Expression and hypermethylation of p27^(kip1) in hepatocarcinogenesis

AIM: To study the expressions of p27kip1 protein and p27mRNA, the hypermethylation of p27kip1 and the relation between them in various stages of hepatocarcinogenesis.METHODS: p27 protein and p27mRNA were detected by immunohistochemical staining and in situ hybridization respectively in 68 cases of normal liver, liver cirrhosis,pericancerous cirrhosis and hepatocellular carcinoma (HCC). The hypermethylation of p27kip1 was detected by methylation-specific PCR (MSP) in 44 cases of normal liver, liver cirrhosis, and HCC.RESULTS: The positive rate of p27 protein was 66.7% (4/6) in normal liver, 60.0% (6/10) in liver cirrhosis, 50.0% (12/24) in pericancerous cirrhosis and 21.4% (6/28) in HCC. There were no statistical differences in normal liver,liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27 protein significantly decreased in HCC compared to that in the other groups (P = 0.006,x2 = 7.664). The positive rate of p27kip1 mRNA was 83.3% (5/6) in normal liver, 70.0% (7/10) in liver cirrhosis, 75.0% (18/24) in pericancerous cirrhosis and 25.0% (7/28) in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27kip1 mRNA also significantly decreased in HCC compared to that in the other groups (P = 0.000,x2 = 16.600). In addition, there was a significant correlation between the expression of p27 protein and p27mRNA in the integrated group of normal liver and liver cirrhosis.However, no significant correlation was found between pericancerous cirrhosis and HCC. Using MSP, we found that 1 HCC in 44 cases (including 6 cases of normal liver,10 cases of liver cirrhosis and 28 cases of HCC) was methylated, whose p27 protein and p27mRNA were negative.CONCLUSION: The reduction or loss of p27 protein and p27mRNA are potentially involved in hepatocarcinogenesis.The hypermethylation of p27 might lead to the loss of p27mRNA transcription.

Role of p53 suppression in the pathogenesis of hepatocellular carcinoma

In the world,hepatocellular carcinoma(HCC)is among the top 10 most prevalent malignancies.HCC formation has indeed been linked to numerous etiological factors,including alcohol usage,hepatitis viruses and liver cirrhosis.Among the most prevalent defects in a wide range of tumours,notably HCC,is the silencing of the p53 tumour suppressor gene.The control of the cell cycle and the preservation of gene function are both critically important functions of p53.In order to pinpoint the core mechanisms of HCC and find more efficient treatments,molecular research employing HCC tissues has been the main focus.Stimulated p53 triggers necessary reactions that achieve cell cycle arrest,genetic stability,DNA repair and the elimination of DNA-damaged cells’responses to biological stressors(like oncogenes or DNA damage).To the contrary hand,the oncogene protein of the murine double minute 2(MDM2)is a significant biological inhibitor of p53.MDM2 causes p53 protein degradation,which in turn adversely controls p53 function.Despite carrying wt-p53,the majority of HCCs show abnormalities in the p53-expressed apoptotic pathway.High p53 in-vivo expression might have two clinical impacts on HCC:(1)Increased levels of exogenous p53 protein cause tumour cells to undergo apoptosis by preventing cell growth through a number of biological pathways;and(2)Exogenous p53 makes HCC susceptible to various anticancer drugs.This review describes the functions and primary mechanisms of p53 in pathological mechanism,chemoresistance and therapeutic mechanisms of HCC.

Study of pathogenic genes in a pedigree with familial dilated cardiomyopathy

BACKGROUND Dilated cardiomyopathy(DCM)is a genetically heterogeneous cardiac disorder characterized by left ventricular dilation and contractile dysfunction.The substantial genetic heterogeneity evident in patients with DCM contributes to variable disease severity and complicates overall prognosis,which can be very poor.AIM To identify pathogenic genes in DCM through pedigree analysis.METHODS Our research team identified a patient with DCM in the clinic.Through invest-igation,we found that the family of this patient has a typical DCM pedigree.High-throughput sequencing technology,next-generation sequencing,was used to sequence the whole exomes of seven samples in the pedigree.RESULTS A novel and potentially pathogenic gene mutation-ANK2p.F3067L-was discovered.The mutation was completely consistent with the clinical information for this DCM pedigree.Sanger sequencing was used to further verify the locus of the mutation in pedigree samples.These results were consistent with those of high-throughput sequencing.CONCLUSIONS ANK2p.F3067L is considered a novel and potentially pathogenic gene mutation in DCM.

