Tumor suppressor genes on frequently deleted chromosome 3p in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is among the most common malignancies in southern China.Deletion of genomic DNA,which occurs during the complex pathogenesis process for NPC,represents a pivotal mechanism in the inactivation of tumor suppressor genes (TSGs).In many circumstances,loss of TSGs can be detected as diagnostic and prognostic markers in cancer.The short arm of chromosome 3 (3p) is a frequently deleted chromosomal region in NPC,with 3p21.1-21.2 and 3p25.2-26.1 being the most frequently deleted minimal regions.In recent years,our research group and others have focused on the identification and characterization of novel target TSGs at 3p,such as RASSF1A,BLU,RBMS3,and CHL1,in the development and progression of NPC.In this review,we summarize recent findings of TSGs at 3p and discuss some of these genes in detail.A better understanding of TSGs at 3p will significantly improve our understanding of NPC pathogenesis,diagnosis,and treatment.

Hepatocellular carcinoma mouse models:Hepatitis B virusassociatedhepatocarcinogenesis and haploinsufficienttumor suppressor genes

The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles in hepatocarcinogenesis still need to be elucidated.Many tumor suppressor genes(TSGs)have been identified as being involved in HCC.These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors:the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele.Hepatitis B virus(HBV)is one of the most important risk factors associated with HCC.Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor,one advantage of mouse models for HBV/HCC research is the numerous and powerfulgenetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs.Here,we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner.Discoveries obtained using mouse models will have a great impact on HCC translational medicine.

Promoter methylation of tumor suppressor genes in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma(ESCC) is a prevalent and fatal cancer in China and other Asian countries.Epigenetic silencing of key tumor suppressor genes(TSGs) is critical to ESCC initiation and progression.Recently,many novel TSGs silenced by promoter methylation have been identified in ESCC,and these genes further serve as potential tumor markers for high-risk group stratification,early detection,and prognosis prediction.This review summarizes recent discoveries on aberrant promoter methylation of TSGs in ESCC,providing better understanding of the role of disrupted epigenetic regulation in tumorigenesis and insight into diagnostic and prognostic biomarkers for this malignancy.

Characterization of six tumorsuppressor genes and microsatellite instability in hepatocellular carcinomain southern African blacks

AIM To analyse cumulative loss of heterozygosity (LOH) of chromosomal regions and tumor suppressor genes in hepatocellular carcinomas (HCCs) from 20 southern African blacks. METHODS p53, RB1, BRCA1, BRCA2, WT1 and E cadherin genes were analysed for LOH, and p53 gene was also analysed for the codon 249 mutation, in tumor and adjacent non tumorous liver tissues using molecular techniques and 10 polymorphic microsatellite markers. RESULTS p53 codon 249 mutation was found in 25% of the subjects, as was expected, because many patients were from Mozambique, a country with high aflatoxin B 1 exposure. LOH was found at the RB1, BRCA2 and WT1 loci in 20%(4/*!20) of the HCCs, supporting a possible role of these genes in HCC. No LOH was evident in any of the remaining genes. Reports of mutations of p53 and RB1 genes in combination, described in other populations, were not confirmed in this study. Change in microsatellite repeat number was noted at 9/*!10 microsatellite loci in different HCCs, and changes at two or more loci were detected in 15%(3/*!20) of subjects. CONCLUSION We propose that microsatellite/genomic instability may play a role in the pathogenesis of a subset of HCCs in black Africans.

EXPRESSION OF NM23-H1 GENE PRODUCT IN NASOPHARYNGEAL CARCINOMA AND ITS CLINICAL SIGNIFICANCE

Objective: The nm23 gene is one of the tumor metastatic suppressor genes. The expression of nm23H1 has been reported to be inversely associated with metastatic potentiality in a number of human carcinomas, including breast, colorectal, gastric, hepatocellular and gallbladder carcinomas. In this study, the immunohistochemical staining of nm23H1 protein in human nasopharyngeal carcinoma (NPC) was examined, and the relationship between nm23H1 and both metastasis and prognosis of patients with NPC was also investigated. Methods: Routine LSAB immunohistochemistry with the nm23H1 monoclonal murine antibody was employed to study the expression of nm23H1 protein in 95 paraffinembedded specimens of NPC treated at our hospital. The clinical pathologic data and results of followup were also retrieved. Comparisons between patients with and without expression of nm23H1 protein with respect to metastasis, locoregional recurrence and survival were performed using Log rank test. Multivariate prognostic analyses were performed by using Cox's regression model. Results: Nm23H1 negative expressive tumors were associated with a higher incidence of lymphnode metastasis (86.7%) than those of nm23H1 positive (48.6%, P<0.01). Nm23H1 negative expressive tumors were associated with a high incidence of recurrence and distant metastasis after radiotherapy (P<0.05). A significant association was found between expression of nm23H1 and prognosis (P<0.01). The expression of nm23H1 indicated favorable prognosis. Conclusion: It was suggested that nm23 H1 negative expression was significantly associated with lymphnode metastasis, recurrence and distant metastasis. Nm23H1 may have value for predicting the prognosis of NPC.

