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Objective@#To examine the correlation between the promoter methylation of Sterol regulatory-element binding protein-2 (SREBP-2) and miR-33a expression as well as serum markers in patients with coronary artery disease (CAD).@*Methods@#The case-control study. 100 participants who underwent coronary angiography from August 2017 to April 2018 in TaiheHospital, Hubei University of Medicine, were recruited in this study.The methylation level of two fragments, including 12 CpG sites in the promoter region of SREBP-2, have been detected by pyrosequencing in 50 patients with coronary artery disease (CAD) and 50 non-CAD controls. Serum miR-33a level and a panel of 15 CAD related biomarkers were examined by qPCR and routine biochemistry methods.@*Results@#Methylation level of one CpG site (F1-4 loci) in SREBP-2 promoter region were significant higher in CAD patients than in controls(4.56%±0.70% vs 3.54%±0.72%, t=-3.864, P<0.001); methylation level of F1-4 site was negatively correlates with the serum miR-33a levels and high-density lipoprotein cholesterol (HDL-C) levels(r=-0.318, P=0.001; r=-0.225, P=0.024, respectively). Furthermore, F1-4 hypermethylation was an independent risk factor of CAD, independent of age, gender, histories of hypertension, hyperlipidemia, and diabetes(OR=2.452, 95%CI=1.398-4.299, P=0.002).@*Conclusion@#These results suggest that DNA methylation and miRNA might cooperate to regulate the lipid metabolism in CAD.
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Objective To investigate the level of mammalian target of rapamycin (mTOR) in serum and the expression of mTOR,nuclear factor-κ B (NF-κ B) and sterol regulatory element binding protein 2 (SREBP2) in placenta among gravidas with preeclampsia.Methods From August 2015 to August 2017,60 gravidas including 40 with severe preeclampsia (SPE) and 20 with mild preeclampsia (MPE) who underwent regular prenatal care and delivered by caesarean section were selected from the Second Xiangya Hospital of Central South University.According to the ratio of 2:1,30 gravidas who delivered through caesarean section due to cephalopelvic disproportion,abnormal fetal position or social factors during the same period were enrolled as the control group.Peripheral blood samples were obtained to determine the concentrations of serum mTOR,high density lipoprotein-cholesterol (HDL-C),low density lipoprotein-cholesterol (LDL-C),triglyceride (TG) and total cholesterol (TC) by enzyme linked immunosorbent assay (ELISA).The expression of mTOR,phospho-mTOR (p-mTOR),NF-κ B and SREBP2 in placenta were measured by Western blot.Clinical datas were statistically analyzed using one-way ANOVA,Bonferroni or Dunnett's T3 test,and Pearson's correlation analysis.Results (1) The serum levels of mTOR and LDL-C in the SPE and MPE group were both higher than that in the control group [mTOR:(11 765.56± 1 698.95) and (8 278.56±1 106.59) vs (4 366.19±716.43) pg/ml;LDL-C:(7.81 ±1.90) and (4.11 ±0.75) vs (2.42±0.45) mmol/L,all P＜0.05].Furthermore the serum levels of mTOR and LDL-C in the SPE group were both higher than those in the MPE group (both P＜0.05).The serum level of HDL-C in the SPE and MPE group were lower than that in the control group [(0.36±0.12) and (0.85±0.11) vs (1.33± 0.16) mmol/L,both P＜0.05],and that in the SPE group was lower than that in the MPE group (P＜0.05).Women in the SPE group showed higher TG level when comparing with the MPE and control group [(46.19± 18.92)vs (35.55±6.54) and (33.24±9.78) nmol/L,both P＜0.05],while the TC levels in the SPE and MPE group were higher than that in the control group[(24.72±7.17) and (21.83±4.19) vs (16.32±3.88) nmol/L,both P＜0.05].(2) The placental expressions of mTOR,p-mTOR,NF-κ B and SREBP2 protein in the SPE and MPE group were higher compared with that in the control group [mTOR:(0.52±0.09) and (0.38±0.08) vs (0.24±0.05);p-mTOR:(0.42±0.08) and (0.26±0.05) vs (0.14±0.03);NF-κ B:(0.58±0.10) and (0.36±0.05) vs (0.21 ± 0.03);SREBP2:(0.52 ± 0.08) and (0.33 ± 0.05) vs (0.20 ± 0.05);all P＜0.05],and those expressions of the SPE group also higher comparing with the MPE group.Otherwise the p-mTOR/mTOR ratios in the SPE group and MPE group were higher than that in the control group [(0.75±0.10) and (0.69±0.14) vs (0.59 ±0.13),both P＜0.05].