Investigation Of Nur77in Regulating Cholesterol Metabolism In HepG2Cells

Background and Objection:Nowadays, more and more people around the world are experiencing cholesterol metabolism disorder due to unreasonable food construction and less of sports. As is known clearly, It is one of the key factors of atherosclerosis and is related to many serious diseases, such as acute myocardial infarction(AMI) and strock. Liver is one of the most important organs which take part in cholesterol metabolism disorder. Abnormal lipid profiles are often found in patients with liver diseases. Therefore, much interest has been attracted to the hepatic cell when investigating cholesterol metabolism disorder.Nur77is also called nuclear receptor subfamily4group A member1(NR4A1)and nerve growth factor I-B(NGFI-B), which is first discovered by Milbrandt etal. in the pheochromocytoma cell line PC12after induced by nerve growth factor in the year of1988. Nur77belongs to the NR4A family of the nuclear receptors which also includes Nurrl (NR4A2) and Nor1(NR4A3). Like other nuclear receptors, it consists of an N-terminal activating-function-1domain, a central DNA binding domain, and a C-terminal ligand-binding domain(LBD). However the LBD of Nur77is not typical and is lack of the classical ligand-binding pocket. Therefore it has been demonstrated that Nur77is regulated by its expression and post-translational modification rather than ligand-binding. It has been reported that Nur77takes part in the regulation of many biological process of which the regulation of apoptosis is the clearest. In response to apoptotic stimuli, Nur77is translocated from the nucleus to the cytoplasm, where it targets mitochondria to induce cytochrome C release and apoptosis. It is also reported that Nur77takes part in the regulation of inflammation, glucose metabolism and energy supply of the skeletal muscle. As for the regulation of lipid metabolism, there is evidence that Nur77participates in hepatic lipid metabolism of the mouse, and can lower the hepatic triglyceride level through suppression of SREBP1c activity. However, There is no significant evidence whether Nur77can regulate the hepatic cholesterol metabolism, especially in human race.Methods and materials1. Optimization of culture conditions and exploration of the Nur77RNA interference conditions of the HepG2cellsHepG2cells were maintained in the DMEM culture with the FBS concentration of10%,15%and20%respectively, and for12h,24h,48h and72h respectively. Thereafter, the morphological characteristics and quantities of the cells were compared. The best serum concentration was confirmed by the result. On the basis of the same primary cell density, the HepG2cells were cultured to the convergence degree of20%,50%,80%,90%,100%,12hrs after100%and24hrs after100%respectively. Then the best passage time point was confirmed by comparing the quantities of live cells, dead cells and the ratio of them. The HepG2cells were trypsinized after rinsing with PBS for0,1,2,3,4and5times respectively. The best passage condition was then confirmed by comparing the digestion period. As for the exploration of the Nur77RNA interference conditions of the HepG2cells, they were transfected with the three pairs of siRNA segments at a concentration of50nM for24hrs respectively. The best siRNA segment was chosen by comparing the depression effect of Nur77mRNA through RT-PCR. Thereafter, HepG2cells were transfected with the chosen siRNA segment at a concentration of50nM for24hrs,36hrs and48hrs respectively. The best transfection time was confirmed by comparing the depression effect of Nur77mRNA through RT-PCR. At last, HepG2cells were transfected with the chosen siRNA for the best time at a concentration of50nM and100nM respectively. Then the best siRNA dose was confirmed by comparing the depression effect of Nur77mRNA through RT-PCR.2. Construction, transfection and overexpression effect identification of the recombinant plasmid pcDNA3.1-Nur77Nur77was cloned from the PCR-XL-TOPO vector with enzyme digestion point EcoRI added to the upstream to form EcoRI-Nur77. EGFP was cloned from the PIRES2-EGFP vector with enzyme digestion point XhoI added to the downstream and joint gene IRES to the upstream to form IRES-EGFP-XhoI. Thereafter, the fragment of EcoRI-Nur77-IRES-EGFP-XhoI was achieved from the EcoRI-Nur77and IRES-EGFP-XhoI by overlap PCR and was linked into pcDNA3.1to achieve the recombinant plasmid. The inserted gene was identified by electrophoresis and sequencing. The recombinant plasmid was then transfected into the hepG2cells by Lipofectamine2000, the over expression effect of Nur77was confirmed by RT-PCR and western blotting.3. Influence of Nur77to the cholesterol metabolism gene expression in HepG2cellsFive groups were included in this part of the experiment:blank