Alteration of tumor suppressor gene p16 and Rb in gastric cancinogesis

IM To study the alterations of tumor suppressor gene p16 and Rb in the carcinogenesis of the stomach. METHODS Different mucosal biopsies were endoscopically obtained, all samples were immediately fixed with 10% buffered formalin, embedded with paraffin and sectioned serielly. Alterations of p16 and Rb protein in 12 cases of superficial gastritis, 15 atrophic gastritis, 20 atypical hyperplasia and 40 cancerous tissues were detected by the immunohistochemical method (ABC). RESULTS Different degrees of nuclear immunostaining of p16 and Rb occurred on gastric epithelium in different stages of lesions. With the lesions progressing, the positive immunostaining rate of p16 protein had a decreasing tendency (833%→733%→300%→275%), and on the other hand, that of Rb protein had an increasing tendency (250%→467%→600%→675%). A negative correlationship was found between these two parameters in the gastric cancer. Of 40 cases of gastric cancer, a negative relationship was observed in 20 cases. In comparison with both positive (9 cases) and both negative tissues (11 cases), there was a significant difference (500%,225%,275%) (P<005).CONCLUSION Abnormal expression of p16 and Rb plays an important role in gastric carcinogenesis.

NDRG2 gene copy number is not altered in colorectal carcinoma

AIM To investigate if the down-regulation of N-myc Downstream Regulated Gene 2(NDRG2) expression in colorectal carcinoma(CRC) is due to loss of the NDRG2 allele(s).METHODS The following were investigated in the human colorectal cancer cell lines DLD-1, Lo Vo and SW-480: NDRG2 mRNA expression levels using quantitative reverse transcriptionpolymerase chain reaction(qRT-PCR); interaction of the MYC gene-regulatory protein with the NDRG2 promoter using chromatin immunoprecipitation; and NDRG2 promoter methylation using bisulfite sequencing.Furthermore, we performed qPCR to analyse the copy numbers of NDRG2 and MYC genes in the above three cell lines, 8 normal colorectal tissue samples and 40 CRC tissue samples.RESULTS As expected, NDRG2 mRNA levels were low in the three colorectal cancer cell lines, compared to normal colon.Endogenous MYC protein interacted with the NDRG2 core promoter in all three cell lines.In addition, the NDRG2 promoter was heavily methylated in these cell lines, suggesting an epigenetic regulatory mechanism.Unaltered gene copy numbers of NDRG2 were observed in the three cell lines.In the colorectal tissues, one normal and three CRC samples showed partial or complete loss of one NDRG2 allele.In contrast, the MYC gene was amplified in one cell line and in more than 40% of the CRC cases.CONCLUSION Our study suggests that the reduction in NDRG2 expression observed in CRC is due to transcriptional repression by MYC and promoter methylation, and is not due to allelic loss.

Aberrant methylation of SPARC in human hepatocellular carcinoma and its clinical implication

AIM:To investigate the methylation status of secreted protein acidic and rich in cysteine(SPARC) in human hepatocellular carcinoma(HCC) and evaluate its clinical implication.METHODS:The methylation status of SPARC was analyzed in one HCC cell line(SMMC-7721) and 60 pairs of HCC and corresponding nontumorous tissues by methylation-specific polymerase chain reaction and bisulfite sequencing.The expression of SPARC mRNA and protein were examined by reverse transcription polymerase chain reaction and immunohistochemistry,respectively.The correlations between the methylation status and the gene expression,the clinicopathological parameters,as well as the prognosis after surgery were analyzed.RESULTS:In the SMMC-7721 cell line,the loss of SPARC expression was correlated with the aberrant methylation and could be reactivated by the demethylating agent 5-aza-2'-deoxycytidine.Methylation frequency of SPARC in HCC was significantly higher than that in the corresponding nontumorous tissues(45/60 vs 7/60,P < 0.001),and it was correlated with the pathological classification(P = 0.019).The downregulation of the SPARC mRNA expression in HCC was correlated with the SPARC methylation(P = 0.040).The patients with methylated SPARC had a poorer overall survival than those without methylated SPARC(28.0 mo vs 41.0 mo,P = 0.043).CONCLUSION:Aberrant methylation is an important mechanism for SPARC inactivation in HCC and SPARC methylation may be a promising biomarker for the diagnosis and prognosis of HCC.

