Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs.

Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathological features of Bloom syndrome, we analyzed mRNA and microRNA (miRNA) expression in fibroblasts from individuals with Bloom syndrome and in BLM-depleted control fibroblasts. We identified mRNA and miRNA expression differences in Bloom syndrome patient and BLM-depleted cells. Differentially expressed mRNAs are connected with cell proliferation, survival, and molecular mechanisms of cancer, and differentially expressed miRNAs target genes involved in cancer and in immune function. These and additional altered functions or pathways may contribute to the proportional dwarfism, elevated cancer risk, immune dysfunction, and other features observed in Bloom syndrome individuals. BLM binds to G-quadruplex (G4) DNA, and G4 motifs were enriched at transcription start sites (TSS) and especially within first introns (false discovery rate ≤ 0.001) of differentially expressed mRNAs in Bloom syndrome compared with normal cells, suggesting that G-quadruplex structures formed at these motifs are physiologic targets for BLM. These results identify a network of mRNAs and miRNAs that may drive the pathogenesis of Bloom syndrome.

Evolutionarily conserved protein ERH controls CENP-E mRNA splicing and is required for the survival of KRAS mutant cancer cells.

Cancers with Ras mutations represent a major therapeutic problem. Recent RNAi screens have uncovered multiple nononcogene addiction pathways that are necessary for the survival of Ras mutant cells. Here, we identify the evolutionarily conserved gene enhancer of rudimentary homolog (ERH), in which depletion causes greater toxicity in cancer cells with mutations in the small GTPase KRAS compared with KRAS WT cells. ERH interacts with the spliceosome protein SNRPD3 and is required for the mRNA splicing of the mitotic motor protein CENP-E. Loss of ERH leads to loss of CENP-E and consequently, chromosome congression defects. Gene expression profiling indicates that ERH is required for the expression of multiple cell cycle genes, and the gene expression signature resulting from ERH down-regulation inversely correlates with KRAS signatures. Clinically, tumor ERH expression is inversely associated with survival of colorectal cancer patients whose tumors harbor KRAS mutations. Together, these findings identify a role of ERH in mRNA splicing and mitosis, and they provide evidence that KRAS mutant cancer cells are dependent on ERH for their survival.

Elevated HERV-K mRNA expression in PBMC is associated with a prostate cancer diagnosis particularly in older men and smokers.

Aberrant expression of subgroup k human endogenous retroviruses (HERV-K) has been observed in prostate cancer. This subgroup is unique because it encodes sequences in the human genome containing open reading frames for near intact retroviruses. We hypothesized that HERV-K reactivation could serve as a non-invasive early disease detection marker for prostate cancer. We evaluated HERV-K gag messenger RNA (mRNA) expression in blood samples of African-American and European-American men using a case-control design via quantitative real-time PCR. Additionally, we examined HERV-K envelope protein expression in prostate tumors by immunohistochemistry. HERV-K envelope protein was commonly upregulated in prostate tumors, but more so in tumors of African-American than European-American patients (61% versus 40%, P < 0.01). Examining HERV-K gag expression in peripheral blood mononuclear cells (PBMC) from 294 cases and 135 healthy men, we found that the abundance of HERV-K gag message was significantly higher in cases than controls and was associated with increased plasma interferon-γ. Men with gag expression in the highest quartile had >12-fold increased odds {odds ratio = 12.87 [95% confidence interval 6.3-26.25]} of being diagnosed with prostate cancer than those in the lowest quartile. Moreover, our results showed that HERV-K expression may perform better as a disease biomarker in older than younger men (whereas the sensitivity of prostate-specific antigen (PSA) testing decreases with age) and in men with a smoking history compared with never smokers. Combining non-invasive HERV-K testing with PSA testing may improve the efficacy of prostate cancer detection specifically among older men and smokers who tend to develop a more aggressive disease.

Germline variation in NCF4, an innate immunity gene, is associated with an increased risk of colorectal cancer.

