGT1A1 gene.2.The Q239 X mutation of UGT1A1 gene did not affect the expression of the gene at DNA level.3.The Q239 X mutation of UGT1A1 gene downregulate the transcription and translation level of the gene,and the homozygous Q239 X mutation downregulate the gene more significantly.4.As an enzyme inducer,phenobarbital can induce the expression of wild-type and Q239 X heterozygous mutant hepatocytes of UGT1A1 gene at the level of transcription and translation,but has no induction effect on the expression of Q239 X homozygous mutant hepatocytes;5.Phenobarbital induced the expression of Q239 X heterozygous mutant hepatocyte of UGT1A1 gene lower than that of wild type.