(3) Pearson's correlation analysis showed that serum level of mTOR and placental expressions of mTOR and p-mTOR in the SPE group were positively correlated with serum LDL-C (r=0.682,0.584 and 0.504,all P＜0.05),TG (r=0.612,0.658 and 0.422,all P＜0.05),while serum level of mTOR and placental expressions of mTOR in the SPE group were positively correlated with TC (r=0.598 and 0.452,all P＜0.05),but were negatively correlated with serum HDL-C (r=-0.375,-0.442 and-0.390,all P＜0.05).The NF-κ B expression in placenta of the SPE group was significantly positively correlated with the mTOR expression in placenta and serum LDL-C (r=0.375 and 0.391,both P＜0.05).Moreover,in the SPE group,the SREBP2 level in placenta was significantly positively correlated with placental expression of mTOR and serum TC level (r=0.364 and 0.392,both P＜0.05).(4) In the MPE group,mTOR level in serum and levels of mTOR and p-mTOR in placenta were significantly positively correlated with serum LDL-C (r=0.813,0.641 and 0.465,all P＜0.05),TG (r=0.646,0.529 and 0.502,all P＜0.05) and TC (r=0.558,0.482 and 0.483,all P＜0.05),while the level of serum mTOR was negatively correlated with the level of serum HDL-C (r=-0.606,P＜0.05).The NF-κ B level in placenta in MPE group was positively correlated with the mTOR in placenta and the serum LDL-C (r=0.458 and 0.595,both P＜0.05),while the SREBP2 level in placenta was significantly positively correlated with mTOR in placenta and serum TC (r=0.580,0.560,respectively;both P＜0.05) in the MPE group.Conclusions mTOR,NF-κ B and SREBP2 may play important roles in the onset and development of preeclampsia by interfering lipid metabolism.
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Objective: To investigate the effects of sterol regulatory element binding protein 2 (SREBP2) on the metabolism of cholesterol as well as the proliferation, apoptosis and migration of normal liver LO2 cells and hepatocellular carcinoma HepG2 cells. Methods: By using pAd-Easy-1 adenovirus vector system, the recombinant adenovirus Ad-SREBP2m (carrying the splicing form of SREBP2) and Ad-GFP (as the control) were constructed, and then infected into LO2 and HepG2 cells, respectively. The total cholesterol level in LO2 and HepG2 cells after infection was detected by a cholesterol quantification kit. The expression levels of SREBP2m, cholesterol synthesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and apoptosis-related proteins (including caspase 3, cleaved-caspase 3 and caspase 12) were detected by Western blotting. The effects of Ad-SREBP2m overexpression on the proliferation, cell cycle, apoptosis and migration of LO2 or HepG2 cells were detected by EdU staining method, FCM and scratch wound healing test, respectively. Results: The recombinant adenovirus Ad-SREBP2m was successfully constructed. Compared with the Ad-GFP group, the expression levels of SREBP2m and its target protein HMGCR were up-regulated (both P 0.05); while in HepG2 cells infected with Ad-SREBP2m, the proportion of G1-phase cells decreased significantly (P < 0.001), but the proportion of S-phase cells increased significantly (P < 0.001). SREBP2m overexpression promoted the proliferation of HepG2 cells (P < 0.001), but had no effect on the proliferation of LO2 cells. The expression levels of total caspase 3, cleaved-caspase 3 and caspase 12 were significantly higher in LO2 cells infected with Ad-SREBP2m than those in Ad-GFP group (all P < 0.001), while the expression levels of total caspase 3, cleaved-caspase 3 and caspase 12 were decreased in HepG2 cells infected with Ad-SREBP2m (all P < 0.05). The apoptosis rate of LO2 cells after Ad-SREBP2m infection was increased by (11.40±0.52)% (P < 0.001), while the apoptosis rate of HepG2 cells in Ad-SREBP2m group was decreased by (4.17±0.47)% as compared with Ad-GFP group (P < 0.05). The migration distance of HepG2 cells in Ad-SREBP2m group was (1.17±0.12) mm more than that in Ad-GFP group (P < 0.05). Conclusion: Overexpression of SREBP2m can promote the proliferation and migration and inhibit the apoptosis of hepatocellular carcinoma HepG2 cells, but it can promote apoptosis of normal liver LO2 cells.