group(Blank), control group of RNA interference (siRNA-NC), RNA interference group(siRNA-Nur77), control group of overexpression(pcDNA3.1-monk) and overexpression group(pcDNA3.1-Nur77).6hrs after transfection of siRNA or recombinant plasmid, a mixture of oleate/palmitate(at a ratio of2:1) was added into the regular DMEM culture to achieve the lipid overloading model of the HepG2cell. The final concentration of the mixture was diluted to0.5nM.48hrs after lipid overloading, different groups of the cells were dissociated with RIPA. The total cholesterol levels(TCHO) were quantified and compared. And the Oil red O staining was carried out to the five groups after the same condition and the same lipid overloading hours. mRNA and protein expression levels of three genes among the five groups were compared48hrs after transfection of siRNA or recombinant plasmid. The three genes are critical in hepatic cholesterol metabolism including low density lipoprotein receptor(LDLR), HMG-CoA Reductase(HMGCR) and liver X receptor a(LxRa).4. Influence of Nur77agonist CsnB to the cholesterol metabolism gene expression in HepG2cells HepG2cells were cultured in the DMEM containing10μg/mLCsnB. Oh,0.75h,1.5hrs,3hrs,6hrs,12hrs and24hrs later the total RNA was extracted from the cells respectively. mRNA relative expression levels of Nur77, LDLR, HMGCR and LXRa were detected. DMSO(solvent of CsnB) was used to replace CsnB in the control group. Changes of the genes were observed in each group, and difference of the genes at the same time point between the two groups was analyzed.5. Statistical analysisAll data was achieved from three independent experiments, and was shown as means±tandard error(x±s). The Levene’s test was used to evaluate homogenity of variances. General linear Model-Univariate was used to compare the cell amounts of different culture time and different serum concentrations. LSD test was used to carry out the post hoc multiple comparisons. One-way ANOVA was used to compare the mRNA relative expression levels, relative intensities of the western blotting bands, and TCHO levels among three or more groups.LSD test was used to carry out the post hoc multiple comparisons. At the case of equal variances not assumed, Welch was used to compare the different groups and Dunnett’s T3test was used to carry out the post hoc multiple comparisons. t test was used to compare the mRNA relative expression levels and relative intensities of the western blotting bands between two groups. The above tests were all carried out by SPSS13.0software. P values less than0.05were considered statistically significant.Results:1. The difference of the cell densities among different serum concentrations was not statistically significant(F=0.008,P=0.992), while the difference of the cell densities among different time points was statistically significant(F=272.9, P<0.001). The differences of the amounts of live cells, dead cells and ratios of them among different convergence degrees were all statistically significant(P<0.001). The live cells increased along with the convergence degree and reached the top at the convergence degree of90%and100%, then decreased afterwards. The dead cells did not change a lot before the convergence degree reached90%, but increased rapidly after the convergence degree reached100%. The ratio of live cells and dead cells reached the top at the convergence degree of80%and90%, and decreased afterwards. It took about5minutes to digest the adherent HepG2cells without rinsing with PBS. However the digestion time decreased to2minutes after rinsing with PBS, and the time did not decrease any more after more rinsing times.As for the optimization of different siRNA segements, siRNA Segementl gained the best suppression effect comparing with the other two. Nur77mRNA relative expression level decreased to about79%after transfection with siRNA Segementl at a dose of50nM for24hrs. As for the optimization of different transfection time, Nur77mRNA relative expression level decreased first and then increased as the trasfection time went along. The suppression effect was best at the36h time point, at which Nur77mRNA relative expression level decreased to about25%. As for the optimization of different siRNA dose, Nur77mRNA relative expression level decreased along with the increase of siRNA dose and reached about10%when using the maximum amount of siRNA dose of100nM. Using the best conditions achieved from above experiments, the Nur77suppression effect of the siRNA-Nur77group arrived to the best(about10%)36hrs after the transfection using the siRNA Segementl with a dose of100nM(p﹤0.001vs. siRNA-NC). Furthermore, the suppression effect was confirmed by western blotting48hrs after transfection. The relative intensity of western blotting bands decreased to about54%.