人肝癌细胞p53基因的表达及其对细小病毒H-1的敏感性

本文利用单链构象多态性分析,17号染色体短臂等位基因杂合性分析,Northern印迹,免疫沉淀,p53基因第7外显子酶切等技术检测了两个中国人肝癌细胞系SMMC-7721,YY-8103和一个自发转化的人肝细胞系L-02的p53基因结构与表达。实验表明,这三个细胞系中没有出现17号染色体短臂等位基因杂合性缺失,第4—9外显子也没发生突变,但其mRNA和蛋白表达水平很低。利用MTT比色分析法研究了这三个细胞系和其他已知p53基因背景的八个人肝癌细胞系(QGY-7703、PLC/PRF/5、Huh-7、Hep3B、FOCUS、Tong/ HCC、SK-Hep-1、HepG2)对自主性细小病毒H-1的敏感性。除HepG2细胞外,其他十个细胞系p53基因的结构和/或表达都不正常。经H-1感染(moi=20)后,其敏感性均高于HepG2细胞。本研究初步表明了p53基因结构或表达的不正常可能导致人肝癌或转化细胞对H-1的敏感性的提高。     

MKi67-siRNA可抑制QGY-7703细胞的生长

为了探讨MKI67在肝癌细胞发生发展中的作用,采用实时定量 PCR 方法检测人肝细胞癌 QGY 7703 细胞中MKI67 基因表达水平, 以及 MKI67在肝细胞癌组织和癌旁正常组织中的表达情况,设计并合成针对MKI67 的siRNA,利用脂质体转染法将其转入QGY-7703 细胞内,通过MTT和细胞集落形成实验观察MKI67-siRNA 对QGY-7703细胞生长活性和增殖能力的影响.实时定量PCR结果表明,MKI67在肝细胞癌组织中的表达水平明显高于癌旁正常组织（P< 0.01）. MTT和细胞集落形成实验结果显示,转染MKI67-siRNA 的QGY-7703细胞生长活性和集落形成率明显低于对照组（P< 0.01）.由此得出结论：MKI67 在肝癌细胞系QGY-7703细胞中的表达水平较高,且它在肝癌组织中的表达水平明显上调. 同时,MKI67-siRNA 可以有效抑制QGY-7703细胞的生长活性和增殖能力,提示MKI67可能与肝细胞癌的发生、发展相关.     

AFP的亚细胞定位及对肿瘤细胞生长的影响

目的观察甲胎蛋白（AFP）在不同肿瘤细胞中的亚细胞定位及对肿瘤细胞生长的影响。方法运用免疫荧光的方法观察内源性AFP在HeI。a细胞、QGY-7703细胞、MCF-7细胞中的亚细胞定位。将构建的表达AFP的质粒pcDNA3-AFP及AFP腺病毒siRNA干涉载体Adv—AFPsiRNA作用于QGY-7703细胞,MCF-7细胞,运用M1Tr,集落形成实验检测细胞增殖状况。结果免疫荧光显示,内源性的AFP在HeLa细胞、QGY-7703细胞、MCF-7细胞均只在细胞质中表达。pcDNA3-AFP使QGY-7703的细胞活性增加了2l％（P〈0．05）及集落形成能力增加了32％（P〈0．01）,MCF-7实验组比对照组细胞活性降低了30％（P〈0．01）．克隆形成能力降低82％（P〈0．01）。Adv—AFPsiRNA使QGY-7703的细胞活性降低了22％（P〈0．05）,平均克隆形成能力降低52％（P〈0．01）,MCF-7细胞活性提高了24．5％（P〈0．05）,克隆形成能力提高了89％（P〈O．01）。结论内源性的AFP只在细胞质中表达。AFP能促进QGY-7703细胞的增殖及克隆形成能力,而在MCF-7细胞中发挥相反的作用。腺病毒介导的内源性的AFP表达的下调能降低QGY-7703的增殖,却增加了MCF-7的细胞活性及克隆形成能力。     

罗格列酮通过PTEN/PI3K/Akt通路影响人肝癌细胞QGY-7701的增殖及凋亡

本研究为了探讨罗格列酮对人肝癌细胞QGY-7701增殖凋亡的影响,阐明其作用机制,以罗格列酮和人肝癌细胞QGY-7701为材料,通过结晶紫染色及CCK8方法,发现罗格列酮对人肝癌细胞QGY-7701有明显的抑制作用,其IC50为83.24μmol/L;通过RT-PCR,发现罗格列酮上调PTEN基因;通过Western blotting,发现罗格列酮能使PCNA、Bcl-2和p-Akt蛋白表达下调,使Bad、PPARγ和PTEN的蛋白表达上调,经PPARγ抑制剂处理后,PTEN和p-Akt表达下调。结果表明,罗格列酮可能通过调节PTEN/PI3K/AKT基因的途径,诱导人肝癌细胞QGY-7701凋亡,并抑制癌细胞的进一步增殖。本研究为临床肝癌研究提供理论基础。     

