CPNE5的A结构域对肿瘤坏死因子α诱导核因子κB活化的影响

[目的]研究CPNE5的A结构域(AD)对肿瘤坏死因子α(TNF-α)激活核因子κB(NF-κB)转录活性的调控作用。[方法]用PCR法扩增AD序列,将其构建EGFP-N1载体上,在真核细胞中表达融合蛋白AD-GFP。以连有κB序列和表达水平受κB序列调控的荧光素酶基因的质粒为报告基因载体,将EGFP-N1、AD-GFP质粒分别与报告基因共转染到HEK293细胞中,经TNF-α处理后,用双荧光素酶报告基因系统测定荧光素酶的活性,检测它们对NF-κB转录活性的影响。[结果]构建了AD-GFP质粒;A结构域能够显著抑制NF-κB转录活性(P<0.05);AD对NF-κB转录活性的抑制具有剂量依赖性。[结论]CPNE5的A结构域能够剂量依赖性抑制NF-κB的转录激活活性。     

MTS1基因β启动子E2F1结合位点序列突变重组质粒的构建与表达

目的：深入研究MTS1基因β启动子的转录激活与E2F1转录因子的相互作用关系，阐明该转录水平的调控机制。方法：用PCR定点突变方法或酶切连接法，构建β启动子0.38kb SacⅡ-Sac I酶切片段中E2F1 A，B，C任意2个位点或3个位点均突变的pGL3重组质粒。用脂质体介导的基因瞬时转染法，将构建的重组质粒转染MTS1基因双等位缺失的急性T淋巴细胞白血病Jurkat细胞，检测pGL3重组质粒中荧光素酶报告基因的表达。结果：构建的E2F1 A，B，C结合位点突变的重组质粒经Sac I或Nae I酶切鉴定和DNA序列分析得到证实。与E2F1位点野生型重组质粒比较，突变型重组质粒在Jurkat细胞中荧光素酶报告基因的表达量减少，以3个位点均突变的重组质粒为明显。结论：构建的E2F1 A，B，C2个或3个结合位点均突变重组质粒成功，可通过基因转染用于研究MTS1基因的功能试验中；MTS1基因β启动子的转录活性可能与E2F1转录因子的反式激活有关。     

人黑素瘤抗原MAGE-A4对p53转录活性的影响

目的:探讨人黑素瘤抗原-A(melanoma antigen-A,MAGE-A)对p53转录活性的影响.方法:选用p53缺失型人肺癌细胞系H1299,采用荧光素酶报告分析法、RT-PCR、Western印迹法、克隆形成实验和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling,TUNEL)等方法研究MAGE-A4对p53转录活性的影响.结果:荧光素酶报告分析结果显示,转染p53基因(25 ng)可以使p21WAF1启动子介导的荧光素酶表达增加(P＜0.01);共转染恒定量(25 ng)的p53基因和不同量的MAGE-A4基因(100、200和400 ng)后, p21WAF1启动子介导的荧光素酶表达均明显高于单独转染p53基因时的表达量(P＜0.01).RT-PCR和Western印迹法检测结果也显示,转染p53基因可以使p21WAF1mRNA和蛋白表达水平增加(P＜0.01),共转染MAGE-A4基因和p53基因后,p21WAF1mRNA和蛋白表达水平均明显高于单独转染p53基因时的表达水平(P＜0.01).克隆形成实验及TUNEL染色结果显示,转染p53基因可以使H1299细胞克隆形成数减少及凋亡细胞数增加(P＜0.01),共转染MAGE-A4基因和p53基因以后,H1299细胞克隆形成数与单独转染p53基因组相比减少,而凋亡细胞数与单独转染p53基因组比较增加(P＜0.01).结论:MAGE-A4可以增强p53的转录活性及功能发挥.     

野生型p53对SMMC-7721细胞中POLD1基因的影响

目的探讨野生型p53基因表达对人肝癌细胞SMMC-7721中POLD1基因的影响。方法设计并构建p53特异性小干扰shRNA绿色荧光真核表达质粒（p53-siRNA）和表达EGFP-p53融合蛋白的p53绿色荧光真核增强表达质粒（pEGFP-p53）,通过稳定转染,将表达pEGFP-p53重组质粒、p53-siRNA转染入SMMC-7721细胞;经G418筛选,获得稳定细胞系7721-p53、7721-p53RNAi。通过RT-PCR检测转染后p53、POLD1的mRNA。结果在人肝癌细胞SMMC-7721中,野生型p53高表达组能够抑制POLD1的基因转录（P〈0.001）;而低表达组能够促进POLD1的基因转录（P〈0.001）。结论在人肝癌细胞SMMC-7721中,p53能够调控POLD1的基因转录。     

