| Objective: To explore the role of HHcy and lipogenic gene ERO1α and its DNA methylationin causing liver lipid metabolism disorders of ApoE-/-mice to clear the effect of HHcy inERO1expression and its DNA methylation in the form of lipid metabolism disorder of ApoE-/-mice.Medthods: Oil red O staining was used to detect lipid deposition in the liver of mice fromeach group, microplate reader was used to detect the changes of the levels of TC and TG in theliver of mice from each group, RT-PCR and Western blot were used to detect the expression ofERO1α mRNA and protein, ntMS-PCR was used to detect the changes of ERO1α gene DNAmethylation and ELISA was used to measure the levels of DNMT1in the liver of mice fromeach group. Construct recombinant plasmid of ERO1α and DNMT1and corresponding siRNAand transfected into the liver cells. RT-PCR was used to detect the changes of DNMT1andERO1α mRNA levels, Western blot was used to detect the expression of ERO1α DNMT1protein, and ntMS-PCR was used to detect the changes of ERO1α gene DNA methylation.Results: The area and levels of lipid in liver of mice in HHcy group was significantly higherthan the intervention group (P<0.01), compared with the control group, the area of liver lipidand the level of TC, TG were increased by approximately4.53,2.21,2.86times. Correlationanalysis showed that, TC were positively correlated with its serum Hcy levels (r2=0.1029,P<0.01), TG were positively correlated with serum Hcy levels (r2=0.1216, P<0.05). Fluorescent RT-PCR results showed that ApoE-/-control group, HHcy group and theintervention group compared with the control group, the expression of ERO1α decreased to76.66%,20.43%and40.14%, the difference was statistically significant (P<0.01). Theintervention group compared with HHcy group, the expression of ERO1α increased1.96-fold,the difference was statistically significant (P<0.01). Result from Western blot showed that Hcyaffected the expression of ERO1α protein was consistent with its mRNA expression. On thecellular level, cells were stimulated with50,100,200,500μmol/L Hcy, compared with thecontrol group, mRNA expression of ERO1α decreased to90.37%, respectively,55.44%,46.88%,36.93%, the difference was statistically significant (P<0.01), folic acid treatmentgroup compared with100μmol/L Hcy group, expression of ERO1α mRNA elevated1.81-fold,the difference was statistically significant (P<0.05). Result from Western blot showed that Hcyaffected the expression of ERO1α protein was consistent with its mRNA expression.Detecting mRNA and protein expression of ERO1α after constructing recombinantplasmid and transfected with liver cells, the results showed that there was no significantchange of mRNA levels of ERO1α in EGFP-N1group compared with the control group, whilethe mRNA levels of EGFP-N1-ERO1α group increased to1.33times, the difference wasstatistically significant (P<0.01), Western blot analysis showed ERO1α protein expression wasconsistent with its mRNA expression. Detect mRNA and protein expression ERO1α afterconstructing ERO1α interference fragment (siRNA1, siRNA2, siRNA3) and transfected livercells, the results showed that compared with control group, mRNA levels of ERO1α werereduced61.06%,38.70%and69.41%in each group, the difference was statistically significant(P<0.01), of which the siRNA2had the most significant difference, western blot analysisshowed ERO1α protein expression were consistent with its mRNA expression. Testingintracellular TC and TG, results showed that compared with the control group, TC and TG ofHcy+EGFP-N1-ERO1α group decreased to51.52%(P<0.05) and60.89%(P<0.01), while siRNA2group increased to1.39times (P<0.05) and1.88fold (P<0.01).Results of promoter activity analysis showed that the core promoter of human ERO1αexist activity, ntMS-PCR analyze DNA methylation of ERO1α gene promoter, results showedthat, DNA methylation in the promoter region of ERO1α gene of HHcy group and interventiongroup increased1.65-fold and1.42-fold compared with the control group, the difference wasstatistically significant (P<0.01), the intervention group compared with HHcy group, ERO1αgene promoter DNA methylation dropped to86.13%, the difference was statisticallysignificant (P<0.01).Detecting mRNA and protein expression of DNMT1and siRNA after constructingrecombinant plasmid and transfected with liver cells, the results showed that the mRNA levelsof EGFP-N1-DNMT1group increased to1.55times, the difference was statistically significant(P<0.01), while the mRNA levels of siRNA group decreased to26.70%, the difference wasstatistically significant (P<0.01), western blot analysis showed ERO1α protein expression wasconsistent with its mRNA expression. Detecting the levels of ERO1α DNA methylation ineach group after transfection, the results showed that compared with the control group, thelevel of ERO1α DNA methylation in EGFP-N1-DNMT1group increased to1.31times, downto72.34%in siRNA group, the difference was statistically significant (P<0.01).Conclusion:1. HHcy can induce liver lipid metabolism in ApoE-/-mouse.2. ERO1α may be the key target gene of lead to HHcy caused lipid metabolism.3.HHcy may cause ApoE-/-mouse ERO1α promoter region demethylation in liver, regulatemetabolism-related genes ERO1α low expression of mRNA and protein this may be one of themechanisms.4. DNMT1may be the important gene of HHcy-induced ERO1α low expression... |
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