Analysis of RIN1 gene expression in colorectal cancer

The RIN1 gene, located on chromosome 11q13.2, is a molecule consisting of a coding region of 2352 bp, has a domain on the 3' side that binds to H-Ras protein, and is presumed to be an important molecule in an intracellular signaling pathway. Since the RIN1 molecule belonging to the effector molecules of H-Ras has not been reported in colorectal or other digestive tract cancers to date, we investigated how the RIN1 gene was involved in colorectal cancer. Fifty-two (51.5%) of 101 colorectal cancer specimens strongly expressed the RIN1 gene compared to the adjacent normal colorectal tissue. The 5-year survival rate of patients positive for the expression of the RIN1 gene was significantly poorer at 55% than that (83%) of patients negative for the expression of the RIN1 gene. Also, we confirmed that RIN1 protein was localized chiefly in the cytoplasm of colorectal cancer cell lines, and bound to 14-3-3 protein, but not to Ras protein. These results indicate that the RIN1 gene serves as an important signal transduction system for evaluating the malignancy of colorectal cancer.     

胃癌细胞周期基因表达谱的变化

目的 检测胃癌MKN45细胞周期基因表达谱的变化,从基因组学角度阐明胃癌细胞增殖的机制。方法 应用双胸腺嘧啶核苷法和胸腺嘧啶核苷-偌考达唑法,分别阻断胃癌细胞株MKN45于G2／M和G1／S交界点后释放;流式细胞仪监测细胞同步化程度;cDNA基因芯片检测细胞周期中G2／M交界点、M／G1过渡期、G1早期、G1晚期、G1／S交界点、S早期、S晚期、G2早期和G2末期的基因表达谱,专业软件进行聚类分析;借助基因数据库,重点对MKN45细胞周期G1末期及G2期上调基因表达进行分析。定量PCR测定cyclinE、cyclinB、plkl、STKl5基因在上述9个时间点的mRNA水平变化。结果 分析基因芯片检测,得到9个时间点均可信的2001个基因,其中959个基因出现改变（上调或下调）,在G1末期或G2期上调基因379个,S期和M期上调基因40个（与G1末期和G2期上调基因重合）,在G1末期上调基因中,功能主要涉及DNA代谢、转录与翻译、蛋白质转运、泛素化和信号转导等;G2期上调基因主要涉及RNA合成与加工、蛋白质转运、细胞骨架合成、凋亡与抑凋亡、转录调节、泛素化、信号转导、有丝分裂调节以及癌基因的表达等。定量PCR检测4个基因的mRNA水平变化趋势,与基因芯片检测结果基本一致。结论 在MKN45细胞周期演进中,DNA复制及染色体分离所需的各种物质准备分别在G1末期及G2期完成,这种准备涉及多种类基因,成为推动MKN45细胞周期进程的主要动力,其中部分基因可能与肿瘤的过度增殖有关。基因芯片检测结果具有较好的可信度,为今后胃癌细胞周期相关基因功能研究提供了帮助。     

Heterogeneous metastasis efficiency of isogenic orthotopic colon cancer xenografts reveals distinctive gene expression profiles.

Hepatic and lung metastases are the leading causes of mortality and major indicators of aggressiveness in colorectal cancer. The underlying molecular mechanisms contributing to the development of metastasis are still unclear. Here, we designed a novel approach to explore gene expression profiles associated with metastasis in human colorectal cancer (hCRC). A series of ten isogenic tumors from three different hCRC models were orthotopically implanted into nude mice. In these series, we analyzed the contribution of dynamic heterogeneity, independently of any intrinsic gene expression program predictive of metastasis. When screened for the presence of disseminated tumor cells in the lung and liver, as the most common host tissues for hCRC metastases, both high- and low-metastatic efficient tumors were found among these isogenic orthotopic series. The metastasis-specific cDNA macroarray analysis of 96 genes, in both tumor populations for each of the three hCRC models, characterized a common differential gene expression within a small group of genes. Our results suggest that, independently of a gene expression profile predictive of metastasis, the progressive acquisition of additional alterations occurs during hCRC tumorigenesis. This dynamic process might determine tumor progression, namely the metastasis dissemination.     