Changes of p53 and Waf1p21 and cell proliferation in esophageal carcinogenesis

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Genes for RNA-binding proteins involved in neuralspecific functions and diseases are downregulated in Rubinstein-Taybi iNeurons

Taking advantage of the fast-growing knowledge of RNA-binding proteins(RBPs)we review the signature of downregulated genes for RBPs in the transcriptome of induced pluripotent stem cell neurons(iNeurons)modelling the neurodevelopmental Rubinstein Taybi Syndrome(RSTS)caused by mutations in the genes encoding CBP/p300 acetyltransferases.We discuss top and functionally connected downregulated genes sorted to“RNA processing”and“Ribonucleoprotein complex biogenesis”Gene Ontology clusters.The first set of downregulated RBPs includes members of hnRNHP(A1,A2B1,D,G,H2-H1,MAGOHB,PAPBC),core subunits of U small nuclear ribonucleoproteins and Serine-Arginine splicing regulators families,acting in precursor messenger RNA alternative splicing and processing.Consistent with literature findings on reduced transcript levels of serine/arginine repetitive matrix 4(SRRM4)protein,the main regulator of the neural-specific microexons splicing program upon depletion of Ep300 and Crebbp in mouse neurons,RSTS iNeurons show downregulated genes for proteins impacting this network.We link downregulated genes to neurological disorders including the new HNRNPH1-related intellectual disability syndrome with clinical overlap to RSTS.The set of downregulated genes for Ribosome biogenesis includes several components of ribosomal subunits and nucleolar proteins,such NOP58 and fibrillarin that form complexes with snoRNAs with a central role in guiding post-transcriptional modifications needed for rRNA maturation.These nucleolar proteins are“dual”players as fibrillarin is also required for epigenetic regulation of ribosomal genes and conversely NOP58-associated snoRNA levels are under the control of NOP58 interactor BMAL1,a transcriptional regulator of the circadian rhythm.Additional downregulated genes for“dual specificity”RBPs such as RUVBL1 and METTL1 highlight the links between chromatin and the RBP-ome and the contribution of perturbations in their cross-talk to RSTS.We underline the hub position of CBP/p300 in chromatin regulation,the impact of its defect on neurons’post-transcriptional regulation of gene expression and the potential use of epidrugs in therapeutics of RBP-caused neurodevelopmental disorders.

Study on the Function of ORF Genes of Porcine Circovirus-like Virus P1

[Objectives]This study was conducted to determine the functions of eight ORF genes of porcine circovirus-like virus P1.[Methods]The double-copy tandem molecular cloning of porcine circovirus-like virus P1 genome was used to construct molecular clones with eight ORFs deleted by DNA site-directed mutagenesis technology.After transfected into PK15 cells for a certain period of time,RNA were extracted and was used to verify whether the eight ORFs were deleted or not and used for gene microarry analysis.The GO functions and KEGG pathway enrichment of differentially expressed genes were analyzed.[Results]P1 ORF1 is mainly involved in the biological processes of defense response to virus,signal transduction,regulation of Rab GTPase activity,and lipid metabolic process,and involved in the molecular functions of protein phosphatase inhibitor activity,phosphatidylinositol phospholipase C activity,2 iron,2 sulfur cluster binding,phosphoric diester hydrolase activity,and Rab GTPase activator activity,and in the KEGG pathways of secretion of digestive gland and nervous system development.P1 ORF2 is mainly involved in the biological processes of positive regulation of leukocyte chemotaxis,positive regulation of cell proliferation,positive regulation of cell migration,defense response to virus,regulation of cell growth,and involved in the molecular functions of insulin-like growth factor binding,and chemokine activity,and in the KEGG pathways of cytosolic DNA-sensing pathway,RIG-I-like receptor signaling pathway,toll-like receptor signaling pathway,chemokine signaling pathway,and cytokines,cytokine-cytokine receptor interaction.The biological processes,molecular functions and related pathways involving P1 ORF3 and ORF5 are basically similar to those of ORF2.P1 ORF8 is mainly involved in the biological processes of purine ribonucleotide biosynthetic process,amino acid transport,defense response to virus,amino acid transmembrane transport,and involved in molecular functions of N6-(1,2-dicarboxyethyl)AMP AMP-lyase(fumarate-forming)activity,iron-sulfur cluster binding,amino acid transmembrane transporter activity.[Conclusions]The analysis of the ORF functions of P1 virus lays a foundation for the study of its pathogenicity and pathogenesis.

