胰岛素对人酰基辅酶A：胆固醇酰基转移酶1基因转录调控的机制研究
摘要
第一部分人酰基辅酶A:胆固醇酰基转移酶1基因P1和P7启动子的克隆及序列分析
目的
克隆并测定人酰基辅酶A:胆固醇酰基转移酶1(acyl coenzyme A: cholesterol acyltransferase1, ACAT1)基因P1和P7启动子的序列。
方法
根据GenBank数据库提供的人ACAT1基因P1和P7启动子核苷酸序列,应用PCR方法从人单核细胞系THP-1细胞扩增分离出ACAT1基因P1和P7启动子全长片段,将PCR产物克隆入T载体,并对所获得的序列进行生物信息学分析。
结果
⑴经琼脂糖凝胶电泳及直接测序鉴定,克隆的人ACAT1基因P1和P7启动子片段碱基序列与GenBank数据库一致,未发现突变。
⑵获得的P1和P7启动子序列中,集中了一些转录因子结合元件的特征序列。结论
成功克隆了人ACAT1基因P1和P7启动子,为研究在动脉粥样硬化过程中ACAT1基因的转录调控机制奠定基础。
第二部分人ACAT1基因P1和P7启动子调控的报告基因载体的构建及在不同细胞系中转录活性的检测
目的
构建人ACAT1基因P1和P7启动子调控的萤光素酶报告基因表达载体,检测其在不同细胞系中的转录活性的差别。
方法
从已构建的克隆载体pMD19-T-P1、pMD19-T-P7中分别双酶切出人ACAT1基因P1和P7启动子全长片段,将该基因亚克隆至表达载体pGL3-Enhancer。分别采用DEAE-dextran、Lipfectamine 2000、FuGENE 6、梭华-Sofast方法将pEGFP-N1转染入人单核细胞系THP-1细胞,用荧光显微镜和流式细胞仪比较它们的转染效率。用DEAE-dextran法将重组质粒pGL3E-P1和pGL3E-P7分别转染入THP-1细胞,用脂质体Lipfectamine 2000将重组质粒pGL3E-P1和pGL3E-P7分别转染入人胚胎肾细胞HEK 293、人子宫颈癌细胞HeLa、人肝母细胞瘤细胞系HepG2, pGL3-Enhancer和pGL3-Control分别作为阴性对照组和阳性对照组,应用双荧光素酶报告基因检测系统检测荧光素酶的表达活性。
结果
⑴PCR及酶切鉴定表明,获得的人ACAT1基因P1和P7启动子的DNA序列已成功载入重组表达载体pGL3E-P1和pGL3E-P7。
⑵与Lipfectamine 2000、FuGENE 6、梭华-Sofast比较,DEAE-dextran介导的THP-1细胞基因瞬时转染效率更高。
⑶pGL3E-P1和pGL3E-P7在THP-1和HepG2细胞中的转录活性高于HEK293和HeLa细胞,在THP-1细胞中转录活性最强,在HepG2细胞中的活性次之,在HEK293、HeLa细胞中活性低或不具有转录活性。
结论
⑴成功构建了含人ACAT1基因P1和P7启动子序列的荧光素酶报告基因载体,并可作为体外研究ACAT1基因转录调控的新手段。
⑵获得效率较高的THP-1细胞基因瞬时转染的方法。
⑶不同类型细胞中ACAT1基因P1和P7启动子的表达活性不同。
第三部分胰岛素对人单核细胞ACAT1基因启动子活性的影响
一、实时荧光定量RT-PCR检测胰岛素对人单核细胞ACAT1 mRNA表达的影响目的
建立SYBR GreenⅠ实时定量RT-PCR方法检测人单核细胞系THP-1细胞中ACAT1 mRNA,探讨胰岛素对THP-1细胞ACAT1 mRNA表达的影响。
方法
体外培养THP-1细胞,用不同剂量的胰岛素与THP-1细胞持续孵育24h,用TRIzol提取总RNA后,将mRNA逆转录成cDNA,再用SYBR GreenⅠ实时定量PCR方法检测ACAT1 mRNA的表达,扩增产物的特异性通过熔解曲线和凝胶电泳双重确认,以ABL为内参照,采用2-△△ct法计算相对表达量。
结果
与未处理组相比,不同剂量胰岛素处理的THP-1细胞ACAT1 mRNA表达水平均显著升高(P<0.05,n=3),并随胰岛素剂量的增加而升高。
结论
⑴SYBR GreenⅠ实时定量RT-PCR是一种快速有效、灵敏度高、特异性良好的定量检测ACAT1 mRNA的方法。
⑵胰岛素可明显促进THP-1细胞的ACAT1 mRNA的表达,其作用呈剂量依赖性。
二、胰岛素对人单核细胞ACAT1基因启动子活性的影响
目的
研究胰岛素对THP-1细胞ACAT1基因P1、P7启动子的活性和P1、P7启动子转录产物表达的影响。
方法
将人ACAT1基因P1和P7启动子调控的报告基因载体pGL3E-P1和pGL3E-P7,通过DEAE-dextran法分别与内参照质粒pRL-TK共同转染入THP-1细胞,恢复生长7小时后给予胰岛素(10-7M)处理,40小时后通过双荧光素酶报告系统观察胰岛素对ACAT1基因P1和P7启动子的调控作用。体外培养THP-1细胞,用10-7M胰岛素与THP-1细胞持续孵育24h,用TRIzol提取总RNA后,应用逆转录聚合酶链反应(RT-PCR)分别检测ACAT1基因P1和P7启动子转录产物表达。