Chronic inflammation has been implicated in the etiology of colorectal adenoma and cancer; however, few key inflammatory genes mediating this relationship have been identified. In this study, we investigated the association of germline variation in innate immunity genes in relation to the risk of colorectal neoplasia. Our study was based on the analysis of samples collected from the prostate, lung, colorectal and ovarian (PLCO) Cancer Screening Trial. We investigated the association between 196 tag single nucleotide polymorphisms (SNPs) in 20 key innate immunity genes with risk of advanced colorectal adenoma and cancer in 719 adenoma cases, 481 cancer cases and 719 controls. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CIs). After Bonferroni correction, the AG/GG genotype of rs5995355, which is upstream of NCF4, was associated with an increased risk of colorectal cancer (OR = 2.43, 95% CI = 1.73-3.39; p < 0.0001). NCF4 is part of the NAPDH complex, a key factor in biochemical pathways and the innate immune response. While not definitive, our analyses suggest that the variant allele does not affect expression of NCF4, but rather modulates activity of the NADPH complex. Additional studies on the functional consequences of rs5995355 in NCF4 may help to clarify the mechanistic link between inflammation and colorectal cancer.

High miR-21 expression from FFPE tissues is associated with poor survival and response to adjuvant chemotherapy in colon cancer.

Colon cancer (CC) is a leading cause of cancer mortality. Novel biomarkers are needed to identify CC patients at high risk of recurrence and those who may benefit from therapeutic intervention. The aim of this study is to investigate if miR-21 expression from RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue sections is associated with prognosis and therapeutic outcome for patients with CC. The expression of miR-21 was measured by quantitative reverse transcriptase-polymerase chain reaction in a Japanese cohort (stage I-IV, n = 156) and a German cohort (stage II, n = 145). High miR-21 expression in tumors was associated with poor survival in both the stage II/III Japanese (p = 0.0008) and stage II German (p = 0.047) cohorts. These associations were independent of other clinical covariates in multivariable models. Receipt of adjuvant chemotherapy was not beneficial in patients with high miR-21 in either cohort. In the Japanese cohort, high miR-21 expression was significantly associated with poor therapeutic outcome (p = 0.0001) and adjuvant therapy was associated with improved survival in patients with low miR-21 (p = 0.001). These results suggest that miR-21 is a promising biomarker to identify patients with poor prognosis and can be accurately measured in FFPE tissues. The expression of miR-21 may also identify patients who will benefit from adjuvant chemotherapy.

Circulating microRNA expression profiles associated with systemic lupus erythematosus.

OBJECTIVE: To evaluate the specificity of expression patterns of cell-free circulating microRNAs (miRNAs) in systemic lupus erythematosus (SLE). METHODS: Total RNA was purified from plasma, and 45 different specific, mature miRNAs were determined using quantitative reverse transcription-polymerase chain reaction assays. A total of 409 plasma samples were obtained from 364 different patients with SLE, healthy control subjects, and control subjects with other autoimmune diseases. The results in the primary cohort of 62 patients with SLE and 29 healthy control subjects were validated in 2 independent cohorts: a validation cohort comprising 68 patients with SLE and 68 healthy control subjects, and a disease control cohort comprising 20 patients with SLE (19 of whom were from the other validation cohort), 46 healthy control subjects, 38 patients with vasculitis, 18 patients with rheumatoid arthritis, and 20 immunosuppressed patients. RESULTS: Seven miRNAs were statistically significantly differentially expressed in plasma from patients with SLE. The expression of miRNA-142-3p (miR-142-3p) and miR-181a was increased, and the expression of miR-106a, miR-17, miR-20a, miR-203, and miR-92a was decreased. In addition, the expression of miR-342-3p, miR-223, and miR-20a was significantly decreased in SLE patients with active nephritis. A predictive model for SLE based on 2 or 4 miRNAs differentiated patients with SLE from control subjects (76% accuracy) when validated independently (P < 2 × 10(-9) ). Use of the 4-miRNA model provided highly significant differentiation between the SLE group and disease controls, except for those with vasculitis. CONCLUSION: Circulating miRNAs are systematically altered in SLE. A 4-miRNA signature was diagnostic of SLE, and a specific subset of miRNA profiles was associated with nephritis. All of the signature miRNAs target genes in the transforming growth factor ß signaling pathways. Other targets include regulation of apoptosis, cytokine-cytokine receptors, T cell development, and cytoskeletal organization. These findings highlight possible dysregulated pathways in SLE and suggest that circulating miRNA patterns distinguish SLE from other immunoinflammatory phenotypes.

Transcription signatures encoded by ultraconserved genomic regions in human prostate cancer.

BACKGROUND: Ultraconserved regions (UCR) are genomic segments of more than 200 base pairs that are evolutionarily conserved among mammalian species. They are thought to have functions as transcriptional enhancers and regulators of alternative splicing. Recently, it was shown that numerous RNAs are transcribed from these regions. These UCR-encoded transcripts (ucRNAs) were found to be expressed in a tissue- and disease-specific manner and may interfere with the function of other RNAs through RNA: RNA interactions. We hypothesized that ucRNAs have unidentified roles in the pathogenesis of human prostate cancer. In a pilot study, we examined ucRNA expression profiles in human prostate tumors. METHODS: Using a custom microarray with 962 probesets representing sense and antisense sequences for the 481 human UCRs, we examined ucRNA expression in resected, fresh-frozen human prostate tissues (57 tumors, 7 non-cancerous prostate tissues) and in cultured prostate cancer cells treated with either epigenetic drugs (the hypomethylating agent, 5-Aza 2'deoxycytidine, and the histone deacetylase inhibitor, trichostatin A) or a synthetic androgen, R1881. Expression of selected ucRNAs was also assessed by qRT-PCR and NanoString®-based assays. Because ucRNAs may function as RNAs that target protein-coding genes through direct and inhibitory RNA: RNA interactions, computational analyses were applied to identify candidate ucRNA:mRNA binding pairs. RESULTS: We observed altered ucRNA expression in prostate cancer (e.g., uc.106+, uc.477+, uc.363 + A, uc.454 + A) and found that these ucRNAs were associated with cancer development, Gleason score, and extraprostatic extension after controlling for false discovery (false discovery rate < 5% for many of the transcripts). We also identified several ucRNAs that were responsive to treatment with either epigenetic drugs or androgen (R1881). For example, experiments with LNCaP human prostate cancer cells showed that uc.287+ is induced by R1881 (P < 0.05) whereas uc.283 + A was up-regulated following treatment with combined 5-Aza 2'deoxycytidine and trichostatin A (P < 0.05). Additional computational analyses predicted RNA loop-loop interactions of 302 different sense and antisense ucRNAs with 1058 different mRNAs, inferring possible functions of ucRNAs via direct interactions with mRNAs. CONCLUSIONS: This first study of ucRNA expression in human prostate cancer indicates an altered transcript expression in the disease.

microRNA and inflammatory gene expression as prognostic marker for overall survival in esophageal squamous cell carcinoma.

MicroRNAs (miRNAs) and inflammatory genes have a role in the initiation and development of esophageal squamous cell carcinoma (ESCC). In our study, we examined the potential of using miRNA and inflammatory gene expression patterns as prognostic classifiers for ESCC. Five miRNAs and 25 inflammatory-related genes were measured by quantitative reverse transcriptase PCR in tumor tissues and adjacent noncancerous tissues from 178 Chinese patients with ESCC. The expression levels of miR-21 (p = 0.027), miR-181b (p = 0.002) and miR-146b (p = 0.021) in tumor tissue and miR-21 (p = 0.003) in noncancerous tissue were associated with overall survival of patients. These data were combined to generate a miRNA risk score that was significantly associated with worse prognosis (p = 0.0001), suggesting that these miRNAs may be useful prognostic classifiers for ESCC. To construct an inflammatory gene prognostic classifier, we divided the population into training (n = 124) and test cohorts (n = 54). The expression levels of CRY61, CTGF and IL-18 in tumor tissue and VEGF in adjacent noncancerous tissue were modestly associated with prognosis in the training cohort |Z-score| > 1.5 and were subsequently used to construct a Cox regression-based inflammatory risk score (IRS). IRS was significantly associated with survival in both the training cohort (p = 0.002) and the test cohort (p = 0.005). Furthermore, Cox regression models combining both miRNA risk score and IRS performed significantly better than models with either alone (p < 0.001 likelihood ratio test). Therefore, miRNA and inflammatory gene expression patterns, alone or in combination, have potential as prognostic classifiers for ESCC and may help to guide therapeutic decisions.

Macrophage migration inhibitory factor induces epithelial to mesenchymal transition, enhances tumor aggressiveness and predicts clinical outcome in resected pancreatic ductal adenocarcinoma.

MIF is a proinflammatory cytokine and is implicated in cancer. A higher MIF level is found in many human cancer and cancer-prone inflammatory diseases, including chronic pancreatitis and pancreatic cancer. We tested the hypothesis that MIF contributes to pancreatic cancer aggressiveness and predicts disease outcome in resected cases. Consistent with our hypothesis we found that an elevated MIF mRNA expression in tumors was significantly associated with poor outcome in resected cases. Multivariate Cox-regression analysis further showed that MIF is independently associated with patients' survival (HR = 2.26, 95% CI = 1.17-4.37, p = 0.015). Mechanistic analyses revealed that MIF overexpression decreased E-cadherin and increased vimentin mRNA and protein levels in pancreatic cancer cell lines, consistent with the features of epithelial-to-mesenchymal transition (EMT). Furthermore, MIF-overexpression significantly increased ZEB1/2 and decreased miR-200b expression, while shRNA-mediated inhibition of MIF increased E-cadherin and miR-200b expression, and reduced the expression of ZEB1/2 in Panc1 cells. Re-expression of miR-200b in MIF overexpressing cells restored the epithelial characteristics, as indicated by an increase in E-cadherin and decrease in ZEB1/2 and vimentin expression. A reduced sensitivity to the chemotherapeutic drug, gemcitabine, occurred in MIF-overexpressing cells. Indicative of an increased malignant potential, MIF over-expressing cells showed significant increase in their invasion ability in vitro, and tumor growth and metastasis in an orthotopic xenograft mouse model. These results support a role of MIF in disease aggressiveness, indicating its potential usefulness as a candidate target for designing improved treatment in pancreatic cancer.

Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer.

Circulating micro-RNA (miR) profiles have been proposed as promising diagnostic and prognostic biomarkers for cancer, including lung cancer. We have developed methods to accurately and reproducibly measure micro-RNA levels in serum and plasma. Here, we study paired serum and plasma samples from 220 patients with early stage nonsmall cell lung cancer (NSCLC) and 220 matched controls. We use qRT-PCR to measure the circulating levels of 30 different miRs that have previously been reported to be differently expressed in lung cancer tissue. Duplicate RNA extractions were performed for 10% of all samples, and micro-RNA measurements were highly correlated among those duplicates. This demonstrates high reproducibility of our assay. The expressions of miR-146b, miR-221, let-7a, miR-155, miR-17-5p, miR-27a and miR-106a were significantly reduced in the serum of NSCLC cases, while miR-29c was significantly increased. No significant differences were observed in plasma of patients compared with controls. Overall, expression levels in serum did not correlate well with levels in plasma. In secondary analyses, reduced plasma expression of let-7b was modestly associated with worse cancer-specific mortality in all patients, and reduced serum expression of miR-223 was modestly associated with cancer-specific mortality in stage IA/B patients. MiR profiles also showed considerable differences comparing African American and European Americans. In summary, we found significant differences in miR expression when comparing cases and controls and find evidence that expression of let-7b is associated with prognosis in NSCLC.

MicroRNAs and inflammation in the pathogenesis and progression of colon cancer.

There is a strong connection between inflammation, altered microRNA (miRNA) expression and colon cancer. Longstanding inflammatory bowel diseases-related colitis leads to increased risk for the development of colorectal cancer (CRC), while sporadic CRC is in part driven by the inflammatory microenvironment. This supports a causative role for inflammation in colon carcinogenesis. miRNAs are a class of small noncoding RNAs that have recently emerged as key players in both inflammation and cancer. Some miRNAs act as inflammatory mediators, others can act as either oncogenes or tumor suppressors depending on the cellular environment in which they are expressed. In particular, miR-21 is an oncogenic miRNA that has been implicated as an inflammatory mediator and may promote inflammation-associated colon carcinogenesis. miRNAs have potential as biomarkers and therapeutic targets in CRC. They are currently being evaluated as early detection biomarkers and prognostic classifiers. Polymorphisms in miRNAs and miRNA-binding sites may alter one's risk of CRC. This review will focus on the role of inflammation and miRNAs in colon carcinogenesis and discuss the potential for miRNAs and inflammatory genes to be used as biomarkers and therapeutic targets of CRC.

MiR-21 is an EGFR-regulated anti-apoptotic factor in lung cancer in never-smokers.