Circulating miRNA expression over the course of colorectal cancer treatment

Colorectal cancer (CRC) is the third-most common cancer type in males and the second-most common cancer type in females, and has the second-highest overall mortality rate worldwide. Approximately 50% of patients in stage I–III develop metastases, mostly localized to the liver. All physiological conditions occurring in the organism are also reflected in the levels of circulating microRNAs (miRNAs/miRs) in patients. miRNAs are a class of small, non-coding, single-stranded RNAs consisting of 18–25 nucleotides, which have important roles in various cellular processes. The aim of the present study was to evaluate a panel of seven circulating miRNAs (miR-106a-5p, miR-210-5p, miR-155-5p, miR-21-5p, miR-103a-3p, miR-191-5p and miR-16-5p) as biomarkers for monitoring patients undergoing adjuvant treatment of CRC. Total RNA was extracted from the plasma of patients with CRC prior to surgery, in the early post-operative period (n=60) and 3 months after surgery (n=14). The levels of the selected circulating miRNAs were measured with the miRCURY LNA miRNA PCR system and fold changes were calculated using the standard ∆∆Cq method. DIANA-miRPath analysis was used to evaluate the role of significantly deregulated miRNAs. The results indicated significant upregulation of miR-155-5p, miR-21-5p and miR-191-5p, and downregulation of miR-16-5p directly after the surgery. In paired follow-up samples, the most significant upregulation was detected for miR-106a-5p and miR-16-5p, and the most significant downregulation was for miR-21-5p. Pathway analysis outlined the role of the differentially expressed miRNAs in cancer development, but the same pathways are also involved in wound healing and regeneration of intestinal epithelium. It may be suggested that these processes should also be considered in studies investigating sensitive and easily detectable circulating biomarkers for recurrence in patients.     

RNA sequencing reveals the expression profiles of circRNA and indicates that circDDX17 acts as a tumor suppressor in colorectal cancer

Background

Circular RNA (circRNA) is a novel class of noncoding RNAs with functions in various pathophysiological activities. However, the expression profiles and functions of circRNAs in colorectal cancer (CRC) remain largely unknown.

Methods

High-throughput RNA sequencing (RNA-seq) was performed to assess circRNA expression profiles in 4 paired CRC tissues, and significantly dysregulated circRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict the potential functions of dysregulated circRNAs. Target miRNAs of circRNAs were predicted using miRanda software, and were further analyzed combining DIANA-miRPath v.3 platform (Reverse Search module) with KEGG pathways of COLORECTAL CANCER and MicroRNAs in cancer (Entry: map05210 and map05206). CircRNA-miRNA interaction networks were constructed using Cytoscape software. Expression levels of a significantly down-regulated circRNA, circDDX17 (hsa_circ_0002211), was detected by qRT-PCR in 60 paired CRC tissues. CircDDX17 was knockdown by siRNA, and the biological functions of circDDX17 were examined in CRC cell lines.

Results

Totally 448 differentially expressed circRNAs were identified, including 394 up-regulated and 54 down-regulated circRNAs. qRT-PCR validation confirmed the reliability of the RNA-Seq data. GO and KEGG analyses revealed that these dysregulated circRNAs were potentially implicated in CRC pathogenesis. Analyses by combining miRanda and miRPath softwares with KEGG pathways suggested that the miRNAs targeted by the top 10 dysregulated circRNAs were associated with the KEGG pathways of COLORECTAL CANCER and MicroRNAs in cancer, indicating that circRNA-miRNA interactions might play important functional roles in the initiation and progression of CRC. The results of qRT-PCR for circDDX17 in 60 paired CRC tissues showed that circDDX17 was significantly down-regulated in CRC tissues and associated with unfavorable clinicopathological parameters. In vitro experiments showed that silencing of circDDX17 promoted CRC cell proliferation, migration, invasion, and inhibited apoptosis.

Conclusions

In conclusion, we have identified numerous circRNAs that are dysregulated in CRC tissues compared with adjacent normal mucosa tissues. Bioinformatic analyses suggested that these dysregulated circRNAs might play important functional roles in CRC tumorigenesis. CircDDX17 functions as a tumor suppressor and could serve as a potential biomarker and a therapeutic target for CRC.

     

cDNA expression array reveals heterogeneous gene expression profiles in three glioblastoma cell lines.