MicroRNA and esophageal carcinoma

Objective：An abundant class of non-coding small RNA molecules, 21-25 nucleotide in length, are widely found in animals and plants and named microRNA （miRNA）. MiRNAs are highly evolutionarily conserved, expressing in specific tissue and timing, and negatively regulate the gene expressions at the posttranscriptional level,and subsequently control crucial physiological processes such as metabolism, amplification, differentiation, development and apoptosis, Therefore, miRNAs could provide an access to many human diseases in theory. Recent evidence demonstrates that miRNAs play an important role in the initiation and progression of human cancer, mainly by interrupting the cell cycle at the cellular level and by interacting with signaling The expression profiling of miRNAs can be used as a tool of diagnosis, staging, prognosis and biotherapy of some tumors, as has already been proven to have superiority to mRNA, in the categorization of tumors. This review focuses on the genesis, mechanism of action of miRNA and its relationship to tumors, detection methods and its potential effect on the diagnosis, staging, and biotherapy in esophageal carcinoma.

Inhibitory effect of IGF-Ⅱ antisense RNA on malignant phenotype of hepatocellular carcinoma

INIRODUCTIONAccording to the therapeutic effect and strategy ofantisense RNA for hepatoccllular carcinoma(HCC),we have specifically synthesized partialcDNA of human insulin-like growth factor Ⅱ(IGF-Ⅱ)and constructed IGF-Ⅱ cDNA antisenseeukaryotic expression vector.The constructedvector was introduced into hepatoma cell lineSMMC-7721 to block the intrinsic IGF-Ⅱexpression.The biological behavior changes ofhepatoma cells were observed.All these

Behind the curtain of non-coding RNAs; long non-coding RNAs regulating hepatocarcinogenesis

Hepatocellular carcinoma(HCC) is one of the most common and aggressive cancers worldwide. HCC is the fifth common malignancy in the world and the second leading cause of cancer death in Asia. Long non-coding RNAs(lncRNAs) are RNAs with a length greater than 200 nucleotides that do not encode proteins. lncRNAs can regulate gene expression and protein synthesis in several ways by interacting with DNA, RNA and proteins in a sequence specific manner. They could regulate cellular and developmental processes through either gene inhibition or gene activation. Many studies have shown that dysregulation of lncRNAs is related to many human diseases such as cardiovascular diseases, genetic disorders, neurological diseases, immune mediated disorders and cancers. However, the study of lncRNAs is challenging as they are poorly conserved between species, their expression levels aren't as high as that of m RNAs and have great interpatient variations. The study of lncRNAs expression in cancers have been a breakthrough as it unveils potential biomarkers and drug targets for cancer therapy and helps understand the mechanism of pathogenesis. This review discusses many long non-coding RNAs and their contribution in HCC, their role in development, metastasis, and prognosis of HCC and how to regulate and target these lncRNAs as a therapeutic tool in HCC treatment in the future.

ISOLATION OF TUMOR DIFERENTIALLY EXPRESSED GENES BY MIXING PROBES LIBRARY SCREEN

Objective: This study was designed to clone candidate tumor suppressor genes down-expressed in Nasopharyngeal Carcinoma (NPC). Methods: Differentially expressed cDNA fragments (AF152605 and AF091517) were labeled by PCR, and Northern blot was used to confirmed transcript length of these genes. Skeleton muscle cDNA library was screened with PCR-labeled probe mixture. Results: 23 positive independent and overlapping positive clones were obtained. By sequencing the positive clones directly, three novel genes (Genbank accession number: AF179285, AF170307 and AF194971), with transcripts of 2.1 Kb, 1.1 Kb and 1.4 Kb respectively, were isolated successfully. Conclusions: Library screening using PCR-labeled probes mixture is an efficient method to get full-length cDNA from multi-cDNA fragment simultaneously and quickly.