人肝癌细胞株QGY—7703的p53基因及其表达研究

本文利用等位基因分析,ＰＣＲ－ＳＳＣＰ,ＲＴ－ＣＰＲ／序列分析,Ｎｏｒｔｈｅｒｎ印迹,免疫组织化学,Ｗｅｓｔｅｒｎ印迹,免疫沉淀等方法对人肝癌细胞株ＱＧＹ－７７０３的Ｐ５３基因背景进行了研究,发现１７号染色体短臂可能存在等位基因缺失,在Ｐ５３基因的编码序列上没有发现任何突变,但发现其ｍＲＮＡ和蛋白表达水平很低,表明在ＱＧＹ－７７０３细胞中Ｐ５３基因的低水平表达可能同细胞的恶性程度有关。     

基于CRISPR/Cas9技术构建CELF6基因敲除细胞系并探讨CELF6与p53基因的关系

目的:构建CELF6基因敲除细胞系并探讨CELF6与p53基因之间的关系。方法:CRISPR/Cas9双载体慢病毒系统通过两个慢病毒载体分别向细胞中导入Cas9蛋白和sg RNA序列表达框,从而实现对CELF6基因的敲除。通过Surveyor(错配酶法)检测sg RNA活性并利用Western blot(蛋白质免疫印迹)检测CELF6的敲除效率。进一步利用CCK-8试剂盒检测敲除CELF6和过表达CELF6对细胞增殖的影响。利用公开可用的癌症基因组图谱(TCGA)数据库分析CELF6在多种肿瘤组织中的表达情况。结果:CELF6基因敲除的HCT116细胞系成功构建。Western blot检测发现CELF6基因敲除的细胞系中p53、p-RB蛋白的表达水平显著下调,而CELF6过表达的细胞中p53、p-RB蛋白的表达水平明显上调。CCK-8实验结果显示敲除CELF6基因后,细胞增殖活性显著增强;而过表达CELF6后,细胞的增殖活性受到明显抑制。生物信息学分析发现CELF6在结肠癌、胶质母细胞瘤、子宫内膜癌、肾嫌色细胞癌、乳腺癌、甲状腺癌等肿瘤组织中显著低表达。结论:推测CELF6是一种新的潜在肿瘤抑制基因,其可能在肿瘤的发生、发展过程中发挥重要作用。     

DEK真核表达载体的构建及其对p53启动子活性的影响

目的：构建DEK的pcDNA3-Flag表达载体,研究其对抑癌基因p53启动子活性的影响。方法：以乳腺文库为模板,PCR扩增DEK编码序列,克隆到pcDNA3-Flag载体,构建成pcDNA3-Flag-DEK,转染293T细胞,Western印迹鉴定peDNA3-Flag载体介导的DEK的表达,萤光素酶报告基因活性实验研究DEK对p53启动子活性的影响。结果：双酶切实验证实得到pcDNA3-Flag-DEK阳性克隆;Western印迹实验发现DEK在293T细胞内表达;转录活性实验表明在ZR75-1乳腺癌细胞中,DEK呈剂量依赖性抑制p53启动子的活性。结论：构建了DEK的真核表达载体,并发现此表达载体能在ZR75-1乳腺癌细胞中抑制p53启动子活性。     

p53过表达抑制同源盒基因NKX3.1启动子活性

NKX3.1是前列腺特异表达的同源盒基因,在前列腺癌的发生发展中起重要作用,而在前列腺癌进展中常会发生p53的基因突变.为研究两者之间的关系,构建NKX-3.1启动子(1 040bp)-荧光素酶报告基因重组质粒（pGL3-1040）及其缺失突变体,瞬时转染前列腺癌细胞LNCaP.通过荧光素酶表达活性分析,检测p53过表达对NKX3.1启动子活性的影响.结果表明：p53在LNCaP细胞中过表达可明显抑制NKX3.1启动子活性;RT-PCR及Western印迹检测p53过表达对NKX3.1表达的影响.结果表明,p53过表达可以明显抑制同源盒基因NKX3.1的表达.通过TRANSFAC软件分析,在NKX3.1基因上游-526至-507区存在一个p53反应元件的5′核心序列.缺失pGL3-1040中的p53反应元件核心序列并不能消除p53对NKX3.1启动子的抑制作用,表明p53不是通过p53反应元件直接抑制NKX3.1启动子活性.进一步通过5′缺失突变分析,发现NKX3.1启动子-140～+8 bp区仍受p53负调控.此148 bp区域中含有一个Sp1和一个CREB元件,瞬时共转染Sp1表达载体或CREB表达载体的结果表明,p53并不是通过与Sp1或CREB相互作用对NKX3.1启动子发挥抑制作用的.上述结果表明,p53过表达可以抑制同源盒基因NKX3.1启动子活性,下调NKX3.1基因的转录,其调控机制有待进一步研究.     

p53 gene expression of human hepatoma cell lines and their sensitivities to parvovirus H-1]

