数据库挖掘法高通量筛选分析前列腺癌相关基因

背景与目的:发现并研究前列腺癌与正常前列腺组织差异表达的基因,不仅可帮助阐明前列腺癌分子病理学机理,而且可提供有价值的诊断及治疗的肿瘤标志物。越来越多的肿瘤基因表达信息被收集在美国癌症基因组解剖计划(CGAP)数据库,给肿瘤研究者提供了一个前所未有的机会去挖掘筛选肿瘤差异表达基因。本研究探讨利用这些公用信息及分析软件去分析挖掘前列腺癌差异表达基因的可行性及具体筛选方法。方法:通过选择合理的数据库及统计参数,利用分析软件SAGEDGED及cDNADGED获得在前列腺癌组织相对正常组织差异表达超过5倍的SAGE标签及cDNA,完成SAGE标签对基因的确认后根据我们制定的筛选原则进行标签筛选;最后通过UniGene、OMIM及Pub/Med等数据库注释候选基因的主要功能及与前列腺癌的关系。结果:通过SAGEDGED得到53条差异表达基因,其中26条为前列腺癌上调表达,27条下调表达。通过cDNADGED得到28条差异表达基因,其中15条前列腺癌上调表达,13条下调表达。结论:合理利用公用生物信息学资源,联合多数据库挖掘肿瘤相关基因是一个简单而可行的方法,可以给进一步的生物学实验以大量的提示。     

Incidentally Detected Adenocarcinoma Prostate in Transurethral Resection of Prostate Specimens: a Hospital Based Study from India

Background: Awareness about prostate cancer has increased in the community, and prostate cancer screening examinations, including prostate specific antigen (PSA) assays, are now widely available. Prior to the PSA era, up to 27% of prostate cancers were detected incidentally at the time of transurethral resection of prostate (TURP). After PSA testing became widely available, the incidence of incidentally detected carcinoma prostate in TURP specimens without prior diagnosis reduced to 5-13%. However, the incidence of incidentally detected carcinoma prostate has been reported to vary across the globe since various factors can influence the identification of this malignancy in TURP specimens. In this paper, we focus on rates of incidentally detected prostate cancer in TURP specimens in our hospital and correlate it with various parameters. Materials and Methods: This retrospective study of histopathological findings of biopsy specimens was conducted for patients undergoing TURP during a period of 5 years from April 2010. The inclusion criteria were patients diagnosed with benign prostatic hyperplasia (BPH) (digital rectal examination (DRE) not showing any abnormally hard areas and normal age adjusted PSA values). Patients with elevated PSA, abnormal DRE, documented urinary tract infection and proved adenocarcinoma prostate (CaP) were excluded from the study. The total weight of prostatectomy specimen, occurrence of carcinoma prostate in the chips, percentage of total tissue resected showing malignancy and Gleason's scores were recorded. Results: A total of 597 patients belonging to the inclusion criteria were studied. The incidence of occult CaP in the study group was 5.2 % (31/597). Out of these, 8 belonged to T1a and 23 belonged to T1b stages. The age group 70 - 79 years had the maximum incidence of occult CaP. It was observed that the clinical grading of prostate did not have a bearing on the incidence of occult CaP whereas the weight of resected specimen correlated with the incidence of CaP. The incidence of occult CaP was greater with low volume prostates ( < 20 g). (P=0.15). Conclusions: The rate of incidentally detected adenocarcinoma prostate in patients undergoing TURP for clinically diagnosed BPH was found to be only 5.2 % in our study which is low when compared with similar studies done elsewhere. The age of the patient and weight of the resected specimen correlated with incidence of occult prostate cancer. The clinical grading of prostate by DRE however, demonstrated no correlation.     

The rate of the founder Jewish mutations in BRCA1 and BRCA2 in prostate cancer patients in Israel

Inherited predisposition occurs in 5-10% of all prostate cancer (CaP) patients, but the genes involved in conferring genetic susceptibility remain largely unknown. Several lines of evidence indicate that germline mutations in BRCA1 and BRCA2 might be associated with an increased risk for CaP. Three mutations in these two genes (185delAG and 5382InsC (BRCA1) and 6174delT (BRCA2) occur in about 2.5% of the general Ashkenazi population, and the 185delAG BRCA1 mutation, in up to 1% of non-Ashkenazi Jews. In order to assess the contribution of these germline mutations to prostate cancer in Jewish Israeli patients, we tested 174 unselected prostate cancer patients (95 of Ashkenazi origin) for these mutations by PCR amplification and modified restriction enzyme digests. Patient's age range was 45-81 years (median 66), and in 24 (14.4%) the disease was diagnosed prior to 55 years of age. Nineteen (11%) and 12 (6.9%) patients had a first or second degree relative with CaP or breast cancer, respectively. Overall, five mutation carriers were detected: 2/152 (1.3%) 185delAG, 2/104 (2%) 5382InsC, and 1/158 (0.6%) 6174delT. In all carriers, the disease was diagnosed after the age of 55, and only one of them had a family history of breast and CaP. In addition, no allelic losses at the BRCA1 locus were demonstrated in 17 patients with a family history of CaP, using seven microsatellite markers. We conclude that the rate of the predominant Jewish BRCA1 and BRCA2 mutations in CaP patients does not significantly differ from that of the general population, and that mutational inactivation of the BRCA1 is rare in familial CaP. Thus, germline BRCA1 and BRCA2 mutations probably contribute little to CaP occurrence, to inherited predisposition, and to early onset disease in Jewish individuals.     