结果
⑴胰岛素处理的THP-1细胞,与对照组相比,ACAT1基因P1启动子的荧光素酶表达活性显著增强,增强到2.6倍,具有显著差异(P<0. 01,n=3)。
⑵与非胰岛素处理的对照组相比,胰岛素增强ACAT1基因P1启动子的转录产物量,升高到1.65倍,具有显著差异(P<0. 01,n=3),而P7启动子的转录产物量不发生显著变化。与上述结果相一致。
结论
胰岛素在人单核细胞中通过激活ACAT1基因P1启动子来上调其表达。
第四部分人ACAT1基因P1启动子的功能分析及其不同长度的缺失体在胰岛素促进THP-1细胞ACAT1基因转录中的作用
目的
构建包含5'-端缺失不同长度人ACAT1基因P1启动子片段的荧光素酶报告基因载体,分析P1启动子的转录激活功能;探讨胰岛素对这一系列ACAT1基因P1启动子缺失体转录活性的影响。
方法
在对人ACAT1基因P1启动子进行生物信息学分析的基础上,设计8种ACAT1基因P1启动子缺失突变体,分别包括ACAT1基因P1启动子-547/+65、-498/+65、-428/+65、-363/+65、-324/+65、-256/+65、-188/+65、-125/+65bp的片段,针对每一种缺失突变体构建相应的萤火虫荧光素酶报告基因载体,分别命名为P1E-2、P1E-3、P1E-4、P1E-5、P1E-6、P1E-7、P1E-8、P1E-9,用DEAE-dextran法瞬时转染THP-1细胞,检测荧光素酶活性。给予胰岛素(10-7M)处理,检测这一系列ACAT1基因P1启动子缺失体转录活性的变化。
结果
⑴PCR、酶切及测序鉴定表明,成功获得8种人ACAT1基因P1启动子缺失突变体的荧光素酶报告基因载体P1E-2、P1E-3、P1E-4、P1E-5、P1E-6、P1E-7、P1E-8、P1E-9。
⑵P1E-9在THP-1细胞中的转录活性均高于其他报告载体。
⑶与非胰岛素处理的对照组相比,胰岛素增强人ACAT1基因P1启动子(P1E-1)的转录活性,升高到2.6倍,具有显著差异(P<0. 01,n=3),而其他8种P1启动子缺失突变体的报告载体转录活性不发生显著变化(P>0.05,n=3)。
结论
⑴成功构建了包含5'-端缺失不同长度人ACAT1基因P1启动子片段的荧光素酶报告基因载体,并为体外研究ACAT1基因P1启动子的转录调控奠定了基础。
⑵人ACAT1基因P1启动子的-125～+65bp区域是它的核心序列。
⑶人ACAT1基因P1启动子5'-端上游-603～-548bp可能存在胰岛素反应元件。
第五部分人ACAT1基因P1启动子的胰岛素反应元件的分析
目的
构建两种包含5'-端缺失不同长度人ACAT1基因P1启动子片段的荧光素酶报告基因载体;探讨胰岛素对这两个ACAT1基因P1启动子缺失体转录活性的影响,确定胰岛素反应元件。
方法
在对人ACAT1基因P1启动子进行生物信息学分析的基础上,针对转录因子c/EBPα、SNF、HNF-3结合元件,设计两种ACAT1基因P1启动子缺失突变体,分别包括ACAT1基因P1启动子-579/+65、-566/+65bp的片段,并构建相应的萤火虫荧光素酶报告基因载体,分别命名为P1E-10、P1E-11,用DEAE-dextran法分别与内参照质粒pRL-TK共同转染THP-1细胞。恢复生长7小时后给予胰岛素(10-7M)处理,40小时后通过双荧光素酶报告系统检测荧光素酶活性。
结果
⑴PCR、酶切及测序鉴定表明,成功获得两种人ACAT1基因P1启动子缺失突变体的荧光素酶报告基因载体P1E-10、P1E-11。
⑵与非胰岛素处理的对照组相比,胰岛素增强人ACAT1基因P1启动子(P1E-1)的转录活性,升高到2.6倍,具有显著差异(P<0. 01,n=3),而另外两种P1启动子缺失突变体的报告载体转录活性不发生显著变化(P>0.05,n=3)。
结论
⑴成功构建了包含5'-端缺失不同长度人ACAT1基因P1启动子片段的荧光素酶报告基因载体,并可作为体外研究ACAT1基因转录调控的新手段。
⑵人ACAT1基因P1启动子5'-端上游-603～-580bp存在胰岛素反应元件,可能为c/EBPα结合位点。
PartⅠCloning and sequencing of P1 and P7 promoters of human acyl coenzyme A: cholesterol acyltransferase1 gene
Objective
To clone P1 and P7 promoters of human acyl coenzyme A: cholesterol acyltransferase1 (ACAT1) gene.
Methods
According 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.
Results
⑴The 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.
Conclusions
In 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 lines
Objective
To 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.
Methods
The 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.
Results
⑴The 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.
Conclusion
⑴We 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 cells
Chapter I Impact of insulin on ACAT1 mRNA expression in THP-1 cells detected with real-time fluorescent quantitative reverse transcription-polymerase chain reaction
Objective
To 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).
Methods
The 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.
Results
The 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).
Conclusions
⑴SYBR 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 cells
Objective
To 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.
Methods
The 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).
Results
⑴The 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.
Conclusions
Insulin up-regulated ACAT1 gene expression by activating P1 promoter. Keywords Insulin; ACAT1; Monocyte; Promoter; Transcriptinal activity; Transcript
PartⅣFunctional analysis of human ACAT1 gene P1 promoter and effect of insulin on the transcripitional activities of P1 promoter and its deletions in THP-1 cells
Objective
To 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.
Methods
According 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.
Results
⑴The 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).
Conclusion
⑴We 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 promoter
Objective
To 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.
Methods
According 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.
Results
⑴The 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).
Conclusion
⑴We 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.
目的
克隆并测定人酰基辅酶A:胆固醇酰基转移酶1(acyl coenzyme A: cholesterol acyltransferase1, ACAT1)基因P1和P7启动子的序列。
方法
根据GenBank数据库提供的人ACAT1基因P1和P7启动子核苷酸序列,应用PCR方法从人单核细胞系THP-1细胞扩增分离出ACAT1基因P1和P7启动子全长片段,将PCR产物克隆入T载体,并对所获得的序列进行生物信息学分析。
结果
⑴经琼脂糖凝胶电泳及直接测序鉴定,克隆的人ACAT1基因P1和P7启动子片段碱基序列与GenBank数据库一致,未发现突变。
⑵获得的P1和P7启动子序列中,集中了一些转录因子结合元件的特征序列。结论
成功克隆了人ACAT1基因P1和P7启动子,为研究在动脉粥样硬化过程中ACAT1基因的转录调控机制奠定基础。
第二部分人ACAT1基因P1和P7启动子调控的报告基因载体的构建及在不同细胞系中转录活性的检测
目的
构建人ACAT1基因P1和P7启动子调控的萤光素酶报告基因表达载体,检测其在不同细胞系中的转录活性的差别。
方法
从已构建的克隆载体pMD19-T-P1、pMD19-T-P7中分别双酶切出人ACAT1基因P1和P7启动子全长片段,将该基因亚克隆至表达载体pGL3-Enhancer。分别采用DEAE-dextran、Lipfectamine 2000、FuGENE 6、梭华-Sofast方法将pEGFP-N1转染入人单核细胞系THP-1细胞,用荧光显微镜和流式细胞仪比较它们的转染效率。用DEAE-dextran法将重组质粒pGL3E-P1和pGL3E-P7分别转染入THP-1细胞,用脂质体Lipfectamine 2000将重组质粒pGL3E-P1和pGL3E-P7分别转染入人胚胎肾细胞HEK 293、人子宫颈癌细胞HeLa、人肝母细胞瘤细胞系HepG2, pGL3-Enhancer和pGL3-Control分别作为阴性对照组和阳性对照组,应用双荧光素酶报告基因检测系统检测荧光素酶的表达活性。
结果
⑴PCR及酶切鉴定表明,获得的人ACAT1基因P1和P7启动子的DNA序列已成功载入重组表达载体pGL3E-P1和pGL3E-P7。
⑵与Lipfectamine 2000、FuGENE 6、梭华-Sofast比较,DEAE-dextran介导的THP-1细胞基因瞬时转染效率更高。
⑶pGL3E-P1和pGL3E-P7在THP-1和HepG2细胞中的转录活性高于HEK293和HeLa细胞,在THP-1细胞中转录活性最强,在HepG2细胞中的活性次之,在HEK293、HeLa细胞中活性低或不具有转录活性。
结论
⑴成功构建了含人ACAT1基因P1和P7启动子序列的荧光素酶报告基因载体,并可作为体外研究ACAT1基因转录调控的新手段。
⑵获得效率较高的THP-1细胞基因瞬时转染的方法。
⑶不同类型细胞中ACAT1基因P1和P7启动子的表达活性不同。
第三部分胰岛素对人单核细胞ACAT1基因启动子活性的影响
一、实时荧光定量RT-PCR检测胰岛素对人单核细胞ACAT1 mRNA表达的影响目的
建立SYBR GreenⅠ实时定量RT-PCR方法检测人单核细胞系THP-1细胞中ACAT1 mRNA,探讨胰岛素对THP-1细胞ACAT1 mRNA表达的影响。
方法
体外培养THP-1细胞,用不同剂量的胰岛素与THP-1细胞持续孵育24h,用TRIzol提取总RNA后,将mRNA逆转录成cDNA,再用SYBR GreenⅠ实时定量PCR方法检测ACAT1 mRNA的表达,扩增产物的特异性通过熔解曲线和凝胶电泳双重确认,以ABL为内参照,采用2-△△ct法计算相对表达量。
结果
与未处理组相比,不同剂量胰岛素处理的THP-1细胞ACAT1 mRNA表达水平均显著升高(P<0.05,n=3),并随胰岛素剂量的增加而升高。
结论
⑴SYBR GreenⅠ实时定量RT-PCR是一种快速有效、灵敏度高、特异性良好的定量检测ACAT1 mRNA的方法。
⑵胰岛素可明显促进THP-1细胞的ACAT1 mRNA的表达,其作用呈剂量依赖性。
二、胰岛素对人单核细胞ACAT1基因启动子活性的影响
目的
研究胰岛素对THP-1细胞ACAT1基因P1、P7启动子的活性和P1、P7启动子转录产物表达的影响。
方法
将人ACAT1基因P1和P7启动子调控的报告基因载体pGL3E-P1和pGL3E-P7,通过DEAE-dextran法分别与内参照质粒pRL-TK共同转染入THP-1细胞,恢复生长7小时后给予胰岛素(10-7M)处理,40小时后通过双荧光素酶报告系统观察胰岛素对ACAT1基因P1和P7启动子的调控作用。体外培养THP-1细胞,用10-7M胰岛素与THP-1细胞持续孵育24h,用TRIzol提取总RNA后,应用逆转录聚合酶链反应(RT-PCR)分别检测ACAT1基因P1和P7启动子转录产物表达。
结果
⑴胰岛素处理的THP-1细胞,与对照组相比,ACAT1基因P1启动子的荧光素酶表达活性显著增强,增强到2.6倍,具有显著差异(P<0. 01,n=3)。
⑵与非胰岛素处理的对照组相比,胰岛素增强ACAT1基因P1启动子的转录产物量,升高到1.65倍,具有显著差异(P<0. 01,n=3),而P7启动子的转录产物量不发生显著变化。与上述结果相一致。
结论
胰岛素在人单核细胞中通过激活ACAT1基因P1启动子来上调其表达。
第四部分人ACAT1基因P1启动子的功能分析及其不同长度的缺失体在胰岛素促进THP-1细胞ACAT1基因转录中的作用
目的
构建包含5'-端缺失不同长度人ACAT1基因P1启动子片段的荧光素酶报告基因载体,分析P1启动子的转录激活功能;探讨胰岛素对这一系列ACAT1基因P1启动子缺失体转录活性的影响。
方法
在对人ACAT1基因P1启动子进行生物信息学分析的基础上,设计8种ACAT1基因P1启动子缺失突变体,分别包括ACAT1基因P1启动子-547/+65、-498/+65、-428/+65、-363/+65、-324/+65、-256/+65、-188/+65、-125/+65bp的片段,针对每一种缺失突变体构建相应的萤火虫荧光素酶报告基因载体,分别命名为P1E-2、P1E-3、P1E-4、P1E-5、P1E-6、P1E-7、P1E-8、P1E-9,用DEAE-dextran法瞬时转染THP-1细胞,检测荧光素酶活性。给予胰岛素(10-7M)处理,检测这一系列ACAT1基因P1启动子缺失体转录活性的变化。
结果
⑴PCR、酶切及测序鉴定表明,成功获得8种人ACAT1基因P1启动子缺失突变体的荧光素酶报告基因载体P1E-2、P1E-3、P1E-4、P1E-5、P1E-6、P1E-7、P1E-8、P1E-9。
⑵P1E-9在THP-1细胞中的转录活性均高于其他报告载体。
⑶与非胰岛素处理的对照组相比,胰岛素增强人ACAT1基因P1启动子(P1E-1)的转录活性,升高到2.6倍,具有显著差异(P<0. 01,n=3),而其他8种P1启动子缺失突变体的报告载体转录活性不发生显著变化(P>0.05,n=3)。
结论
⑴成功构建了包含5'-端缺失不同长度人ACAT1基因P1启动子片段的荧光素酶报告基因载体,并为体外研究ACAT1基因P1启动子的转录调控奠定了基础。
⑵人ACAT1基因P1启动子的-125～+65bp区域是它的核心序列。
⑶人ACAT1基因P1启动子5'-端上游-603～-548bp可能存在胰岛素反应元件。
第五部分人ACAT1基因P1启动子的胰岛素反应元件的分析
目的
构建两种包含5'-端缺失不同长度人ACAT1基因P1启动子片段的荧光素酶报告基因载体;探讨胰岛素对这两个ACAT1基因P1启动子缺失体转录活性的影响,确定胰岛素反应元件。
方法
在对人ACAT1基因P1启动子进行生物信息学分析的基础上,针对转录因子c/EBPα、SNF、HNF-3结合元件,设计两种ACAT1基因P1启动子缺失突变体,分别包括ACAT1基因P1启动子-579/+65、-566/+65bp的片段,并构建相应的萤火虫荧光素酶报告基因载体,分别命名为P1E-10、P1E-11,用DEAE-dextran法分别与内参照质粒pRL-TK共同转染THP-1细胞。恢复生长7小时后给予胰岛素(10-7M)处理,40小时后通过双荧光素酶报告系统检测荧光素酶活性。
结果
⑴PCR、酶切及测序鉴定表明,成功获得两种人ACAT1基因P1启动子缺失突变体的荧光素酶报告基因载体P1E-10、P1E-11。
⑵与非胰岛素处理的对照组相比,胰岛素增强人ACAT1基因P1启动子(P1E-1)的转录活性,升高到2.6倍,具有显著差异(P<0. 01,n=3),而另外两种P1启动子缺失突变体的报告载体转录活性不发生显著变化(P>0.05,n=3)。
结论
⑴成功构建了包含5'-端缺失不同长度人ACAT1基因P1启动子片段的荧光素酶报告基因载体,并可作为体外研究ACAT1基因转录调控的新手段。
⑵人ACAT1基因P1启动子5'-端上游-603～-580bp存在胰岛素反应元件,可能为c/EBPα结合位点。
PartⅠCloning and sequencing of P1 and P7 promoters of human acyl coenzyme A: cholesterol acyltransferase1 gene
Objective
To clone P1 and P7 promoters of human acyl coenzyme A: cholesterol acyltransferase1 (ACAT1) gene.