DNA structure and expression of p53 gene in human hepatoma cell lines SMMC-7721, YY-8103 and a spontaneously transformed liver cell line L-02 were analysed using the following method: analysis of allelic losses on chromosome 17p, PCR/SSCP, Northern blot and immunoprecipitation. There was no point mutation found in the exons 4-9 of the p53 gene, and a low level of expression of p53 gene was detected in the three cell lines. These observations were in agreement to the reported results of the relevant experiment using the human hepatoma cell line QGY-7703. Sensitivities of these cell lines and other eight human hepatoma cell lines (QGY-7703, PLC/PRF/5, Tong/HCC, Huh-7, FOCUS, Hep3B, SK-Hep-1, HepG2) with known p53 backgrounds to parvovirus H-1 was assayed using MTT method. Abnormality in the structure and/or function was observed in all of the cell lines examined except HepG2. The cell line HepG2 with normal structure and function of the p53 gene was found to be the least sensitive to H-1 in comparison to all the cell lines which have defeated structure and/or function of the p53 gene. The present study serves as a preliminary evidence that enhancement of the sensitivity of human hepatoma cell lines to H-1 is correlated to the abnormality of the structure and/or function of the p53 gene.     

Lack of expression of the protein p53 gene in a human T-cell leukemia line

Expression of the gene encoding the nuclear phosphoprotein p53 (a proto-oncogene classified in the same functional family as c-myc and E1a adenovirus gene) was examined in a human T-cell leukemia (KE-37R cell line). No p53 (or a modified product) could be detected by immunoprecipitation with monoclonal antibodies P Ab 421 and P Ab 122 in KE-37R cell extracts, and no p53-specific RNA was characterized by Northern blot analysis. Southern blot using a murine p53 cDNA clone as a probe, did not reveal any gross rearrangement in the structure of the gene. However, this molecular probe was not suited for investigating the 5' end of the gene which contains the promoter and the non coding exon 1. It is interesting to notice that in KE-37R cells, c-myc has been activated by a t(8; 14) (q24; q11) translocation, suggesting that the c-myc product might substitute to some functions normally requiring p53.     

Identification of differential proteins in nasopharyngeal carcinoma cells with p53 silence by proteome analysis

Although mutation of p53 tumor-suppressor gene is rare in nasopharyngeal carcinoma (NPC), NPC has a high frequency of overexpression of p53 protein. There seem to be complex mechanisms of inactivation and stabilization of p53 in NPC. To detect proteins associated with the function of p53 in high throughout screening, we succeeded in establishing p53 knockdown human NPC CNE2 cell line (CNE2sip53) using stable RNA interference, and compared the proteomic changes between CNE2sip53 and control cell line CNE2/pSUPER using two-dimensional gel electrophoresis. Twenty-two differentially expressed proteins between the two cell lines were identified by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3sigma, etc.), and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, several differential proteins including HSP27, HSP70, GRP75 and GRP78 were verified as p53 interacting proteins in NPC by immunoprecipitation and Western blot analysis, and the suppression of HSP27 expression by HSP27 antisense oligonucleotides could decrease the p53 protein level. Our data suggest that these differential proteins may be associated with the function of p53 in NPC, and provide new clues to elucidate the mechanisms of inactivation and stabilization of p53 in NPC.     

肽类生长因子对抗癌基因的诱导作用

观察了肽类生长因子对２ＢＳ细胞中抗癌基因表达的影响。结果表明,ＥＧＦ或ＦＧＦ均可明显诱导２ＢＳ细胞中Ｒｂ基因的表达,其幅度分别为３０５％、２４３％;ＥＧＦ也可诱导２ＢＳ细胞中ＴＧＦβ１基因表达增加３０－５０％,但ＦＧＦ对ＴＧＦβ１的表达无明显影响;ＥＧＦ或ＦＧＦ对ｐ５３基因的表达均无明显诱导作用。上述结果为生长因子作用机制研究及生长因子与抗癌基因的关系提供了新的线索。     

Involvement of Annexin A2 in p53 induced apoptosis in lung cancer

Tumor suppressor p53 plays important roles in cell cycle regulation, apoptosis and DNA repair in different cell types including lung cancer. There are different p53 apoptotic pathways in high and low metastatic ability lung cancer cells. However, the exactly mechanism in the pathway is still unclear. Here we found that Annexin A2, a Ca²⁺-dependent phospholipid-binding protein, is involved in p53-mediated apoptosis. First, by using mRNA differential display technique, down-regulated Annexin A2 expression was found in all cell lines transfected of Ad-p53 (adenoviral expression construct encoding wild type p53 gene) especially in highly metastatic Anip973 lung cancer cells. Then, decreased expression of Annexin A2 was further confirmed by Northern blot and Western blot analysis. At last, knock down of Annexin A2 by siRNA inhibited cellular proliferation in BE1 cell line with highly metastatic ability. Taken together, our results suggested that Annexin A2 may play roles in p53 induced apoptosis and it is also involved in regulation of cell proliferation. The authors Yun Huang, Yan Jin and Cheng-hui Yan contributed equally to this work.