The Regulation Mechanisms Of Insulin On Human Acyl Coenzyme A: Cholesterol Acyltransferase1 Gene Expression

PartⅠCloning and sequencing of P1 and P7 promoters of human acyl coenzyme A: cholesterol acyltransferase1 geneObjectiveTo clone P1 and P7 promoters of human acyl coenzyme A: cholesterol acyltransferase1 (ACAT1) gene.MethodsAccording to the complete DNA nucleotide sequences of P1 and P7 promoters from GenBank, the desired DNA segments of 668 bp and 762 bp were obtained respectively from human monocytic leukemia cell line (THP-1) by polymerase chain reaction (PCR) method. After the PCR products were inserted into pMD19-T simple vector, the positive clones containing P1 and P7 fragments were selected and confirmed by agarose gel electrophoresis and sequencing, then analyzed by methods of biological information.Results⑴The DNA sequences of cloned P1 and P7 promoters were accordant with GenBank data by agarose gel electrophoresis and sequencing, and no mutation.⑵The obtained P1 and P7 promoters contained some specific DNA sequences that were recognized by transcription factors.ConclusionsIn this study P1 and P7 promoters of human ACAT1 gene were cloned successfully. These results make an important basis for studying the transcriptional regulation mechanisms of ACAT1 gene during development of atherosclerosis.PartⅡConstruction of firefly luciferase report vectors containing human ACAT1 gene P1 and P7 promoters and identification the transcriptional activities in different cell linesObjectiveTo construct luciferase report vectors containing P1 and P7 promoters of human ACAT1 gene and investigate the transcriptional activities of P1 and P7 promoters in different cell lines.MethodsThe ACAT1 gene P1 and P7 promoters were cut from pMD19-T-P1 and pMD19-T-P7 by restriction enzyme digestion, then subcloned into Firefly luciferase report vector pGL3-Enhancer to produce the constructs named pGL3E-P1 and pGL3E-P7. pEGFP-N1 was transfected into THP-1 cells respectively with DEAE-dextran, Lipfectamine 2000, FuGENE 6 or Sofast reagent, then their transfection efficiency were compared by fluorescence microscope and FACS analysis. pGL3E-P1 and pGL3E-P7 were separately transfected into human monocytic leukemia cell line (THP-1) by DEAE-dextran transfection reagent, and separately transfected into human embryonic kidney cell line (HEK 293), human cervical adenocarcinoma cell line (HeLa) and human hepatocellular carcinoma cell line (HepG2) by Lipofectamine 2000 liposome. Cells were also transfected with pGL3-Enhancer and pGL3-Control which served as negative and positive controls respectively. The activities of luciferase, which reflect the transcriptional activities of the ACAT1 gene P1 and P7 promoters, were detected by dual-Luciferase reporter assay system after 48 hours of transfection.Results⑴The recombinant Firefly luciferase report vectors pGL3E-P1 and pGL3E-P7 were confirmed by PCR and restriction enzyme digestion.⑵The efficiency of gene transfection into THP-1 cells by DEAE-dextran was higher than by Lipfectamine 2000, FuGENE 6 or Sofast reagent.⑶The activities of pGL3E-P1 and pGL3E-P7 in THP-1 and HepG2 cells were higher than in HEK293 and HeLa cells. The activities of both promoters were the highest in THP-1 cells. There were low activities or no activity detected from HEK293 and HeLa cells transfected with pGL3E-P1 and pGL3E-P7.Conclusion⑴We successfully constructed luciferase reporter vectors containing human ACAT1 gene P1 and P7 promoters, and established a new means to study the transcriptional regulation mechanisms of ACAT1 during development of atherosclerosis.⑵DEAE-dextran used in transient transfection of THP-1 cells was a high efficiency reagent.⑶The transcriptional activities of human ACAT1 gene P1 and P7 promoters varied in different cell lines.PartⅢImpact of insulin on the transcriptional activities of human ACAT1 gene P1 and P7 promters in THP-1 cellsChapter I Impact of insulin on ACAT1 mRNA expression in THP-1 cells detected with real-time fluorescent quantitative reverse transcription-polymerase chain reactionObjectiveTo establish a SYBR GreenⅠreal-time reverse transcription-polymerase chain reaction (RT-PCR) for quantitating human ACAT1 mRNA and to study the impact of insulin on ACAT1 mRNA expression in human monocytic leukemia cell line (THP-1).MethodsThe THP-1 cells were incubated in vitro, exposed to different concentrations of insulin for 24h. Total RNA was extracted with TRIzol, and mRNA was transcribed reversely into cDNA. SYBR GreenⅠreal-time PCR was used to quantitate mRNA expression of ACAT1 and reference gene ABL in THP-1 cells. The specific amplicons were measured by gelelectrophoresis with ethidium bromide staining, and the characteristics of their melting temperatures(Tm) were analysed by melting curves.ResultsThe relative expression level of ACAT1 mRNA was significantly up-regulated in dose-dependent manner in insulin-treated cells in comparison with untreated THP-1 cells (P<0.05, n=3).Conclusions⑴SYBR GreenⅠreal-time quantitative RT-PCR is a rapid, sensitive method with high specificity to detect ACAT1 mRNA.⑵Insulin increased the ACAT1 mRNA expressin in THP-1 cells with dose-dependent manner.ChapterⅡImpact of insulin on the transcriptional activities of human ACAT1 gene P1 and P7 promters in THP-1 cellsObjectiveTo investigate the influence of insulin on the transcriptional activities of ACAT1 gene P1 and P7 promoters in THP-1 cells. To study the impact of insulin on the expression of ACAT1 gene P1 and P7 promoter transcripts in THP-1 cells.MethodsThe luciferase report vectors containing ACAT1 gene P1 and P7 promoters were respectively transfected into THP-1 cells with internal control plasmid pRL-TK by using the DEAE-dextran method. 