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[ ABSTRACT] AIM:To explore the effect of sterol regulatory element-binding protein 2 ( SREBP-2) on tunicamy-cin-induced endoplasmic reticulum stress ( ERS) in chondrocytes.METHODS:After isolation of human normal chondro-cytes and osteoarthritis ( OA) chondrocytes, the normal cells were cultured and treated with tunicamycin and SREBP-2 siR-NA.After 24 h treatment, fluorescent quantitative RT-PCR ( RT-qPCR) was applied to quantify microRNA-185 ( miR-185) levels.The cell apoptotic rate was determined by flow cytometry.The expression of SREBP-2 and ERS-related pro-teins, C/EBP homologous protein (CHOP), phosphorylated eukaryotic initiation factor-2α(p-eIF2α) and activating tran-scription factor 4 (ATF4), and the expression of apoptosis-related proteins, Bcl-2, Bax and caspase-3, were determined by Western blot.The caspase-3 activity kit was used to determine the caspase-3 activity.RESULTS: Compared with hu-man normal chondrocytes, both SREBP-2 up-regulation and miR-185 down-regulation were observed in OA chondrocytes (P<0.05).SREBP-2 siRNA transfection enhanced tunicamycin-inhibited miR-185 level (P<0.05).miR-185 overex-pression reduced tunicamycin-induced SREBP-2 expression ( P <0.05 ) .OA control group and tunicamycin treatment group consistently resulted in ERS and cell apoptosis with concomitant enhancement of CHOP, p-eIF2αand ATF4 proteins, increases in Bax and caspase-3 proteins, and reduction of Bcl-2 (P<0.05).However, SREBP-2 silencing significantly re-versed these effects ( P<0.05) .The apoptotic rates were consistent with the expression tendency of apoptosis-related pro-teins (P<0.05).SREBP-2 siRNA transfection markedly down-regulated tunicamycin-induced caspase-3 activity, which was notably blocked by miR-185 inhibition (P<0.05).CONCLUSION:SREBP-2 silencing may inhibit tunicamycin-in-duced ERS and cell apoptosis via up-regulating miR-185 expression.
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Objective:To study the relationship between rs 2228314 polymorphism in sterol regulatory element binding protein 2 gene (SREBP2) and obesity, serum lipid levels in children and adolescents . Methods:In our study , 2 030 children and adolescents aged from 7 to 18 years participated .Anthropo-metric measurements, including height and weight, were performed.Their serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol ( HDL-C) were detected .The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS ) was used to detect rs2228314 genotypes.Results: The GC/CC genotypes of rs2228314 polymorphisms had lower HDL-C levels than GG genotype [(0.10 ±0.35) mmol/L vs. (0.14 ±0.36) mmol/L, P=0.020].The rs2228314 polymorphism was associated with the abnormal HDL-C level under the dominant model after adjustment for study samples , sex and age ( OR=1.400, 95%CI:1.027-1.907, P=0.033).The rs2228314 polymorphism was not associated with obesity un-der the dominant model after adjustment for study samples , sex, age and HDL-C level ( OR=1.178, 95%CI: 0 .971 -1 .430 , P =0 .096 ) . Conclusion: The GC/CC genotype carriers of SREBP2 rs2228314 polymorphism have higher risk of abnormal HDL-C level than the individuals with GG geno-type among children and adolescents .