ALK5激活型突变体在肾癌细胞中的表达及功能

目的:构建ALK5激活型真核表达质粒Flag-ALK5 T204D,证实融合蛋白在肾癌细胞内表达,并验证其对TGF-β信号通路的激活作用。方法:以野生型ALK5质粒为模板,采用重叠延伸PCR方法进行定点突变;将构建的重组质粒测序并转染到肾癌细胞ACHN中,提取细胞蛋白进行Western blot检测;利用双荧光素酶报告基因分析,检测该突变体对TGF-β信号通路典型靶基因启动子的激活作用。结果:对ALK5的表达序列进行定点突变,构建了ALK5激活型真核表达质粒Flag-ALK5 T204D;Western blot检测到融合蛋白的表达,分子量约为58kDa;Flag-ALK5 T204D能够上调TGF-β信号通路典型靶基因启动子活性。结论:成功构建了Flag-ALK5 T204D并验证了其对TGF-β信号通路的激活作用,为进一步研究ALK5的生物学特性及其功能奠定了基础。     

Gemin3基因通过抑制p53表达阻碍细胞凋亡

目的 探讨Gemin3基因表达在细胞凋亡中的作用及途径.方法 采用免疫共沉淀、谷胱甘肽-S转移酶共沉淀实验确定Gemin3与p53两种蛋白在体内、体外相互结合及相瓦作用的结构域.荧光素酶报告基因检测转染Gemin3基因对p53基因的影响,依靠慢病毒载体介导小干扰RNA敲低Gemin3基因的表达,并经嘌呤霉素筛选获得稳定的Gemin3低表达细胞系,实时定量PCR检测Gemin3敲低对p53及其下游基因在转录水平的影响,流式细胞术检测Gemin3敲低对细胞凋亡的影响.结果 Gemin3的C端与p53的中部DNA部位相互结合.将p53报告基因、p53蛋白基因以及逐渐增量的Gemin3基因共转染Saos-2细胞,随着Gemin3质粒转染浓度的增高,p53介导的报告基因表达水平逐渐降低,Gemin3在转录水平抑制p53基因的表达.实时定量PCR结果显示,与对照细胞比较,Gemin3敲低细胞中,p53、p21和Bax mRNA的表达水平明显增高.Western blot检测结果显示,Gemin3敲低细胞中,p53的表达明显升高.流式细胞术检测结果显示,敲低Gemin3基因的表达导致细胞凋亡的增加.结论 Gemin3与p53相互结合并通过抑制p53的表达阻碍细胞凋亡.     

人卵巢癌相关候选基因HE4(WFDC2)的转录调控主要由Sp1与位于-71和-48的Egr-1位点结合所介导

目的阐明HE4基因转录调控机制的细节.方法1.用半定量RT-PCR的方法评估HE4基因在肿瘤细胞株中的表达情况;2.以荧光素酶报告基因载体为基础,对HE4基因的上游顺序构建了3'、5'缺失突变和一系列连接体扫描突变;3.通过瞬时转染/体外双荧光素酶报告系统对这些重组质粒中插入片段进行启动子活性的分析;4.针对蛋白和关键顺式区域的结合的特异性和能力,开展泳动滞后和抗体介导的超迁移分析.结果通过对(-1860/ 29)的HE4基因上游片段及其剪切体的分析,将HE4基因最小启动子确定为-107/ 15的DNA片段.通过对连接体扫描突变体的分析,将关键的顺式作用元件确定在W45(-71/-48)片段,生物信息学提示该区存在两个Egr-1位点.通过用已知的反式作用因子与报告基因质粒分别进行共转染,提示Sp1是最为有效的反式作用因子,而不是Egr-1.通过泳动滞后和抗体介导的超迁移实验,证实Sp1确实是参与作用的转录因子.结论HE4基因的转录活性主要是由转录因子Sp1与位于(-71/-48)区域的两个Egr-1位点结合所介导的.     