A novel biomarker for staging human prostate adenocarcinoma: overexpression of matriptase with concomitant loss of its inhibitor, hepatocyte growth factor activator inhibitor-1.

BACKGROUND: Matriptase, a type II transmembrane serine protease is involved in angiogenesis, degradation of extracellular matrix, and in the progression of some epithelial cancers. Here, we establish the clinical significance of matriptase and its inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1), during the progression of human prostate cancer (CaP). METHODS: The expression patterns of matriptase and HAI-1 were determined in primary cultures of normal human prostate epithelial (NHPE) cells, human CaP cells LNCaP, DU-145, CWR22Rnu1, and PC-3, and in tissue samples of 172 patients with normal prostate, benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and adenocarcinoma of different tumor grades. RESULTS: The protein and mRNA levels of matriptase were significantly higher in all carcinoma cells as compared with NHPE cells. Conversely, all CaP cells exhibited a reduced expression of HAI-1 as compared with NHPE cells. A progressive increase in the protein levels of matriptase was observed with increasing tumor grade in CaP specimens as compared with normal and BPH tissue specimens. Tissue samples of normal prostate exhibited a high constitutive protein level of HAI-1 compared with BPH and low-grade cancer with a progressive loss with increasing tumor grade. CONCLUSION: The increased expression of matriptase and loss of HAI-1 may be an important event during the progression of CaP in humans. We suggest that the ratio of these two gene products may serve as a promising biomarker for CaP progression and a potential marker for establishing the efficacy of therapeutic and chemopreventive interventions.     

Expression of the human cachexia-associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia-associated protein (HCAP) was recently identified from a breast cancer library based on the available 20-amino acid sequence of proteolysis-inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP-associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ-confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC-3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia.     

Is urokinase gene 3'-UTR polymorphism associated with prostate cancer?

Urokinase gene is believed to play a key role in tissue degradation and cell migration under various normal and pathological conditions, including cancer invasion and metastasis. It may be responsible in the development of prostate cancer (CaP), although there is lack of genetic evidence. Our aim was to study single nucleotide polymorphism (C/T) in 3'-untranslated region to investigate the possibility. DNA was extracted from blood samples of 103 CaP patients and 107 normal controls. Polymerase chain reaction (PCR) based restriction analysis was used to identify the C/T polymorphism of the urokinase gene. Significant difference in the frequency distribution of CT and TT genotypes in CaP patients as compared to normal was observed (p=0.04). Two folds risk for prostate cancer with T alleles in north Indian population was apparent. We also observed significant association for TT genotypes with higher Gleason score of tumors in CaP patients (p<0.05). A positive association was also evident in tobacco users having T alleles with risk of CaP. Our findings demonstrated a positive association of T allele of 3'UTR of urokinase gene with the risk of prostate cancer. We therefore hypothesize that C/T polymorphism may influence the etiology of CaP and is likely to become another new marker.     

Metallothionein is up-regulated under hypoxia and promotes the survival of human prostate cancer cells

Tumor hypoxia is a common feature of several cancers, including prostate cancer, and is associated with tumor progression, acquisition of anti-apoptotic potential and therapeutic resistance. We explored hypoxia-inducible genes and examined the effect of knockdown of a target molecule with small interference RNA (siRNA) on the proliferation of human prostate cancer cells. Human prostate cancer cell lines (LNCaP and PC-3) were cultured in normoxia (21% O2) or hypoxia (0.5% O2). Hypoxia-inducible genes were identified by cDNA microarray analysis. Metallothionein (MT) expression was assessed by real-time RT-PCR, Western blot analysis and immunohistochemical staining. siRNA was transfected to knock down MT expression, and the cell cycle and apoptosis were evaluated by flow cytometry analysis. In cDNA microarray analysis, 22 genes (including MT) were up-regulated under hypoxia. MT-1X and MT-2A were up-regulated in real-time RT-PCR. In particular, MT-2A was increased 3-fold in LNCaP and 8-fold in PC-3. The siRNA-MT-2A treatment resulted in a 20% inhibition of cell growth and induced apoptosis in both LNCaP and PC-3. In human prostate tissue, intense staining of MT was observed in cancer cells and residual cancer cells after androgen ablation therapy, while normal tissue was only stained in patches. In conclusion, MT was up-regulated under hypoxia in prostate cancer cells and overexpressed in prostate cancer tissue and residual cancer cells after androgen ablation therapy. As down-regulation of MT by siRNA inhibited cell growth and induced cell death, MT may be a new molecular target for the treatment of human prostate cancer.     