Fifteen percent of lung cancer cases occur in never-smokers and show characteristics that are molecularly and clinically distinct from those in smokers. Epidermal growth factor receptor (EGFR) gene mutations, which are correlated with sensitivity to EGFR-tyrosine kinase inhibitors (EGFR-TKIs), are more frequent in never-smoker lung cancers. In this study, microRNA (miRNA) expression profiling of 28 cases of never-smoker lung cancer identified aberrantly expressed miRNAs, which were much fewer than in lung cancers of smokers and included miRNAs previously identified (e.g., up-regulated miR-21) and unidentified (e.g., down-regulated miR-138) in those smoker cases. The changes in expression of some of these miRNAs, including miR-21, were more remarkable in cases with EGFR mutations than in those without these mutations. A significant correlation between phosphorylated-EGFR (p-EGFR) and miR-21 levels in lung carcinoma cell lines and the suppression of miR-21 by an EGFR-TKI, AG1478, suggest that the EGFR signaling is a pathway positively regulating miR-21 expression. In the never-smoker-derived lung adenocarcinoma cell line H3255 with mutant EGFR and high levels of p-EGFR and miR-21, antisense inhibition of miR-21 enhanced AG1478-induced apoptosis. In a never-smoker-derived adenocarcinoma cell line H441 with wild-type EGFR, the antisense miR-21 not only showed the additive effect with AG1478 but also induced apoptosis by itself. These results suggest that aberrantly increased expression of miR-21, which is enhanced further by the activated EGFR signaling pathway, plays a significant role in lung carcinogenesis in never-smokers, as well as in smokers, and is a potential therapeutic target in both EGFR-mutant and wild-type cases.

Predictive models of molecular machines involved in Caenorhabditis elegans early embryogenesis.

Although numerous fundamental aspects of development have been uncovered through the study of individual genes and proteins, system-level models are still missing for most developmental processes. The first two cell divisions of Caenorhabditis elegans embryogenesis constitute an ideal test bed for a system-level approach. Early embryogenesis, including processes such as cell division and establishment of cellular polarity, is readily amenable to large-scale functional analysis. A first step toward a system-level understanding is to provide 'first-draft' models both of the molecular assemblies involved and of the functional connections between them. Here we show that such models can be derived from an integrated gene/protein network generated from three different types of functional relationship: protein interaction, expression profiling similarity and phenotypic profiling similarity, as estimated from detailed early embryonic RNA interference phenotypes systematically recorded for hundreds of early embryogenesis genes. The topology of the integrated network suggests that C. elegans early embryogenesis is achieved through coordination of a limited set of molecular machines. We assessed the overall predictive value of such molecular machine models by dynamic localization of ten previously uncharacterized proteins within the living embryo.

Inflammation and cancer: interweaving microRNA, free radical, cytokine and p53 pathways.

Chronic inflammation and infection are major causes of cancer. There are continued improvements to our understanding of the molecular connections between inflammation and cancer. Key mediators of inflammation-induced cancer include nuclear factor kappa B, reactive oxygen and nitrogen species, inflammatory cytokines, prostaglandins and specific microRNAs. The collective activity of these mediators is largely responsible for either a pro-tumorigenic or anti-tumorigenic inflammatory response through changes in cell proliferation, cell death, cellular senescence, DNA mutation rates, DNA methylation and angiogenesis. As our understanding grows, inflammatory mediators will provide opportunities to develop novel diagnostic and therapeutic strategies. In this review, we provide a general overview of the connection between inflammation, microRNAs and cancer and highlight how our improved understanding of these connections may provide novel preventive, diagnostic and therapeutic strategies to reduce the health burden of cancer.

A small protein encoded by a putative lncRNA regulates apoptosis and tumorigenicity in human colorectal cancer cells.

Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal-tract-specific lncRNA (LINC00675) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP (FOXA1-Regulated Conserved Small Protein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity, and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells.

FDA Approval: Blinatumomab for Patients with B-cell Precursor Acute Lymphoblastic Leukemia in Morphologic Remission with Minimal Residual Disease.

On March 29, 2018, the FDA granted accelerated approval for blinatumomab (Blincyto; Amgen, Inc.) for the treatment of adults and children with B-cell precursor acute lymphoblastic leukemia (BCP ALL) in first or second complete remission with minimal residual disease (MRD) greater than or equal to 0.1%. Blinatumomab is a CD3xCD19 bispecific antibody approved previously for the treatment of relapsed or refractory BCP ALL. The basis for this accelerated approval was a single-arm trial. For the 86 patients in first and second complete remission with MRD ≥ 0.1%, conversion to MRD < 0.01% was achieved after one cycle of blinatumomab by 85.2% [95% confidence interval (CI): 73.8%, 93.0%] and 72.0% (95% CI: 50.6%, 87.9%), respectively, and the estimated median hematologic relapse-free survivals (RFS) were 35.2 months (95% CI: 0.4-53.5) and 12.3 months (95% CI: 0.7-42.3), respectively. Hematologic RFS was considered substantial independent of whether patients underwent subsequent allogeneic stem cell transplantation. The safety profile for blinatumomab was established in prior studies, and no new safety signals were observed in the new population. Cytokine release syndrome and neurotoxicity remain significant risks. The FDA is requiring confirmation of clinical benefit in a randomized trial.