has-miR-223表达载体构建及其对靶基因ARTN的调控

目的:构建hsa-miR-223真核表达载体,并在人食管癌细胞KYSE-150中验证hsa-miR-223对靶基因artemin(ARTN)的调控作用.方法:以基因组DNA为模板,RT-PCR扩增包括hsa-miR-223前体序列在内的基因序列,并将其克隆到载体pCD?NA3.1(+)上,通过实时定量PCR方法检测hsa-miR-223在人胚肾HEK293细胞中的表达.根据生物信息学预测软件TargetScan、mirBase和PicTar对hsa-miR-223靶基因进行预测,将靶基因ARTN的3'UTR区融合到pMIR荧光素酶基因下游,通过双荧光素酶报告基因检测hsa-miR-223 对靶基因ARTN 的3'UTR 区的调控作用.将pCDNA3.1-miR-223 表达质粒转染人食管癌细胞KYSE150,通过Western blot检测对ARTN蛋白表达的影响.结果:成功构建hsa-miR-223表达载体pCDNA3.1-miR-223.实时定量PCR 验证表明pCDNA3.1-miR-223 在人胚肾HEK293 中能够明显地过表达成熟miR-223.生物信息学预测ARTN 是hsa-miR-223的靶基因,并构建融合ARTN的3'UTR区表达质粒pMIR-ARTN,在此基础上对ARTN的3'UTR"种子区"进行定点突变.双荧光素酶报告基因分析表明hsa-miR-223能够作用于ARTN的3'UTR.Western blot法进一步证实ARTN是miR-223的靶基因.结论:hsa-miR-223作用于ARTN的3'UTR可在转录后水平上调控ARTN蛋白的表达.     

人SOX7启动子荧光素酶报告质粒构建及HMGA2对其活性影响的研究

目的 研究发现,HMGA2促进肿瘤细胞发生EMT与其下游基因SOX7有关.为进一步验证转录因子HM-GA2与其下游基因SOX7之间的调控关系,本研究通过构建人SOX7基因启动子荧光素酶报告质粒,采用荧光素酶报告实验观察HMGA2对SOX7基因启动子荧光素酶活性的影响,为研究HMGA2转录表达的调控机制提供必要的实验基础.方法 采用PCR技术扩增人SOX7基因启动子序列(-1 549～+79nt),将SOX7基因启动子插入荧光素酶报告基因载体pGL3-basic中构建质粒pGL3-SOX7-promoter;再分别将pGL3-SOX7-promoter、pGL3-basic-SOX7-promoter和对照质粒(pLV-UbC-IRES2-EGFP)、pGL3-SOX7-promoter和HMGA2表达质粒(pLV-UbC-HMGA2-IRES2-EGFP)3组质粒共转染293T细胞,培养48 h后,检测3组荧光素酶的表达情况,观察HMGA2对SOX7基因启动子的调控作用.结果 通过菌落PCR和核酸测序证实,人SOX7基因启动子荧光素酶报告质粒pGL3-SOX7-promoter构建成功;将pGL3-SOX7-promoter和HMGA2表达质粒(pLV-UbC-HMGA2-IRES2-EGFP)共转染293T细胞48 h后,与对照组相比,SOX7基因启动子介导荧光素酶的活性降低约31％,受到明显抑制(P=0.003),提示HMGA2可能调控SOX7基因启动子的活性.结论 本研究成功构建了SOX7基因启动子荧光素酶报告质粒,初步验证了SOX7是HMGA2下游的作用靶点.     

转录因子STAT5A启动子荧光素酶报告基因质粒的构建及鉴定

目的： 利用分子克隆的方法构建转录因子STAT5A启动子荧光素酶报告基因质粒并鉴定其效应。方法： 以乳腺癌MCF7细胞基因组DNA为模板,采用PCR方法扩增STAT5A基因的启动子序列,并将其克隆至荧光素酶报告基因质粒pGL3-Enhancer中,经过菌液扩增、酶切、测序等方法获得目的质粒,并通过双荧光报告基因实验进一步验证其功能。结果： 构建的STAT5A启动子荧光素酶报告基因质粒pGL3-STAT5A-pro-luc-E序列正确,且具有转录活性。在双荧光报告基因实验中发现重组载体pGL3-STAT5A-pro-luc-E荧光素酶的相对活性约为阴性对照pGL3-Enhancer的8倍（P<0.01）,而pGL3-STAT5A-pro-N-luc-E荧光素酶的相对活性约是阴性对照pGL3-Enhancer的6倍（P<0.01）。结论： 本项目成功构建了转录因子STAT5A启动子荧光素酶报告基因质粒,为进一步研究STAT信号转导及转录激活蛋白信号通路提供了重要的研究工具。     