Methods
According 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.
Results
⑴The 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.
Conclusions
In 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 lines
Objective
To 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.
Methods
The 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.
Results
⑴The 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.
Conclusion
⑴We 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 cells
Chapter I Impact of insulin on ACAT1 mRNA expression in THP-1 cells detected with real-time fluorescent quantitative reverse transcription-polymerase chain reaction
Objective
To 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).
Methods
The 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.
Results
The 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).
Conclusions
⑴SYBR 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 cells
Objective
To 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.
Methods
The 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).
Results
⑴The 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.
Conclusions
Insulin up-regulated ACAT1 gene expression by activating P1 promoter. Keywords Insulin; ACAT1; Monocyte; Promoter; Transcriptinal activity; Transcript
PartⅣFunctional analysis of human ACAT1 gene P1 promoter and effect of insulin on the transcripitional activities of P1 promoter and its deletions in THP-1 cells
Objective
To 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.
Methods
According 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.
Results
⑴The 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).
Conclusion
⑴We 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 promoter
Objective
To 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.
Methods
According 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.
Results
⑴The 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).
Conclusion
⑴We 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.
引文
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12. Puglielli L, Konopka G, Pack-Chung, et al. Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid-peptide. Nature Cell Biololy, 2001, 3: 905-912.
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4. Beillard E, Pallisgaard N, van der Velden VHJ, et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using‘real-time’quantitative reverse-transcriptase polymerase chain reaction (FQ-PCR)- a Europe against cancer program. Leukemia, 2003, 17(12): 2474-2486.
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2. Yang JB, Duan ZJ, Yao W, et al. Synergistic transcriptional activation of human Acyl-coenzyme A: cholesterol acyltransterase-1 gene by interferon-gamma and all-trans-retinoic acid THP-1 cells. J Biol Chem, 2001, 276(24): 20989-20998.
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3. Batetta B, Mulas MF, Petruzzo P, et al. Opposite pattern of MDRI and caveolin-1 gene expression in human atherosclerotic lesions and proliferating human smooth muscle cells. Cell Mol Life Sci, 2001, 58(8): 1113-1120.
4. Petruzzo P, Mulas MF, Brotzu G, et al.Lipid metabolism and molecular changes in normal and atherosclerotic vessels. Eur J Vasc Surg, 2001, 22(1): 31-36.
5. Maung K, Miyazaki A, Nomiyama H, et al. Induction of acyl-coenzyme A:cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D(3) or 9-cis-retinoic acid in undifferentiated THP-1 cells. J Lipid Res, 2001, 42(2):181-187.
6. Cheng W, Kvilekval KV, Abumrad NA. Dexamethasone enhances accumulation of cholesteryl esters by human macrophages. Am J Physiol, 1995, 269: E642-648.
7. Hori M, Miyazaki A, Tamagawa H, et al. Up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 by transforming growth factor-beta1 during differentiation of human monocytes into macrophages. Biochem Biophys Res Commun, 2004, 320(2): 501-505.
8. Bai Z, Cheng B, Yu Q, et al. Effects of leptin on expression of acyl-coenzymea: cholesterol acyltransferases-1 in cultured human monocyte-macrophages. J Huazhong Univ Sci Technolog Med Sci, 2004, 24(6): 563-5, 590.
9. Suguro T, Watanabe T, Kanome T, et al. Serotonin acts as an up-regulator of acyl- coenzyme A:cholesterol acyltransferase-1 in human monocyte-macrophages. Atherosclerosis, 2006, 186(2): 275-281.
10. Watanabe T, Suguro T, Kanome T, et al. Human urotensin II accelerates foam cell formation in human monocyte-derived macrophages. Hypertension, 2005, 46(4): 738-744.
11. Furukawa K, Hori M, Ouchi N, et al. Adiponectin down-regulates acyl-coenzyme A:cholesterol acyltransferase-1 in cultured human monocyte-derived macrophages. Biochem Biophys Res Commun, 2004, 317(3): 831-836.