树舌多糖对小鼠HepA P16 P27 Rb基因表达的影响

目的:探讨树舌多糖抗肿瘤作用的机制. 方法:通过免疫组化、ELISA法分别测定瘤体和血清中抑癌基因P16,P27,Rb表达蛋白的量;通过透射电镜观察凋亡小体是否存在. 结果:树舌多糖作用后P16,P27,Rb基因表达蛋白的量与盐水组有差异具有统计学意义,免疫组化结果:bP<0.01, vs(1)(2);aP<0.05,vs(2)(3);dP<0.01,vs(3).ELISA结果: aP<0.05,vs(3);bP<0.01,cP<0.05,vs(4);dP<0.01,vs (3)(4);fp<0.01,vs(4);gP<0.05,hP<0.01,vs(3).且P16, P27表达的量与Rb呈负相关mr=-0.094(1),nr=-0.446 (1),再有观察到凋亡小体的存在. 结论:树舌多糖通过激活抑癌基因,抑制肿瘤的生长促进肿瘤细胞凋亡.

ALV-A p27基因的原核表达及其鼠源多克隆抗体的制备

试验拟通过构建A亚群禽白血病病毒(ALV-A) p27基因原核表达载体,从而制备抗p27蛋白鼠源多克隆抗体。将感染ALV-A毒株的DF-1细胞反复冻融后提取RNA并反转录为cDNA,利用PCR扩增获得ALV-A p27基因,将目的基因克隆至pColdⅠ载体,并转化入大肠杆菌BL21(DE3)菌中进行诱导表达,经镍柱纯化后免疫28～32日龄的雌性小鼠,采用间接ELISA法测定抗体效价,Western blot检测抗体特异性。试验经PCR扩增出750 bp左右的特异性条带,并成功构建pColdⅠ-ALV-A-p27原核表达载体,加入终浓度为1 mmol/L的IPTG于18℃诱导表达18～24 h成功得到重组蛋白,该蛋白主要以包涵体形式存在。间接ELISA检测显示,制备的鼠源多克隆抗体血清抗体效价为1∶3 200;Western blot检测结果表明,制备的多克隆抗体能特异性识别原核表达的p27蛋白。试验结果在ALV的抗原分析、血清学诊断等方面有着重要的应用价值。

Effects of endotoxin on expression of ras, p53 and bcl-2 oncoprotein in hepatocarcinogenesis induced by thioacetamide in rats

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河北省蛋鸡群ALV p27抗原ELISA检测

禽白血病（Avian Leukusis,AL）是由反转录病毒科禽白血病/肉瘤病毒群病毒引起的禽类多种肿瘤性疾病的总称,可引起感染鸡免疫抑制和多组织器官发育迟缓、萎缩以及肿瘤等,造成鸡的生产性能下降甚至死亡,由鸡分离的ALV划分为A、B、C、D、E、J六个亚群。自2002年蛋鸡群发现禽白血病以来,全国各地频发,特别是自2009年以来,湖北、山东等地鸡白血病疑似病例频发,鸡群ALV的感染率高达60%,病死率高达50%以上[1]。

SIV p27单克隆抗体的制备和鉴定

目的建立SIVp27杂交瘤细胞株，并对其分泌的SIVp27单克隆抗体进行初步鉴定。方法使用基因重组的SIVp27蛋白免疫BALB/c小鼠，采用杂交瘤技术使用半固体培养基法建立杂交瘤细胞株，制备单克隆抗体。通过染色体核型对杂交瘤细胞株进行鉴定；采用Western blot、免疫荧光法、酶联免疫吸附法确定单克隆抗体的交叉反应性、相对亲和力、抗原识别表位、免疫球蛋白的类型和亚类。对单克隆抗体进行鉴定。结果获得4株可稳定分泌SIVp27单克隆抗体的杂交瘤细胞，IC3、286为IgG1类，2E12为IgG2b类，3G3为IgG2a类。4株单抗均能识别SIV的p27蛋白，与逆转录病毒SRV、STLV无交叉反应，286、2E12与H1Vp24有交叉反应。免疫荧光法检测腹水效价为1：10240～1：40960。1C3、286、2E12、3G3染色体平均数分别为103、97、96、101。2E12与3G3识别不同的抗原表位。结论成功地制备出4株SIVp27单克隆抗体，均具有良好的特异性和亲和力，为进一步建立免疫分析方法，进行SIV／SAIDS及其艾滋病相关研究，奠定了基础。