Frequent Down-regulation and Deletion of KLF6 in Primary Hepatocellular Carcinoma

Kruppel-like factor 6 (KLF6) was reported as tumor suppressor in multiple cancers. However, loss of chromosomal locus spanning KLF6 is relatively infrequent in previous published studies. To explore the role of KLF6 in hepatocellular carcinoma (HCC), we examined the gene for expression change, loss of heterozygosity (LOH) and mutation in 26 HCC samples. The expression levels of KLF6 were significantly down-regulated in HCCs, as detected by qRT-PCR. LOH occurred in 11 (52%) of 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 24%, and sequence changes located in activation domain of KLF6. Furthermore, MTT assay showed a significant antiproliferative effect of the wt KLF6 transfected in HepG2 hepatoblastoma cells. Fluorescence-activated cell sorting analysis revealed that KLF6 could induce apoptosis. These findings indicate that deregulation of KLF6, together with genetic abnormalities of allelic imbalance and mutations, may play a role in HCC pathogenesis.

Detailed Deletion Mapping of Chromosome 9p21-22 in Nasopharyngeal Carcinoma

Objective: To further refine the extent of deletion on chromosome 9p21-22 in nasopharyngeal carcinoma (NPC) and provide evidence for discovering new tumor suppressor gene. Methods: Loss of heterozygosity (LOH) on chromosome 9p21-22 was analyzed in 25 paired blood and tumor samples by using 11 high-density microsatellite polymorphic markers. Results: 17 of 25 cases (68.0%) showed LOH at one or more loci. Higher frequencies of LOH were found at four loci: D9S161 (35.0%), D9S1678 (31.5%), D9S263 (33.3%) and D9S1853 (33.3%), where 6 cases had a contiguous stretch of allelic loss. Conclusion: The minimal common region of deletion might be defined between D9S161 and D9S1853 (estimated about 2.7 cM in extent) at 9p21.1, suggesting that inactivation of one or more tumor suppressor genes located in this region may be an important step in NPC.

鼻咽癌分子标志物研究

鼻咽癌严重危害人类健康,寻找其早期诊断及预后相关的分子标志物迫在眉睫.在总结本课题组运用基因组学、转录组学、蛋白质组学和组织微阵列等高通量技术对不同分化阶段、不同组织类型和不同临床分期的鼻咽癌标本进行大规模筛选工作的基础上,结合近年国际上有关进展,初步构建了鼻咽癌不同发病阶段的分子靶标系统:a.证实SPLUNC1、p16、EBER-1、p27、RASSF1A和CDH13是鼻咽癌早期诊断的理想分子靶标;b.鼻咽癌上调基因RB1,STMN1和DSP及下调基因SERPINB6,AGTRL1和SYTL2的分类预测模型是区分正常鼻咽上皮和鼻咽癌的分子靶标;c.NGX6、Ezrin、LTF、OPN、THY1和Tiam-1是鼻咽癌侵袭与转移预测的候选分子靶标;d.Cyclin D1、Survivin和HPA是与鼻咽癌预后相关的候选分子标志物;e.证实Bcl-2、EGFR和Ki67是预测鼻咽癌放疗敏感与否的候选分子靶标;f.SAA和cox-2是监测鼻咽癌复发的候选分子标志物;g.发现BRD7、NGX6、NOR1和UBAP1的6个SNP改变是鼻咽癌遗传易感风险因子;h.建立了由139个基因组成的鼻咽癌不同临床分期分子靶标系统.这些在大样本基础上的分子靶标筛选为鼻咽癌分子分型研究奠定了重要工作基础.

抑癌基因PTEN在子宫内膜癌组织中的表达

目的 :了解抑癌基因PTEN在子宫内膜癌中的表达及与子宫内膜癌临床病理因素的相关性 ,探讨其与子宫内膜癌发生发展的关系 .方法 :运用免疫组织化学SABC法测定PTEN蛋白在 77(子宫内膜样腺癌 72 ,子宫浆液性癌 5 )例子宫内膜癌组织和 2 5例正常子宫内膜中的表达 ,并比较PTEN表达变化与组织学类型、手术临床分期、病理分级、子宫肌层浸润程度、淋巴结转移及患者年龄等临床病理因素之间的相关性 .结果 :子宫内膜癌组织中PTEN表达缺失率为 6 0 % ,与正常子宫内膜相比 (0 % )存在显著性差异 (P =0 .0 0 0 0 ) .子宫内膜样腺癌中PTEN的失表达率为 6 4 % ,而子宫浆液性癌为0 % ,二者差异显著 (P =0 .0 0 4 8) .PTEN的失表达率随内膜癌恶性程度的增加而增加 (P =0 .0 0 34) ,但与手术临床分期、肌层浸润程度、淋巴结转移及患者年龄等因素无关 (P >0 .0 5 ) .结论 :抑癌基因PTEN是在子宫内膜癌发生过程中占重要地位的突变基因 ,PTEN基因的失表达与子宫内膜癌 。