Hormone therapy failure in human prostate cancer: analysis by complementary DNA and tissue microarrays.

BACKGROUND: The molecular mechanisms underlying the progression of prostate cancer during hormonal therapy have remained poorly understood. In this study, we developed a new strategy for the identification of differentially expressed genes in hormone-refractory human prostate cancer by use of a combination of complementary DNA (cDNA) and tissue microarray technologies. METHODS: Differences in gene expression between hormone-refractory CWR22R prostate cancer xenografts (human prostate cancer transplanted into nude mice) and a xenograft of the parental, hormone-sensitive CWR22 strain were analyzed by use of cDNA microarray technology. To validate the data from cDNA microarrays on clinical prostate cancer specimens, a tissue microarray of specimens from 26 prostates with benign prostatic hyperplasia, 208 primary prostate cancers, and 30 hormone-refractory local recurrences was constructed and used for immunohistochemical detection of protein expression. RESULTS: Among 5184 genes surveyed with cDNA microarray technology, expression of 37 (0.7%) was increased more than twofold in the hormone-refractory CWR22R xenografts compared with the CWR22 xenograft; expression of 135 (2.6%) genes was reduced by more than 50%. The genes encoding insulin-like growth factor-binding protein 2 (IGFBP2) and 27-kd heat-shock protein (HSP27) were among the most consistently overexpressed genes in the CWR22R tumors. Immunohistochemical analysis of tissue microarrays demonstrated high expression of IGFBP2 protein in 100% of the hormone-refractory clinical tumors, in 36% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). Overexpression of HSP27 protein was demonstrated in 31% of the hormone-refractory tumors, in 5% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). CONCLUSIONS: The combination of cDNA and tissue microarray technologies enables rapid identification of genes associated with progression of prostate cancer to the hormone-refractory state and may facilitate analysis of the role of the encoded gene products in the pathogenesis of human prostate cancer.     

Differential gene expression profiles and identification of the genes relevant to clinicopathologic factors in colorectal cancer selected by cDNA array method in combination with principal component analysis

The clinical outcome of patients with colorectal cancer frequently varies even if they are at the same clinicopathologic stage. Alternative superior tumor markers of colorectal cancer are needed for prediction of clinical outcome. To clarify the regulatory factors in colorectal cancers, we examined differential expression profiles using cDNA macroarray technique with surgically resected specimens obtained from the patients with colorectal cancer. The gene profiles by an average-linkage hierarchical clustering analysis were found to be almost separable into two groups: tumor group and normal mucosa group. The relationship between several clinicopathologic factors and cancer related genes were investigated by using statistical analyses including principal component analysis (PCA). c-myc-binding protein MM-1, and c-jun proto-oncogene were identified as possible markers of tumor histology and clinical prognosis and early growth response protein 1 (EGR1) was selected to play an important role in progression of clinical stage. We conclude that, with PCA method, we successfully selected the genes relevant to clinicopathologic factors using limited population of clinical samples.     

基因芯片分析前列腺癌细胞系中转移相关基因

目的:通过基因芯片技术检测具有不同转移潜能的前列腺癌细胞系LNCaP和C4-2中表达差异的基因,寻找前列腺癌转移相关基因.方法:采用TRIzol一步法提取LNCaP和C4-2细胞的总RNA,并纯化mRNA,反转录合成荧光分子标记的cDNA探针,与基因芯片杂交.采用Genepix Pro 6.0图像分析软件进行芯片图像分析,把图像信号转化为数字信号,然后以差异大于等于2倍的标准来确定差异表达基因.结果:在LNCaP与C4-2细胞中,表达差异的基因共有417个,上调基因301个,下调基因116个.分析显示差异表达基因分别与细胞增殖、代谢、细胞因子、细胞转移等相关,其中发现7个基因与肿瘤细胞的转移相关,分别为EGFR、EGF、PIK3R1、Cyclin E、HYAL1、GNAI1、AZGP1.结论:筛选出LNCaP与C4-2细胞系中7个转移相关基因,为进一步研究前列腺癌转移机制奠定了基础.     

