人鼻咽与鼻咽癌组织p53调节基因差异表达的研究

目的 用cDNA Array比较鼻咽癌组织及正常组织基因表达谱,研究鼻咽癌组织内p53调节基因表达差异。方法 Atlas人类肿瘤cDNA表达阵列7742－1滤膜杂交后,用AtlasImage 1.01a分析膜杂交结果,RT－PCR反应验膜杂交结果,免疫组化证实基因在蛋白质水平的表达改变。结果 在588个肿瘤相关基因中,共有134个基因表达上调,88个基因表达下调。膜上有p53调节基因32种,其中13种显示差异表达,有11个表达上调,2个表达下调。结论 在鼻咽癌组织内,p53的功能失控,MDM2、p21和Bax可能对鼻咽癌细胞的生长起重要的调控作用。     

人鼻咽与鼻咽癌cDNA阵谱中DNA修复相关基因表达差异的初步研究

目的 用cDNA阵卤咽癌组织及正常组织的基因表达谱,研究鼻咽癌组织内修复相关基因的表达差异。方法 Atlas human cancer cDNA expression array 7742-1杂交后,用Atlas Image 1.01a分析滤膜杂交结果,RT－PCR反应验证滤膜杂交结果。结果 在588个肿瘤相关基因中,共有134个基因表达上调,88个基因表达下调。在有DNA损伤应答、修复、重组相关基因的C区中,表达上调的基因有21个,与修复相关的有6个;表达下调的有44个,与修复相关的则有12个。结论 DNA修复相关基因的改变可能在鼻咽癌病理生理过程中起一定作用。     

Comprehensive analysis of differentially expressed genes reveals the promotive effects of UBE2T on colorectal cancer cell proliferation

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Via analysis using The Cancer Genome Atlas database, the present study identified 1,835 genes that were differentially expressed in CRC, including 811 upregulated and 1,024 downregulated genes. Enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery tool revealed that these differentially expressed genes were associated with the regulation of CRC progression by modulating multiple pathways, such as ‘Cell Cycle, Mitotic’, ‘DNA Replication’, ‘Mitotic M-M/G1 phases’ and ‘ATM pathway’. To identify the key genes in CRC, protein-protein interaction (PPI) network analysis was performed and the hub modules in upregulated and downregulated PPI networks were identified. Ubiquitin-conjugating enzyme E2 T (UBE2T), a member of the E2 family, was identified to be a key regulator in CRC. To the best of our knowledge, the present study was the first to demonstrate that UBE2T expression was upregulated in CRC samples compared with normal tissues. Kaplan-Meier analysis revealed that higher expression levels of UBE2T were associated with worse prognosis compared with lower UBE2T expression levels in CRC. Additionally, the present study demonstrated that knockdown of UBE2T inhibited CRC cell proliferation. Flow cytometry assays revealed that UBE2T knockdown induced cell cycle arrest at G₁ phase and apoptosis in vitro. These results suggested that UBE2T may be a novel potential biomarker for CRC.     

下调PPP2R5C表达对Jurkat细胞GSK-3β、MDM2、pTEN、TP53通路相关调控基因表达谱的影响

目的 利用基因芯片技术研究RNA干扰下调PPP2R5C对Jurkat细胞中GSK-3β、MDM2、pTEN、TP53通路相关基因表达的影响。方法 利用核转染技术将PPP2R5C-siRNA799转导Jurkat细胞,培养48 h后,提取细胞总RNA,纯化mRNA后反转录制备目标序列,与Affymetrix基因表达谱芯片3’IVT杂交,以Affymetrix GeneChip Scanner 3000扫描仪扫描得到原始图像数据,并应用GeneSpringGX 11.0软件进行差异表达谱分析。结果 基因芯片分析GSK-3β、MDM2、pTEN、TP53信号通路相关的47个基因,发现全部基因发生了差异表达,其中上调基因有22个,下调基因有25个。明显上调的基因有SNAI1、APC、CDKN1A、ATM、MDM2和pTEN,而明显下调的基因只有GSK-3β。结论 下调Jurkat细胞PPP2R5C基因时,在一定程度上选择性调节涉及细胞增殖和凋亡相关的GSK-3β、MDM2、pTEN和TP53信号通路基因的表达,从而抑制Jurkat细胞增殖。     

Deregulation of the G1/S phase transition in cancer and squamous intraepithelial lesions of the uterine cervix: a case control study