Tumor cell lines are an indispensable tool for cancer research. However, among cell lines of the same pathological group, heterogeneity has been detected in gene expression, gene mutation, and cellular response to various treatments. In this study, we systematically investigated the extent of heterogeneity of gene expression in three glioblastoma cell lines using cDNA array technology in which the expression of 588 cellular genes is studied simultaneously. Comparison of the expression profiles revealed substantial qualitative and quantitative heterogeneity. Among the 588 genes, 197 genes were expressed in all three lines and 56 genes were not expressed in any of the three lines; total of 222 genes were expressed in only two of the three cell lines, and 113 genes were expressed in only one of the three cell lines. These results provide molecular evidence that cell lines of the same pathological origin can be highly heterogeneous.     

Functional analysis of the gene expression profiles of colorectal cancer cell lines in relation to oxaliplatin and cisplatin cytotoxicity

The objective was to relate the gene expression profiles of colorectal cancer cells in culture to the in vitro cytotoxicity of cisplatin and oxaliplatin. We studied the gene expression profiles of six human colorectal cancer cell lines, using the Atlas Plastic Human 8K Microarray from Clontech, and related it to the in vitro cytotoxicities of oxaliplatin and cisplatin obtained by inhibition of exponential growth of cells. We calculated the Pearson's coefficients of correlation (r) between gene expression and drug IC50. A functional analysis was performed using the Gene Ontology Consortium database. Results were validated on a series of representative genes by real-time quantitative PCR. Validation of the significance of the coefficients of correlation was also performed using a leave-one-out analysis. We identified 394 genes whose expression was significantly correlated (P<0.05) to oxaliplatin cytotoxicity and 40 with cisplatin cytotoxicity. Three major functions were preferentially involved in oxaliplatin activity: protein synthesis, cell energetics and response to oxidative stress. No significant correlation was observed between oxaliplatin or cisplatin cytotoxicity and the expression of genes involved in DNA repair, cell proliferation or cell adhesion. A strongly significant correlation was found between the microarray and the rt-PCR approaches (r=0.968, P<10(-6)). The leave-one-out analysis showed that the same functions still appeared significantly involved in the activity of both drugs. Based on the functional analysis, we hypothesized that oxaliplatin would specifically form protein adducts during synthesis, thus exposing their thiol groups, which are known to be especially vulnerable to reactive oxygen species.     

Correlation of c-MET expression with clinical characteristics and the prognosis of colorectal cancer

BackgroundThe proto-oncogene c-MET (mesenchymal-epithelial transition factor gene) plays a critical role in cellular proliferation, survival, migration, and invasion in cancers. The aim of this study is to explore the relationship between c-MET expression and the clinicopathological characteristics of colorectal cancer (CRC) patients.MethodsA total of 337 enrolled patients were collected in present study. Here, the c-MET and EGFR expression were detected by immunohistochemistry (IHC). The mutational statuses of KRAS in exons 2, 3, and 4, NRAS in exons 2, 3, and 4, and BRAF in exon 15 from formalin-fixed sections were detected by direct DNA sequencing.ResultsOur results showed that high c-MET expression was significantly associated with tumor perineural invasion (P=0.007) and gender (P=0.016). High level c-MET expression (c-MET-high) in the primary tumors was observed in 68.2% of patients. In the 337 enrolled patients, 43.2% of patients had KRAS mutations, 3.3% of patients had NRAS mutations, and 4.7% of patients had BRAF mutations. However, KRAS, NRAS, and BRAF gene mutations had no association with c-MET protein levels in primary tumors. Additionally, c-MET protein expression had a strong correlation with EGFR expression (P=0.002). The survival time was not significantly longer for patients with c-MET-high primary tumors than for those with c-MET-low primary tumors.Conclusionsc-MET immunohistochemistry was significantly higher in primary tumors with perineural invasion, female gender, and EGFR high expression. However, c-MET-high in the primary tumors was not significantly associated with longer survival compared with c-MET-low tumors. Further studies are required to investigate c-MET as potential molecular marker of progression and to test the possibility of its incorporation as a new therapeutic target.     