HIV-1 p24和SIV p27两种ELISA试剂盒检测SIV p27抗原的比较

目的由于检测SIV p27抗原试剂盒来源困难,有时不稳定,鉴于HIV-1 p24与SIVp27有较强的交叉抗原,本研究比较HIV-1 p24和SIV p27两种ELISA试剂盒检测SIV p27抗原得出的结果是否存在一定的相关性。方法 HIV-1 p24和SIV p27两种ELISA试剂盒定性和定量检测样品中SIV p27抗原,并对检测结果进行回归和相关分析。结果 HIV-1 p24和SIV p27两种ELISA试剂盒检测SIV p27抗原的灵敏度分别是150 pg/mL和62.5 pg/mL。两种试剂盒检测病毒液和血浆中SIV p27抗原的定性结果一致。定量结果的统计分析得出病毒液的直线回归决定系数R2=0.857,直线相关系数r=0.926,P<0.01,直线正相关程度较高;血浆的直线回归决定系数R2=0.512,直线相关系数r=0.716,P<0.05,直线正相关程度较低。结论 HIV-1 p24 ELISA试剂盒能够替代SIVp27 ELISA试剂盒定性检测病毒液和血浆中SIV p27抗原,但只能定量检测病毒液中SIV p27抗原。

Detecting the polymorphism sites of p53 and Fas genes of Han population in Zhejiang province

BACKGROUND： It is of significance for single nucleotide polymorphisms （SNPs）, a difference of rank, which exists widely in biology, genetics and other fields. OBJECTIVE： To detect polymorphism sites in exon-4 of p53 gene, promotor of Fas gene and intron-7 of Fas gene of healthy people in Han nationality in Zhejiang province. DESIGN： Simple random sampling. SETTING： Department of Surgery of the 118 Hospital of Chinese PLA.PARTICIPANTS： A total of 80 healthy people in Han nationality were selected from hospitals in Zhejiang province from August 2005 to January 2006. There were 43 males and 37 females aged from 3 to 78 years with the mean age of 39.5 years, and all subjects were consent. DNA which was used in genetic analysis was selected from peripheral venous blood of all subjects and maintained at -20℃.METHODS： Polymorphism sites in exon-4 of p53 gene, promotor of Fas gene and intron-7 of Fas gene were detected with directly DNA sequencing technique. MAIN OUTCOME MEASURES ： Polymorphism sites in exon-4 of p53 gene, promotor of Fas gene and intron-7 of Fas gene of healthy people in Han nationality in Zhejiang province. RESULTS： A total of 80 samples were involved in the final analysis. SNPs sites were found at the 119^th base of exon-4 of p53 gene （the 72^nd codon of p53 gene）, the 670^th base of upper start codon in promotor of Fas gene （Fas-670）, and the 995^th base of intron-7 of Fas gene, especially SNPs in the 995^th base of intron-7 pf Fas gene, i.e. C→A transversion, was a new site.CONCLUSION ： One unknown SNPs site is discovered in intron-7 of Fas gene of people in Han nationality in Zhejiang province. This study also proves that the 72^nd codon exists in p53 gene and the -670 polymorphism site exists in promotor of Fas gene.

DNA methylation and carcinogenesis in digestive neoplasms

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PDRG1 at the interface between intermediary metabolism and oncogenesis

PDRG1 is a small oncogenic protein of 133 residues. In normal human tissues, the p53 and DNA damageregulated gene 1(PDRG1) gene exhibits maximal expression in the testis and minimal levels in the liver. Increased expression has been detected in several tumor cells and in response to genotoxic stress. High-throughput studies identified the PDRG1 protein in a variety of macromolecular complexes involved in processes that are altered in cancer cells. For example, this oncogene has been found as part of the RNA polymerase Ⅱ complex, the splicing machinery and nutrient sensing machinery, although its role in these complexes remains unclear. More recently, the PDRG1 protein was found as an interaction target for the catalytic subunits of methionine adenosyltransferases. These enzymes synthesize S-adenosylmethionine, the methyl donor for, among others, epigenetic methylations that occur on the DNA and histones. In fact, downregulation of S-adenosylmethionine synthesis is the first functional effect directly ascribed to PDRG1. The existence of global DNA hypomethylation, together with increased PDRG1 expression, in many tumor cells highlights the importance of this interaction as one of the putative underlying causes for cell transformation. Here, we will review the accumulated knowledge on this oncogene, emphasizing the numerous aspects that remain to be explored.