High-risk types of HPV express the oncoproteins, E6 and E7, that can inactivate TP53 and RB1, respectively, and thus take control of both cell cycle and apoptosis. Herein, the mRNA expression profiles of 24 G1/S checkpoint genes were analysed in cancer and squamous intraepithelial lesions (SIL) of the uterine cervix. In total 35 squamous cervical carcinomas, 26 high-grade SIL (HSIL), 33 low-grade SIL (LSIL) tissues, and 28 normal uterine cervix specimens as controls were assessed by RT-PCR. Five genes were found to be upregulated only in tumours, RBL2, E2F2, CDK6, CCNE1 and MYC; eight in tumours and HSILs, E2F1, E2F3, E2F5, CCND1, CDK2, CDKN1B, PCNA and POLA, and five in tumours, HSILs and LSILs, TP53, E2F4, CDKN1A, CDKN2A and DHFR. MDM2 was found to be upregulated in SIL, while RBL1 was found to be downregulated in all three groups of cases. TP73 exhibited lower levels in carcinomas; however, its exon 13-containing isoforms were increased and exon 2-containing isoforms were reduced in both cancer and HSIL. Three genes, RB1, CDK4 and CDKN2D, did not exhibit any significant alteration in gene expression. Hierarchical clustering revealed that this set of G1/S checkpoint genes was able to discriminate the total 122 samples into groups of disease and non-disease with only 8 exceptions (6.6%). Our data suggest that deregulation of G1/S phase transition in cervical carcinogenesis is a progressive process. Certain clusters of genes are activated very early in pre-cancerous SILs while others are activated later, during malignant transformation. The ability of this array of markers to identify disease status suggests that it could be used for diagnostic purposes.     

Epigenetic downregulation of the ISG15–conjugating enzyme UbcH8 impairs lipolysis and correlates with poor prognosis in nasopharyngeal carcinoma

We identified the UBE2L6 gene, encoding the ISG15-conjugating enzyme UbcH8, as one gene significantly downregulated by promoter hypermethylation in nasopharyngeal carcinoma (NPC). Reduced expression of the UbcH8 protein correlated with poor outcome in NPC patients. Restored expression of UBE2L6 suppressed proliferation and colony formation in NPC cells, while inducing apoptosis. Of particular interest, we found that aberrant lipid turnover was controlled by UbcH8 in NPC through ISG15-conjugation of valosin-containing protein (VCP). Tumor tissue and NPC cell lines showed conspicuously strong accumulation of lipid droplets (LDs) compared to control nasopharyngeal epithelium and non-cancerous cell lines. We demonstrated that UbcH8 counteracts degradation of adipocyte triglyceride lipase (ATGL), a key enzyme in lipid catabolism.     

肺癌变基因表达差异cDNA微阵列的研究

背景与目的：肺癌发病率和死亡率均较高,其发病的分子机制尚不清楚。本实验旨在研究人肺癌组织、癌旁组织、正常肺组织及淋巴结转移癌组织的基因差异表达情况,寻找肺癌组织中相对高表达基因,为肺癌的早期诊断和治疗提供可能的理论依据。方法：将新鲜肺癌组织、癌旁组织、正常肺组织及淋巴结转移癌组织用液氮速冻,提取总的RNA后逆转录标记cDNA探针,与含588个基因的cDNA微阵列膜杂交,通过放射自显影、灰度扫描杂交信号强弱获得差异表达基因。结果：肺癌组织中差异表达的基因有40个,其中早期生长反应蛋白1(early growth response protein 1,EGR1)、分泌性凋亡相关蛋白1(secreted apoptosis related protein 1．SARP1)等36个基因表达上调,诱导骨髓白细胞分化蛋白1(myeloid cell leukemia protein 1,MCL1)等4个基因表达下调,以癌基因／抑癌基因、细胞周期调节、生长因子及凋亡相关基因上调为主;癌旁组织有33个差异表达基因,基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)等20个基因上调,而下调的基因有MCLI、内皮素2(endothelin 2,ET2)等13个;转移淋巴结组织有21个基因表达差异,CD40受体相关因子1(CD40 receptor-associated factor 1,CRAF1)等15个基因下调。而上调的基因仅有6个,主要以细胞粘附分子、基质金属蛋白酶、胶原蛋白等上调为主。结论：EGR1、SARP1等基因可能是肺癌变的关键基因,MMP-9、血小板反应蛋白2(thombospondin 2．TSP2)等基因可能在肺癌浸润和转移中起重要作用。     