大肠癌组织及结肠癌细胞系中FHIT基因的异常表达

目的：探讨ＦＨＩＴ基因与大肠癌的关系。方法：采用ＲＴ－ＰＣＲ及ＰＣＲ产物直接测序法,对３０例大肠癌组织及其配对正常组织,４个结肠癌细胞系的ＦＨＩＴ基因进行检测。结果：在８例（２６．７％）大肠癌组织和２个（５０％）结肠癌细胞系中检测到异常ＦＨＩＴ转录本,配对正常组织均无异常ＦＨＩＴ转录本;测序证实异常转录本缺失１￣３个ＦＨＩＴ外显子。异常ＦＨＩＴ转录本与大肠癌患者的年龄,性别、血清ＣＥＡ水平、肿瘤部     

原位杂交检测新基因ST13（SNC6）在大肠癌中的表达及临床意义

目的 确定新基因ST13在大肠癌中的组织学定位,观察它在大肠癌发生、发展不同阶段的表达情况,探讨其表达与大肠癌临床病理特征的关系。方法 用RNA－RNA原位杂交的方法检测ST13基因在30例大肠癌病人的正常肠黏膜、癌旁黏膜、腺瘤以及癌组织中的表达状况。结果 ST13基因表达在大肠的正常腺上皮结构上,着色颗粒位于胞浆,而胞核无或着色较淡,癌组织中没有或有少量表达,且一般较正常黏膜染色为淡;在间质和肌层中未见明显阳性信号。正常黏膜标本中阳性率为20／30;癌旁黏膜中15／30;腺瘤中4／4;癌组织中9／30。ST13基因在同一大肠癌病人正常黏膜和癌组织中表达之间存在显著性差异（P＜0．01）。在癌组织中ST13有表达和无表达病例组之间Dukes各期分布有差别,有表达者中属DukesA期和B期为多,C期为少;而无表达者中C期较多,A、B期为少,但差异无显著性（P＝0．052）。同时,还发现有淋巴结转移者ST13在癌组织中表达阳性率（3／17）低于无淋巴结转移者（6／13）,但差异无显著性（P＝0．099）。结论 ST13基因在大肠癌中的表达率低于相应的正常黏膜,此种低表达多见于Dukes C期及淋巴结转移者,ST13基因可能在大肠癌的发生、发展中起作用。     

Proto-oncogene abnormalities in human breast cancer: correlations with anatomic features and clinical course of disease

DNAs from fifty-three primary breast cancers were hybridized with 16 different proto-oncogene or oncogene probes. Abnormalities of one or more of five proto-oncogenes were found in fifty-eight percent of tumors at the time of mastectomy. Amplification of c-myc and c-erbB-2, and allelic deletions of c-ras-Ha and c-myb were the most common abnormalities. The presence of altered proto-oncogenes correlated with clinical stage of the cancers. Fifteen of 43 evaluable tumors of stages I to III recurred, and four of five evaluable stage IV tumors progressed within 16 to 24 months of surgery. All but one of the cancers that recurred or progressed had detectably altered proto-oncogenes (P less than .001). Analysis of proto-oncogenes may have prognostic value in breast cancer.     

Mass spectrometry protein expression profiles in colorectal cancer tissue associated with clinico-pathological features of disease

Background  

Studies of several tumour types have shown that expression profiling of cellular protein extracted from surgical tissue specimens by direct mass spectrometry analysis can accurately discriminate tumour from normal tissue and in some cases can sub-classify disease. We have evaluated the potential value of this approach to classify various clinico-pathological features in colorectal cancer by employing matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS).     

Long noncoding RNA profiles identify five distinct molecular subtypes of colorectal cancer with clinical relevance

Colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behavior and response to therapy. Increasing evidence suggests that long noncoding RNAs (lncRNAs) are frequently aberrantly expressed in cancers, and some of them have been implicated in CRC biogenesis and prognosis. Using an lncRNA-mining approach, we constructed lncRNAs expression profiles in approximately 888 CRC samples. By applying unsupervised consensus clustering to LncRNA expression profiles, we identified five distinct molecular subtypes of CRC with different biological pathways and phenotypically distinct in their clinical outcome in both univariate and multivariate analysis. The prognostic significance of the lncRNA-based classifier was confirmed in independent patient cohorts. Further analysis revealed that most of the signature lncRNAs positively correlated with somatic copy number alterations (SCNAs). This lncRNAs-based classification schema thus provides a molecular classification applicable to individual tumors that has implications to influence treatment decisions.