Molecular classification of green tea catechin-sensitive and green tea catechin-resistant prostate cancer in the TRAMP mice model by quantitative real-time PCR gene profiling

We previously found that human prostate cancer (CaP) progression is accompanied by differential expression of a panel of 8 informative genes, some of which are metabolically related. Gene profiling focused on this 8-gene pack by northern blot analysis in combination with standard clinical information provided reliable prognostic prediction of human CaP. For a better insight into the potential of this 8-gene signature in tumor detection/classification and therapeutic response, we determined, by qPCR, the expression of these informative genes in the TRAMP mice model of CaP progression. The 8-genes signature resulted effective in discriminating, by linear discriminant analysis (LDA), the prostate of wild type mice from transgenic TRAMP mice developing CaP (P < 0.0002). Since it is known that Green Tea Catechin (GTC) administration to TRAMP mice results in a substantial delay of CaP progression in 80% of the animals, while 20% remain unresponsive, we determined the 8-gene signature in the prostates of GTC-sensitive and GTC-resistant mice. LDA discriminated benign tissue from CaP (i.e. wild-type + chemoprevented, GTC-sensitive TRAMP mice, in which CaP progression was delayed, was discriminated from TRAMP mice + GTC-resistant TRAMP mice, in which CaP developed irrespective of GTC administration; P < 0.01). Moreover, GTC-sensitive TRAMP mice bearing CaP were discriminated from GTCs-resistant ones, (P = 0.0001). These results show that qPCR gene profiling, based on the signature of the 8-genes selected by us, could represent an appropriate means for studying the biological behavior of CaP, which may lead to identifying new tools of potential prognostic value, in that a molecular classification for the presence/absence of cancer and for discriminating GTCs-responsive from GTC-resistant CaP is provided.     

cDNA microarray analysis with amplified RNA after isolation of intact cellular RNA from neoplastic and non-neoplastic prostate tissue separated by laser microdissections

Laser microdissection is a valuable tool to prepare pure cell populations from complex tissues for further analyses. Gene expression studies by real-time RT-PCR and cDNA arrays of microdissected tissues are becoming widely used methods. The integrity and quantity of prepared RNA must be proven to ensure reliable results in subsequent applications such as quantitative RT-PCR and cDNA-arrays. In the present study we used RNAlater trade mark protected prostate tissue for laser microdissection of tumor and tumor-free tissues. RNA quality and quantity was assessed using automated capillary gel electrophoresis. By using quantitative real time-RT-PCR before and after mRNA amplification the insulin-like growth factor binding protein-3 (IGFBP-3) gene expression was shown to be down-regulated in three out of five cases and DD3 was up-regulated in cancer tissues in all cases. The up-regulation of DD3 and the down-regulation of IGFBP-3 gene expression in cancer tissue were conserved after RNA amplification. A cDNA microarray also revealed an IGFBP-3 down-regulation in cancer tissue as well as several genes known to be differerentially expressed in prostate cancer. Taken together, we present a novel method for the isolation of intact RNA from laser microdissection-derived prostate cancer tissue useful for downstream applications of real-time RT-PCR and cDNA microarrays.     

Knockin of SV40 Tag oncogene in a mouse adenocarcinoma of the prostate model demonstrates advantageous features over the transgenic model

Prostate cancer (CaP) is the most common cancer in adult men in North America. Since there is no naturally occurring prostate cancer in the mouse, preclinical studies stipulate for the establishment of a genetically manipulated mouse CaP model with features close to the human situation. In view of the limitations of transgenic technique-derived CaP models, herein we report the first application of knockin technology to establish a new mouse adenocarcinoma prostate model (PSP-KIMAP) by targeting of SV40 Tag to a prostate tissue-specific gene, PSP94 (prostate secretory protein of 94 amino acids). In order to demonstrate its novelty, we compared KIMAP to a PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP) model. The CaP development of the PSP-KIMAP mice started almost immediately after puberty at 10 weeks of age from mouse prostatic intraepithelial neoplasia (mPIN) with microinvasion to well-differentiated CaP, and demonstrated a close-to-human kinetics of prolonged tumor growth and a predominance of well and moderately differentiated tumors. The invasive nature of KIMAP model was demonstrated by multitissue metastases (lymph node, lung and liver etc) and also by immunohistochemical study of multiple invasive prostate tumor markers. PSP-KIMAP model is responsive to androgen deprivation (castration). The knockin technology in our KIMAP model demonstrates highly predictive CaP development procedures and many advantageous features, which the traditional transgenic technique-derived CaP models could not reach for both basic and clinical studies. These features include the high stability of both phenotype and genotype, highly synchronous prostate cancer development, high and precise prostate tissue targeting and with no founder line variation. The differences between the two CaP models were attributed to the introduction of a single endogenous knockin mutation, resulting in a CaP model self-regulated and controlled by a prostate gene promoter/enhancer of PSP94.