Abnormality of the DNA double-strand-break checkpoint/repair genes, ATM, BRCA1 and TP53, in breast cancer is related to tumour grade

The role of the DNA double-strand-break (DSB) checkpoint/repair genes, ATM, BRCA1 and TP53, in sporadic breast cancer requires clarification, since ATM and BRCA1 mutations are rare in sporadic tumours. In an attempt to explain this phenomenon, we postulated that (i) in addition to genetic deletion, abnormal expression of DSB checkpoint/repair proteins might abolish the function of these genes and (ii) there might be a combined effect of individual defective genes during breast cancer pathogenesis. Using a largely homogenous group of 74 specimens of early-onset (< or =35 years of age) infiltrating ductal carcinomas, we examined associations between pathological grade and genetic deletion and/or abnormal protein expression of ATM, BRCA1 and TP53. The results showed that high-grade tumours displayed a high frequency of loss of heterozygosity (LOH) at, and/or abnormal expression of, ATM, BRCA1 and TP53. Multigenetic analysis showed abnormalities in BRCA1 to be independently associated with high-grade tumours. ATM and TP53 appeared to play an assistant role, abnormalities in these genes significantly increasing the possibility of poor differentiation in tumours with abnormalities in BRCA1. Furthermore, a higher number of abnormalities (LOH or abnormal expression) in these three genes correlated with poor tumour differentiation. Thus, this study suggests that combined changes in several DSB checkpoint/repair genes belonging to a common functional pathway are associated with breast cancer pathogenesis.     

儿童骨肉瘤中TP53突变与免疫分子基因表达的相关性分析

目的:筛选儿童骨肉瘤中突变基因,并研究其表达与免疫反应的关系。方法:从癌症基因组图谱（The Cancer Genome Atlas,TCGA）下载全部234例骨肉瘤病例数据集,从基因表达数据库（Gene Expression Omnibus,GEO）下载数据集GSE36001（19个肿瘤样本和6个正常样品）和GSE39055（37例肿瘤样本和12个正常样本）,包括基因突变数据、转录组数据和临床数据,进行生物信息学分析。收集11例骨肉瘤手术样本,实时荧光定量PCR验证临床样本中基因表达。结果:67.09%的骨肉瘤患者存在突变,其中错义突变、单核苷酸变异和C>T突变较多,突变基因排在前10位的分别为TP53、TTN、ATRX、MUC16、RB1、PCLO、RYR2、CSMD1、USH2A、MUC17。在数据集GSE36001和GSE39055中,骨肉瘤组织中TP53均过表达（P＜0.000 1）。在11例病例样本中,骨肉瘤组织中TP53表达高于正常组织（P=0.002）。TCGA中,TP53突变型表达高于TP53野生型（P=0.015）。在11例病例样本中,TP53突变型表达高于TP53野生型（P=0.038）。TCGA中,突变型TP53表达与DNA甲基化呈显著负相关（P=0.003）。在TP53突变的骨肉瘤样本中,B细胞、T细胞CD8+相关的免疫应答显著下调,CD274和CTLA4显著高表达（P＜0.05）。结论:TP53突变与骨肉瘤的发生及免疫反应密切相关,有望成为骨肉瘤诊断和治疗的作用靶点。     

Combination of microdissection and microarray analysis to identify gene expression changes between differentially located tumour cells in breast cancer

Comparison of gene expression changes between cancer cells at the periphery and in the centre of breast cancers was performed using a combination of microdissection and microarray analysis. Cancer cells from the two areas were pooled separately from five patients with ductal carcinoma in situ and separately from five patients with frankly invasive cancer. Limited total RNA, 100-200 ng, from this microdissected tissue required use of the Atlas SMART trade mark Probe Amplification Kit to synthesize and amplify cDNA and make (33)P-labelled probes. Probes were then hybridized to Atlas Human Cancer 1.2 Arrays containing 1176 known genes. Triplicate analysis revealed that 22 genes changed their expression levels in the periphery relative to the central region: 15 upregulated and seven downregulated (arbitrary threshold of 1.5-fold or greater). Differences in RNA levels were confirmed by quantitative real-time PCR for two of the genes and by changes in protein levels, detected by immunohistochemistry, for a couple of representative gene products. Thus, changes in gene expression associated with variation in microanatomical location of neoplastic cells can be detected within even small developing tumour masses.     