(P=0.004vs. siRNA-NC) The result agreed with that of the RT-PCR analysis.2. Electrophoresis and sequencing results indicated that the recombinant plasmid pcDNA3.1-Nur77was successfully constructed. What,s more, it can be successfully transfected into the HepG2cells. The transfection rate can reach80%48hrs after the transfection using a plasmid-Lipofectamine2000ratio of1:2.5(μg:μL). Comparing with the "pcDNA3.1-monk" group, the Nur77mRNA relative expression level raised to about500times(P<0.001), and the relative intensity of western blotting bands raised to about3.7times(P=0.001), which indicated that the overexpression effect of the recombinant plasmid was rather satisfying.3. The difference of hepatic cholesterol levels among the four groups was statistically significant after corrected with total protein’s level and the blank group(F=107.5, P﹤0.001). The TCHO level in hepG2cells rised to1.3times after suppression of Nur77(1.34±0.04vs.1.04±0.03, P<0.001). On the opposite side, it decreased to76%after over expression of Nur77(0.74±0.01vs.0.97±0.06, P﹤0.001). The changes were statistically different. The results of Oil red O staining agreed with the TCHO quantification results. As for LDLR, the mRNA relative expression level raised to1.7times(1.836±0.104vs.1.108±0.056) and the relative intensity of western blotting bands raised to1.5times(1.167±0.105vs.0.797±0.075) comparing with the "siRNA-NC" group. While the mRNA relative expression level decreased to45%(0.412±0.067vs.0.925±0.068) and the relative intensity of western blotting bands decreased to46%(0.300±0.046vs.0.653±0.045) comparing with the "pcDNA3.1-monk" group. The differences were all statistically different. As for HMGCR, the mRNA relative expression level raised to1.8times(1.608±0.105vs.0.914±0.041) and the relative intensity of western blotting bands raised to1.4times (1.593±0.125vs.1.103±0.081) comparing with the "siRNA-NC" group. While the mRNA relative expression level decreased to51%(0.570±0.081vs.1.116±0.054) and the relative intensity of western blotting bands decreased to54%(0.510±0.046vs.0.943±0.075) comparing with the "pcDNA3.1-monk" group. The differences were all statistically different. However, as for LxRa, the mRNA relative expression level and the relative intensity of western blotting bands did not change obviously with that of Nur77.4. Nur77mRNA relative expression level reached to the top after1.5hrs’ stimulation with CsnB at a final dose of10μg/mL, which was about2.65times of that of Oh. Then it begun to go down and decreased to the basic line at12hrs. Expression levels of LDLR decreased along with the stimulation time of CsnB, which was particularly obvious at the12hrs and24hrs point. The difference among different time points was statistically different(F=60.7, P<0.001). LDLR also decreased along with the stimulation time of DMSO at the same dose, but the decreasing trend was much smoother. The difference of the LDLR mRNA relative expression level between the CsnB group and DMSO group was statistically different(t=-6.6, P=0.003) at the time point of24h. As for HMGCR, relative expression levels of HMGCR mRNA also decreased along with the stimulation time of CsnB, and the difference among different time points was also statistically different(F=5.0, P=0.003). HMGCR mRNA of the CsnB group also decreased greatly comparing with that of DMSO group(F=4.6, P=0.042). However, the expression level of LXRa did not change greatly after stimulation with CsnB(F=0.7, P=0.612). What’s more, the difference of the LXRa mRNA relative expression level between the CsnB group and DMSO group was neither statistically different(F=1.3, P=0.266).Conclusion:1. As for the HepG2cells we achieved, they should be cultured in DMEM with10%FBS concentration, and should be subcultured when the convergence degree reaches90%. The best effect can be achieved when using PBS to rinse the cells once before digestion and subculture. siRNA Segementl was the most effective segement among the three siRNA segements. The best suppression effect of Nur77was achieved after transfection at a final siRNA concentration of100nM for36hrs in the HepG2cells.2. The recombinant plasmid pcDNA3.1-Nur77was successfully constructed by subcloning. Good overexpression effect can be achieved using the recombinant plasmid to transfect the HepG2cells with a plasmid-Lipofectamine2000ratio of1:2.5(μg:μL) for48hrs.3. Nur77takes part in the regulation of hepatic cholesterol metabolism and reduces cholesterol level in hepatocyte. This regulation may lie in that Nur77can reduce hepatic expression of LDLR and HMGCR. 4. CsnB can induce the increase of Nur77expression in HepG2cells effectively. LDLR and HMGCR decrease with the CsnB stimulation time, but LxRa dosen’t change a lot. This agree with the effect of Nur77overexpression recombinant plasmid.