食管癌中抑癌基因PTEN的表达及临床意义

目的探讨抑癌基因PTEN在食管癌中的表达及临床意义。方法用免疫组织化学方法检测80例食管癌及其相应的手术远端正常食管组织中PTEN蛋白的表达水平。结果PTEN在食管鳞癌中的表达率明显低于癌旁正常食管黏膜组织(P<0.01),而且PTEN蛋白表达与肿瘤分化程度、浸润深度、淋巴结转移相关(P<0.01)。结论从蛋白水平证明PTEN基因表达缺失或突变在食管鳞癌的发生发展中可能起重要作用,PTEN蛋白表达的检测可作为临床治疗和判断预后的依据。

抑癌基因PTEN在食管癌中的表达

目的 研究抑癌基因PTEN在食管癌组织的表达 ,探讨PTEN与食管癌发生发展之间的关系。方法 应用免疫组织化学SABC法对食管癌及癌旁食管粘膜PTEN的表达进行检测。结果 PTEN在食管鳞癌 (4 8 2 2 % )中的表达率明显低于癌旁正常食管粘膜组织 (90 6 3% ) ,有显著性差异 (P <0 0 1)。而且从食管鳞癌Ⅰ至Ⅲ级PTEN表达率逐渐降低 ,鳞癌Ⅰ至Ⅲ级PTEN的表达率分别是 :78 5 7%、 6 0 0 %、 2 7 2 7% ,有显著性差异 (P <0 0 1)。结论 从癌旁正常食管鳞状上皮组织 ,到Ⅰ～Ⅲ级食管鳞癌组织的PTEN表达依次降低 ,表明PTEN在食管癌的发生以及进展过程中有表达缺失。

PTEN蛋白在食管癌中的表达及临床意义

目的研究PTEN蛋白在食管癌中的表达,探讨PTEN与食管癌的发生发展之间的关系。方法采用免疫组化法检测60例食管癌组织及癌旁正常食管上皮组织PTEN的表达情况。结果食管癌组织中PTEN蛋白的阳性表达率为65.0%,显著低于癌旁正常食管上皮组织94.4%的阳性率,统计学分析表明两者差异有显著性意义(P<0.05)。PTEN蛋白表达与肿瘤的分化程度,浸润深度,淋巴结转移及TNM分期相关,其在食管癌高、中、低分化组的阳性率逐渐降低,分别为88.9%、69.6%、36.8%,差异有显著性意义(P<0.05),随癌组织浸润程度的加深而明显降低(P<0.05),有淋巴结转移组明显低于无淋巴结转移组(P<0.05)。结论PTEN蛋白表达的降低在食管癌的发生发展中可能起重要的作用,PTEN蛋白表达的检测有望成为食管癌辅助诊断和判断预后的参考指标之一。

SEMA3B基因在鼻咽癌组织中的表达、杂合性丢失和甲基化分析

SEMA3B基因定位于鼻咽癌高频缺失区域3p21.3上,最近被证明具有抑瘤基因的功能.分析了鼻咽癌组织中SEMA3B基因的表达、杂合性丢失(LOH)和甲基化情况.首先应用逆转录-聚合酶链式反应(RT-PCR)方法检测了33例鼻咽癌组织和15例慢性鼻咽炎组织中SEMA3B基因的表达,结果显示75.8%(25/33)鼻咽癌组织中SEMA3B基因表达缺失或下调,显著低于慢性鼻咽炎组织中的表达(P=0.001).进一步选取3个微卫星位点D3S1568、D3S1621和D3S4597分析了20例鼻咽癌组织中SEMA3B基因LOH的情况,结果表明3个位点的丢失率分别为10%、20%和15%,总的丢失率为45%,统计分析发现LOH与基因表达之间存在明显相关(P=0.023).最后,采用甲基化特异性PCR方法分析了SEMA3B基因启动子区甲基化,结果发现在100%的鼻咽癌组织和73.3%的慢性鼻咽炎组织中检测到SEMA3B基因启动子区高甲基化.由此得出结论,SEMA3B基因在鼻咽癌组织中表达缺失或下调,LOH是引起其表达异常的原因之一.