7 h after incubation, cells were treated with or without insulin(10-7M). 40 h later, then the activities of the luciferase were determined by dual-Luciferase reporter assay system.The THP-1 cells were incubated in vitro, exposed to 10-7M insulin for 24h. Total RNA was extracted with TRIzol, then ACAT1 gene P1 transcript and P7 transcript levels were detected by Reverse transcription polymerase chain reaction (RT-PCR).Results⑴The transcriptinal activity of ACAT1 gene P1 promoter significantly inceased in insulin-treated THP-1 cells compared with untreated cells (P<0. 01, n=3).⑵The relative expression level of ACAT1 gene P1 transcript was significantly up-regulated in insulin-treated THP-1 cells in comparison with untreated cells (P<0. 01, n=3), no effect on P7 promoter.ConclusionsInsulin up-regulated ACAT1 gene expression by activating P1 promoter. Keywords Insulin; ACAT1; Monocyte; Promoter; Transcriptinal activity; TranscriptPartⅣFunctional analysis of human ACAT1 gene P1 promoter and effect of insulin on the transcripitional activities of P1 promoter and its deletions in THP-1 cellsObjectiveTo construct luciferase report vectors containing eight different fragments of human ACAT1 gene P1 promoter and analyze the transcriptional function of human ACAT1 gene P1 promoter; to investigate the effects of insulin on transcripitional activities of P1 promoter and its deletions in THP-1 cells.MethodsAccording to the analysis of biological information, eight different kinds of human ACAT1 gene P1 promoter deletions were separately cloned into Firefly luciferase report vector pGL3-Enhancer to produce the constructs named P1E-2, P1E-3, P1E-4, P1E-5, P1E-6, P1E-7, P1E-8, P1E-9, which included -547/+65, -498/+65, -428/+65, -363/+65, -324/+65, -256/+65, -188/+65, -125/+65bp respectively. The transcriptional activity of each construct was detected after transciently transfecting into THP-1 cells by DEAE-dextran transfection reagent. 7 h after incubation, cells were treated with or without insulin(10-7M). 40 h later, the luciferase activities in insulin-treated cells were compared with untreated cells.Results⑴The recombinant Firefly luciferase report vectors P1E-2, P1E-3, P1E-4, P1E-5, P1E-6, P1E-7, P1E-8, P1E-9 were confirmed by PCR, restriction enzyme digestion and sequencing, which included eight different kinds of human ACAT1 gene P1 promoter deletions respectively.⑵T he transcripitional activity of P1E-9 was higher in comparison with other deletion mutants in THP-1 cells.⑶The transcriptinal activity of ACAT1 gene P1 promoter (P1E-1) significantly inceased in insulin-treated cells compared with untreated cells (p<0.01, n=3), no effect observed on other eight P1 promoter deletions (P>0.05, n=3).Conclusion⑴We successfully constructed luciferase reporter vectors containing eight different fragments of human ACAT1 gene P1 promoter, made an important basis for studying the transcriptional regulation mechanisms of ACAT1 gene P1 promoter during development of atherosclerosis.⑵T he core sequence of human ACAT1 gene P1 promoter was implied between -125 and +65bp.⑶An insulin-responsive element maybe located in the -603 and -548bp sequence of human ACAT1 gene P1 promoter. PartⅤIdentification of an insulin-responsive element in human ACAT1 gene P1 promoterObjectiveTo construct luciferase report vectors containing two different fragments of human ACAT1 gene P1 promoter; to investigate the effects of insulin on the transcripitional activities of P1 promoter and its deletions in THP-1 cells, and to identify the insulin-responsive element in human ACAT1 gene P1 promoter.MethodsAccording to the analysis of biological information, aiming directly at c/EBPα, SNF, HNF-3 binding site, two different kinds of human ACAT1 gene P1 promoter deletions were separately cloned into Firefly luciferase report vector pGL3-Enhancer to produce the constructs named P1E-10, P1E-11, which included -579/+65, -566/+65bp respectively. The luciferase report vectors P1E-1, P1E-10 and P1E-11 were respectively transfected into THP-1 cells with internal control plasmid pRL-TK by using the DEAE-dextran method. 7 h after incubation, cells were treated with or without insulin(10-7M). 40 h later, the luciferase activities were determined by dual-Luciferase reporter assay system.Results⑴The recombinant Firefly luciferase report vectors P1E-10, P1E-11 were confirmed by PCR, restriction enzyme digestion and sequencing, which included two different kinds of human ACAT1 gene P1 promoter deletions respectively.⑵The transcriptinal activity of ACAT1 gene P1 promoter (P1E-1) significantly inceased in insulin-treated cells compared with untreated cells (p<0.05, n=3), no effect observed on other two P1 promoter deletions (P>0.1, n=3).Conclusion⑴We successfully constructed luciferase reporter vectors containing two different fragments of human ACAT1 gene P1 promoter, and made an important basis for studying the transcriptional regulation mechanisms of ACAT1 gene P1 promoter during development of atherosclerosis. ⑵We identified an insulin-responsive element located in the -603~-580bp sequence of human ACAT1 gene P1 promoter, which could be a c/EBPαbinding site.