Association of Epstein-Barr virus antibody levels with precancerous gastric lesions in a high-risk cohort.

We evaluated associations between Epstein-Barr virus (EBV) antibody levels and precancerous gastric lesions (chronic atrophic gastritis, intestinal metaplasia, and dysplasia) in 183 subjects from Linqu, China. Immunoglobulin G antibody titers to EBV nuclear antigen (EBNA) and viral capsid antigen (VCA) were determined by two-fold serial dilution using immunofluorescence assays. Histological progression and regression were assessed by gastroscopic examination at the time of phlebotomy and at follow up 2 years later. Antibody titers did not differ significantly among histological diagnoses determined at the time of phlebotomy. However, subjects with dysplasia at follow up had significantly higher geometric mean antibody titers for both anti-VCA and anti-EBNA. Subjects with greater than median antibody levels were more likely to progress between examinations, especially in the subgroup with intestinal metaplasia at the time of phlebotomy (odds ratios [95% confidence intervals]: 5.7 [1.6-20] for anti-EBNA >or=1:320; 3.8 [1.0-15] for anti-VCA >or=1:640). Our findings suggest a possible role for EBV reactivation at an early phase of gastric carcinogenesis.

MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma.

CONTEXT: MicroRNAs have potential as diagnostic biomarkers and therapeutic targets in cancer. No study has evaluated the association between microRNA expression patterns and colon cancer prognosis or therapeutic outcome. OBJECTIVE: To identify microRNA expression patterns associated with colon adenocarcinomas, prognosis, or therapeutic outcome. DESIGN, SETTING, AND PATIENTS: MicroRNA microarray expression profiling of tumors and paired nontumorous tissues was performed on a US test cohort of 84 patients with incident colon adenocarcinoma, recruited between 1993 and 2002. We evaluated associations with tumor status, TNM staging, survival prognosis, and response to adjuvant chemotherapy. Associations were validated in a second, independent Chinese cohort of 113 patients recruited between 1991 and 2000, using quantitative reverse transcription polymerase chain reaction assays. The final date of follow-up was December 31, 2005, for the Maryland cohort and August 16, 2004, for the Hong Kong cohort. MAIN OUTCOME MEASURES: MicroRNAs that were differentially expressed in tumors and microRNA expression patterns associated with survival using cancer-specific death as the end point. RESULTS Thirty-seven microRNAs were differentially expressed in tumors from the test cohort. Selected for validation were miR-20a, miR-21, miR-106a, miR-181b, and miR-203, and all 5 were enriched in tumors from the validation cohort (P < .001). Higher miR-21 expression was present in adenomas (P = .006) and in tumors with more advanced TNM staging (P < .001). In situ hybridization demonstrated miR-21 to be expressed at high levels in colonic carcinoma cells. The 5-year cancer-specific survival rate was 57.5% for the Maryland cohort and was 49.5% for the Hong Kong cohort. High miR-21 expression was associated with poor survival in both the training (hazard ratio, 2.5; 95% confidence interval, 1.2-5.2) and validation cohorts (hazard ratio, 2.4; 95% confidence interval, 1.4-3.9), independent of clinical covariates, including TNM staging, and was associated with a poor therapeutic outcome. CONCLUSIONS: Expression patterns of microRNAs are systematically altered in colon adenocarcinomas. High miR-21 expression is associated with poor survival and poor therapeutic outcome.

Gene clustering based on RNAi phenotypes of ovary-enriched genes in C. elegans.

Recently, a set of 766 genes that are enriched in the ovary as compared to the soma was identified by microarray analysis [1]. Here, we report a functional analysis of 98% of these genes by RNA interference (RNAi). Over half the genes tested showed at least one detectable phenotype, most commonly embryonic lethality, consistent with the expectation that ovary transcripts would be enriched for genes that are essential in basic cellular and developmental processes. We find that essential genes are more likely to be conserved and to be highly expressed in the ovary. We extend previous observations and find that fewer than the expected number of ovary-expressed essential genes are present on the X chromosome. We characterized early embryonic defects for 161 genes and used time-lapse microscopy to systematically describe the defects for each gene in terms of 47 RNAi-associated phenotypes. In this paper, we discuss the use of these data to group genes into "phenoclusters"; in the accompanying paper, we use these data as one component in the integration of different types of large-scale functional analyses. We find that phenoclusters correlate well with sequence-based functional predictions and thus may be useful in predicting functions of uncharacterized genes.