Gene expression changes induced by the human carcinogen aristolochic acid I in renal and hepatic tissue of mice

Aristolochic acid (AA) is the causative agent of urothelial tumors associated with AA nephropathy and is also implicated in the development of Balkan endemic nephropathy‐associated urothelial tumors. These tumors contain AA‐characteristic TP53 mutations. We examined gene expression changes in Hupki (human TP53 knock‐in) mice after treatment with aristolochic acid I (AAI) by gavage (5 mg/kg body weight). After 3, 12 and 21 days of treatment gene expression profiles were investigated using Agilent Whole Mouse 44K Genome Oligo Array. Expression profiles were significantly altered by AAI treatment in both target (kidney) and nontarget (liver) tissue. Renal pathology and DNA adduct analysis confirmed kidney as the target tissue of AAI‐induced toxicity. Gene ontology for functional analysis revealed that processes related to apoptosis, cell cycle, stress response, immune system, inflammatory response and kidney development were altered in kidney. Canonical pathway analysis indicated Nfκb, aryl hydrocarbon receptor, Tp53 and cell cycle signaling as the most important pathways modulated in kidney. Expression of Nfκb1 and other Nfκb‐target genes was confirmed by quantitative real‐time PCR (qRT‐PCR) and was consistent with the induction of Nfκb1 protein. Myc oncogene, frequently overexpressed in urothelial tumors, was upregulated by AAI on the microarrays and confirmed by qRT‐PCR and protein induction. Collectively we found that microarray gene expression analysis is a useful tool to define tissue‐specific responses in AAI‐induced toxicity. Several genes identified such as TP53, Rb1, Mdm2, Cdkn2a and Myc are frequently affected in human urothelial cancer, and may be valuable prognostic markers in future clinical studies.     

肺腺癌癌旁组织及胚胎肺的基因表达谱研究

目的 利用基因芯片技术研究肺腺癌组织、癌旁组织及胚胎肺组织中的基因表达差异。方法 分别抽取肺腺癌组织、癌旁组织和胚胎肺组织的总RNA,并纯化mRNA。采用逆转录的方法,制成cDNA链,并以两种荧光Cy5和Cy3标记后做探针,与含有1152条人类全长基因芯片进行杂交。以ScanArray 4000荧光扫描仪扫描芯片上两种荧光信号,并用计算机处理和分析。结果 4例肺腺癌组织和癌旁组织标本中,共同表达差异基因25个,其中上调基因3个,下调基因22个;胚胎肺组织和癌旁组织标本中,表达差异基因316个,其中上调基因192个,下调基因124个;胚胎肺组织和癌旁组织中表达差异基因与肺腺癌组织和癌旁组织比较,共同表达差异基因16个,其中上调基因12个,下调基因4个。结论 肺腺癌与癌旁组织共同表达差异的25个基因可能与肺癌的发生、发展有关;胚胎肺组织与癌旁组织表达差异的316个基因与生长发育环境有关;胚胎肺组织和癌旁组织与肺腺癌组织和癌旁组织共表达差异的16个基因可能与早期肺腺癌启动有关。     

Human tumor cells segregate into radiosensitivity groups that associate with ATM and TP53 status

We seek to determine whether cellular radiosensitivity in nineteen human colorectal tumor cell lines and three human glioblastoma tumor cell lines segregate into statistically distinct groups and whether such groups correlate with gene expression. We measure clonogenic survival in 22 cell lines that vary in radiosensitivity and in expression of selected genes: ATM, TP53, CDKN1A, 14-3-3σ, Ki-ras and DNA mismatch repair genes. We describe and compare radiosensitivity in these cell lines by one-parameter or two parameter analysis. Radiosensitivity varies among and between colorectal tumor cell lines and glioblastoma cell lines. When compared directly using survival, or using two-parameter analysis of radiosensitivity, cell lines distribute into four statistically-significant radiosensitivity groups. These groups associate strongly with the status of two genes, ATM and TP53, but do not associate with CDKN1A, 14-3-3σ, Ki-ras and DNA mismatch repair genes. Intrinsic cellular radiosensitivity of 22 colorectal and glioblastoma cell lines fall into four radiosensitivity groups that associate with expression of ATM and TP53. These analyses suggest multiple mechanisms underlay intrinsic cellular radiosensitivity.     