PTEN P27 SKp2在上皮性卵巢癌中的表达及其临床意义

目的:探讨PTEN、P27和SKp2在上皮性卵巢癌中的表达及临床意义。方法:阅读国内外有关文献并进行综述。结果:PTEN、P27和SKp2基因表达异常可能在上皮性卵巢癌的发生、发展过程中起关键作用,PTEN、P27和SKp2基因可作为卵巢癌预后的重要指标。结论:PTEN、P27和SKp2可能参与上皮性卵巢癌的发生、发展,PTEN、P27和SKp2可能是卵巢癌治疗靶点。

细胞周期素E P27^(KIP1)多参数测定在胃癌早期诊断中的应用

目的探讨细胞周期素E(cyclinE)、P27KIP1的表达在胃癌早期诊断中的价值。方法本研究共选取34例胃癌、10例癌前病变及10例正常胃黏膜,机械法或胶原酶和透明质酸酶混合消化法制备单细胞悬液。异硫氰酸荧光素(FITC)标记的二抗和藻红蛋白(PE)标记的单抗经多参数流式细胞术的方法分别检测cyclinE、P27KIP1的表达。结果胃癌cyclinE和P27KIP1的表达率分别为(32±15)%、(6±5)%,与正常胃黏膜及癌前病变相比较,差异有统计学意义(P<0.01)。结论CyclinE过表达和P27KIP1低表达使G1/S转换点调节失控,与胃癌的发生、进展密切相关。二者的联合测定有利于胃癌的早期诊断。

PROMOTION OF CHEMICAL CARCINOGENESIS AND P53 EXPRESSION BY REDUCTION OF SUPEROXIDE DISMUTASE ACTIVITY IN THE LUNG OF RAT IN V

In the present experiment,an inhibitor of superoxide dismutase(SOD),diethyldithiocarbamate(DETC),was used to decrease SOD activity for the observation of the relation between SOD activity and carcinogenesis and the expression of P53 protein in vivo.144 Wistar rats were used for the Present study.The results showed that the SOD activity reduction by DETC resulted markedly in the promotion of the carcinogenesis and the expression of P53 protein in the lung tissues,but the increase of SOD activity by the addition of plus SOD inhibited the pathological changes significantly.The frequency of the pathological lesions and Positive P53 expression are 36/42 and 8/42 in the animals without DETC and SOD:16/52 and 4/52 in the animals with SOD and 46/50 and 26/50 in the animals with DETC respectively.The results reported in this Paper suggest that:(1) the decrease of SOD activity enhanced the carcinogenesis induced by chemical carcinogen;(2) P53 gene may be associated with the process of tumorigenesis;and(3) at the same time the abnormal expression of P53 protein may be associated with the transition from premalignant lesions to carcinoma.

肾上腺皮质肿瘤中PCNA p27和TOPOⅡα的表达与其生物学行为的关系

目的:探讨肾上腺皮质肿瘤中增殖相关的抗原p27、细胞增殖核抗原(PCNA)、DNA拓扑异构酶2(TOPOⅡα)的表达水平与肿瘤生物学行为间的关系。方法:免疫组化SP法。结果:与肾上腺皮质腺瘤相比,在肾上腺皮质腺癌中p27的表达减少(P<0.01),PCNA和TOPOⅡα的表达增多(P<0.01)。在正常组和腺瘤组间三个指标的表达均无显著性差异(P>0.05)。PCNA与TopoⅡα的表达呈正相关,两者与p27的表达均呈负相关。结论:p27的表达减少和PCNA、TOPOⅡα的表达增多可作为判断肾上腺肿瘤的恶性程度的参考指标。三者联合检测对评价肾上腺皮质肿瘤的生物学行为有重要意义。