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13. Yang L, Yang JB, Chen J, et al. Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone. Cell Res, 2004, 14(4): 315-323.
1. Cases S, Novak S, Zheng Y, et al. ACAT-2, a second mammalian acyl CoA: chelesterol acyltransferase: its cloning, expression, and characterization. J Biol Chem, 1998, 273: 26755-26764.
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6. Lee O, Chang CC, Lee W, et al. Immunodepletion experiments suggest that acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) protein plays a major catalytic role in adult human liver, adrenal gland, macrophages, and kidney, but not in intestines. J Lipid Res, 1998, 39(8):1722-7.
7. Naomi S, Akira M, Motohiro T, et al. Localization of Human Acyl-Coenzyme A: Cholesterol Acyltransferase-1 (ACAT-1) in Macrophages and in Various Tissues . Am J Pathol, 2000, 156:227–236.
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10. Jeffery LS, Kavitha R, Robert S, et al. Quantitative analysis of the expression of ACAT1 genesin human tissues by real-time PCR. J Lipid Res, 2004, 45:686–696.
11. Chang TY, Chang CCY, Lu X, et al. Catalysis of ACAT may be completed within the plane of the membrane: a working hypothesis. J Lipid Res, 2002, 42: 1933-1938.
12. Puglielli L, Konopka G, Pack-Chung, et al. Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid-peptide. Nature Cell Biololy, 2001, 3: 905-912.
1. Batetta B, Mulas MF, Petruzzo P, et al. Opposite pattern of MDRI and caveolin-1 gene expression in human atherosclerotic lesions and proliferating human smooth muscle cells. Cell Mol Life Sci, 2001, 58(8): 1113-1120.
2. Petruzzo P, Mulas MF, Brotzu G, et al.Lipid metabolism and molecular changes in normal and atherosclerotic vessels. Eur J Vasc Surg, 2001, 22(1): 31-36.
3. Ball TB, Plummer FA, Hayglass KT, et al. Improved mRNA Quantitation in LightCycler RT-PCR. Int Arch Allergy Immunol, 2003, 130: 82–86.
4. Beillard E, Pallisgaard N, van der Velden VHJ, et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using‘real-time’quantitative reverse-transcriptase polymerase chain reaction (FQ-PCR)- a Europe against cancer program. Leukemia, 2003, 17(12): 2474-2486.
5. Brandfass C and Karlovsky P. Simultaneous detection of Fusarium culmorum and F. graminearum in plant material by duplex PCR with melting curve analysis. BMC Microbiol, 2006, 6: 4.
6. HsuehWA, Law R. The central role of fat and effect of peroxisome proliferator- activated receptor-gamma on progression of insulin resistance and cardiovascular disease. Am J Cardio, 2003, 92: 3J - 9J.
7. Lijnen HR. Extracellular proteolysis in the development and progression of atherosclerosis. Biochem Soc Trans, 2002, 30 (2): 163-167.
8. Rewers M, Zaccaro D, Agostino RD, et al. Insulin sensitivity, insulinemia, and coronary artery disease: the insulin resistance atherosclerosis study. Diabetes Care, 2004, 27: 781-787.
9. Goff DC Jr, D'Agostino RB Jr, Haffner SM, et al. Insulin resistance and adiposity influence lipoprotein size and subclass concentrations. Results from the Insulin Resistance Atherosclerosis Study. Metabolism, 2005, 54(2): 264-270.
10. EZ Jia, ZJ Yang, B Yuan, et al. Relationship between true fasting plasma insulin level and angiographic characteristics of coronary atherosclerosis. Clin Cardiol, 2006, 29(1): 25-30.
11. Vaidyula VR, Boden G, Rao AK, et al. Platelet and monocyte activation by hyperglycemia and hyperinsulinemia in healthy subjects. Platelets. 2006, 17(8): 577- 585.
12. MJ Muis, ML Bots, HJ Bilo, et al. High cumulative insulin exposure: a risk factor of atherosclerosis in type 1 diabetes? Atherosclerosis, 2005, 181(1): 185-192.
13. Lisa OR, Line MG, Stephen JY, et al. Glucose-dependent regulation of cholesterol ester metabolism in macrophages by insulin and leptin. J Biol Chem, 2002, 277(45): 42557-42562.
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