Downregulated NM23-H1 expression is associated with intracranial invasion of nasopharyngeal carcinoma

Because the focus of nasopharyngeal carcinoma (NPC) is very close to intracranial organs, it often makes incursions into cranial cavity. Identification of intracranial invasion-associated indicators will provide potential therapeutic targets for NPC patients with intracranial invasion. In this regard, Human Xpro HC-plus cancer-related gene chip was utilised to screen intracranial invasion-associated genes for NPC from the biopsied primary focus tissue samples. In all, 8 upregulated and 23 downregulated genes were obtained. VEGF165 and MMP-9, the two upregulated genes, and NM23-H1, the downregulated one, were further confirmed by immunohistochemistry, quantitative real-time PCR and western blot. Invasion-associated cellular and nude mouse models were subsequently employed to study the biological properties of NM23-H1. NM23-H1 expression was significantly lower in 5-8F cells compared with that in 6-10B cells. Moreover, patch-clamp and transwell chamber were adopted to investigate the invading potential-associated biological dynamic mechanisms in the two cell lines, and Ca(2+) current and motility were significantly elevated in 5-8F cells compared with that in 6-10B cells. Berberine, an inhibitor of Ca(2+) current, could substantially increase the expression of NM23-H1 and decrease 5-8F cell motility. The specificity of berberine on NM23-H1 and cell motility was confirmed by RNAi assay.     

cDNA Array Analysis of SPARC-modulated Changes in Glioma Gene Expression

We have demonstrated that secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas and it promotes glioma invasion and delays tumor growth in vitro and in vivo. cDNA array analyses were performed to determine whether SPARC, which interacts at the cell surface, has an impact on intracellular signaling and downstream gene expression changes, which might account for some of its effects on invasion and growth. Using a doxycycline (dox)-controlled gene expression system, two cDNA array analyses were performed using a parental U87T2 clone (–SPARC) transfected with the dox-controlled transactivator and a U87T2 parental-derived SPARC-transfected clone, A2b2 (+SPARC). Array analysis performed between the parental and the SPARC-transfected clone (–dox) identified 13 upregulated genes and 14 downregulated genes. With the exception of PAI-1 and MMP2, the identified genes are novel with respect to SPARC's mechanism of action. Array analysis performed using the SPARC-transfected clone (±dox) identified 2 types of gene regulation; one reversible upon SPARC suppression, the other irreversible. Two of the SPARC-induced genes, BIGH3 (irreversible by dox) and PAI-1 (reversible by dox) were further studied in additional SPARC-transfected clones, human astrocytoma tissues, and human glioma cell lines by RT-PCR and Northern blot analyses. The results indicate that: (1) the array results were validated, (2) the dox regulation was validated, and (3) the differential expression identified by the array analyses was present between normal brain and in human astrocytoma tissues and cell lines. Therefore, we conclude that these cDNA array analyses provide candidate genes involved in SPARC-mediated effects on glioma cell cycle progression, signaling, and migration, and that SPARC may induce reversible and irreversible gene expression changes. Further investigation of these candidates may shed insights into SPARC's role in glioma cell proliferation and invasion, and potential use as a therapeutic target.     

Mechanisms of paclitaxel-induced apoptosis in an ovarian cancer cell line and its paclitaxel-resistant clone

BACKGROUND: To understand the complicated network of paclitaxel (PTX)-induced apoptosis pathways and to elucidate mechanisms of drug resistance in ovarian cancer, we looked at PTX-induced apoptosis by using cDNA microarray. We also quantitated the changes in apoptosis-related proteins in the process of apoptosis. METHODS: An ovarian cancer cell line KF, and its PTX-resistant clone KFTX, were treated with PTX or carboplatin (CBDCA). After exposure to PTX or CBDCA, the induction of apoptosis was examined by internucleosomal DNA fragmentation. Changes in mRNA expression after 12 h of exposure to PTX were studied using cDNA microarray and RT-PCR. Changes in P53 and Bcl-2 levels were also measured over 24 h by ELISA. RESULTS: With increased doses of PTX or CBDCA, an increase in apoptosis was noted in both cell lines. cDNA microarray revealed that PTX treatment upregulated expression of caspase 1, 2, 3, 4, 6, 9, 10, their activator apaf-1, and stress reaction-related genes, gadd34, gadd153 in KF, although most of them were unchanged or downregulated in KFTX. bag-1 and hsc70 were markedly upregulated in KFTX. p53 and bcl-2 were not upregulated in either cell line. Results from protein studies also supported the cDNA microarray data. CONCLUSIONS: p53-independent mitochondrial pathways and stress-reaction-induced pathways play critical roles in PTX-induced apoptosis in ovarian cancer cells. Suppression of those pathways and upregulation of bag-1 and hsp-70 played an important role in acquiring resistance to PTX.