RNAi沉默p53基因对HeLa细胞核转录因子3表达及其对细胞增殖和侵袭能力的影响

目的：探讨RNAi沉默p53基因对宫颈癌HeLa细胞核转录因子3（STAT3）的表达,及对HeLa细胞增殖和侵袭能力的影响。方法：构建3种靶向p53基因的shRNA1~3序列,并转染HeLa细胞,采用逆转录PCR和Western blot检测细胞p53 mRNA和蛋白的表达,以筛选有效沉默p53的shRNA序列。将筛选的序列转染HeLa细胞后,采用逆转录PCR和Western blot检测STAT3 mRNA和蛋白的表达;采用四甲基噻唑蓝法（MTT）检测细胞增殖;采用Transwell检测细胞侵袭能力。结果：构建的3种p53-shRNA均可沉默宫颈癌HeLa细胞p53的表达,尤以shRNA3的沉默效果最好,转染shRNA3后,HeLa细胞p53 mRNA和蛋白的表达量分别为0.23±0.05和0.41±0.11,显著低于未转染组、空质粒组、p53-shRNA1组和p53-shRNA2组（P均 < 0.05）,故后续试验均以shRNA3转染HeLa细胞。将shRNA3序列转染HeLa细胞沉默p53基因后,p53-shRNA3组STAT3 mRNA和蛋白表达量分别为1.09±0.21和1.46±0.25,显著高于未转染组（0.53 ±0.10和0.74±0.14）及空质粒组（0.55±0.11和0.73±0.15）（P均 < 0.05）。MTT法分析结果显示,在孵育24、36、48和72 h时,p53-shRNA3组HeLa细胞增殖能力均显著大于未转染组和空质粒组（P均 < 0.05）。Transwell法分析结果显示,转染12 h后p53-shRNA3组穿膜细胞数目（59.36±5.33）显著高于未转染组（15.73±1.29）和空质粒组（16.01±1.34）（P均 < 0.05）。结论：RNAi沉默p53基因能够上调宫颈癌HeLa细胞STAT3的表达,并促进细胞的增殖及侵袭能力。     

Epigenetic modification of RhoE expression in gastric cancer cells

RhoE is a unique member of Rho family of GTPases without detectable intrinsic GTPase activity. Our previous study showed that RhoE is a tumor suppressor gene and its expression is down-regulated in gastric cancer. However, the mechanism underlying the down-regulated expression of RhoE in gastric cancer has not been elucidated yet. In the present study, the effect of epigenetic modification on the RhoE expression in gastric cancer cells was investigated. The mRNA and protein expression of RhoE were detected by real-time RT-PCR and Western blotting, respectively. Results showed RhoE was significantly down-regulated in three gastric cancer cell lines. A promoter (2980 bp) of RhoE and its five truncated mutants were cloned into vector pGL-3Basic for the activity analysis by luciferase reporter assay. Treatment with trichostatin A, a histone deacetylation inhibitor, enhanced not only the activity of RhoE promoter, but also the mRNA and protein expression of RhoE in three gastric cancer cell lines, whereas treatment with 5-Aza-2'-deoxycytidine, a DNA methylation inhibitor, affected neither RhoE promoter activity nor RhoE expression. No synergistic effect was observed in cells treated with both drugs. Our results suggested that RhoE expression in gastric cancer cells was regulated by histone deacetylation, but not by DNA methylation, at the epigenetic level.     

Anti-apoptotic activity of p53 maps to the COOH-terminal domain and is retained in a highly oncogenic natural mutant.

The tumour suppressor p53 plays a complex role in the regulation of apoptosis. High levels of wild type p53 potentiate the apoptotic response, while physiological range, low levels of the protein have an anti-apoptotic activity in serum starved immortalized fibroblasts. Here we report that primary fibroblast-like cells that show normal growth control are also efficiently protected from apoptosis by the endogenous p53 activity. The capacity to inhibit apoptosis is not restricted to the wild type protein: the R-->H175 p53 mutant fully retains the anti-apoptotic activity of the wild type p53, providing a possible explanation for its high oncogenicity. Using a series of point and deletion mutants of p53 under the control of tetracycline-regulated promoter we show that certain mutants, like the wild type, protect cells at low levels but lead to apoptosis when overexpressed. This latter effect is lost upon deletion of a proline-rich domain in the NH2 part of the protein. The anti-apoptotic activity can be mapped to the extreme carboxy-terminal part of the protein and is therefore independent of other well characterized p53 activities. Our results add a new level of complexity to the network of interactions mediated by p53 in normal physiology and pathology.     

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene.

The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a ''gain of function'' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-small-cell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms'' overexpression is indicative of a ''gain of function'' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas.