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1.  Polo-like Kinase 1 regulates cell proliferation and is targeted by miR-593* in esophageal cancer 
Polo-like kinase 1 (PLK1) is overexpressed in various human cancers. However, the biological functions and the post-transcriptional regulations of PLK1 in esophageal cancer (EC) are still unknown. The purposes of this study are to determine whether PLK1 can be a molecular target of EC therapy, and to identify a microRNA targeting PLK1. We performed loss-of- and gain-of-function experiments regarding cell proliferation, cell cycle, apoptosis, in vivo tumor formation, and luciferase reporter assays, using siRNAs against PLK1 and microRNA. PLK1 protein was expressed in all 11 EC cell lines, but not in normal esophageal epithelial cells (HEEpiC). Knock-down of PLK1 in EC cells induced G2/M arrest (p<0.001) in cell cycle assay, and reduced cell proliferation (p=0.019) and tumor formation ability in vivo (p<0.0001). MiR-593*, identified as a microRNA targeting PLK1 by a database search, was less expressed especially in six EC cell lines than HEEpiC cells. Moreover, miR-593* expression level was inversely correlated with PLK1 mRNA level in 48 clinical tissue specimens of EC (p=0.006). Introduction of synthetic miR-593* suppressed PLK1 expression by 69–73%, reduced cell proliferation (p=0.008), and increased cell proportion of G2/M phase (p=0.01) in HSA/c (an EC cells), whereas a miR-593* inhibitor up-regulated PLK1 expression by 11–55%. Additionally, luciferase assay demonstrated that miR-593* interacted two binding sites in the PLK1 3′-UTR and reduced 56.8–71.5% of luciferase activity by degrading luciferase mRNA in HSA/c cells. In conclusion, PLK1 is post-transcriptionally regulated by miR-593*, and could be a promising molecular target for EC treatment.
doi:10.1002/ijc.25874
PMCID: PMC3176391  PMID: 21170987
esophageal cancer; PLK1; microRNA; miR-593*
2.  Relation Between Normal Rectal Methylation, Smoking Status, and the Presence or Absence of Colorectal Adenomas 
Cancer  2010;116(19):4495-4501.
BACKGROUND
Colorectal cancer (CRC) is 1 of the leading causes of death in the Western world. CRC develops from premalignant lesions, chiefly colorectal adenomas. Currently, the most accurate and recommended screening method for finding colorectal adenomas is colonoscopy performed on all individuals aged >50 years. However, the costs and risks associated with this procedure are relatively high. The objectives of the current study were to correlate epigenetic alterations that occur in normal rectal mucosa, smoking status, and age with the presence or absence of concomitant colorectal adenomas and to assess the potential clinical value of methylation in normal rectal biopsies as a screening assay for the presence of polyps and, hence, the need for a full colonoscopy.
METHODS
One hundred thirteen normal rectal mucosal biopsies from 113 patients were studied. DNA was extracted, modified with sodium bisulfite, and subjected to real-time quantitative, methylation-specific polymerase chain reaction analysis for the following genes: adenomatous polyposis coli (APC); cadherin 1, type 1, E-cadherin (epithelial) (CDH1); estrogen receptor 1 (ESR1); cytokine high in normal 1 (HIN1); hyperplastic polyposis protein 1 (HPP1); O-6 methylguanine-DNA methyltransferase (MGMT); neural epidermal growth factor-like 1 (NELL1); splicing factor 3B, 14-kDa subunit (p14); cyclin-dependent kinase (CDK) inhibitor 2B (inhibits CDK4) (p15); retinoic acid receptor beta (RARβ); somatostatin (SST); tachykinin, precursor 1 (TAC1); and tissue inhibitor of metalloproteinase (TIMP) metallopeptidase inhibitor 3 (TIMP3). Data were then analyzed using several proprietary software programs.
RESULTS
By using several sets of genes, clinical characteristics, and multivariate analyses, the authors developed a prediction model for the presence of concomitant colorectal adenomas at the time of rectal biopsy. They also observed strong correlations between smoking status and rectal methylation pattern and between smoking status and the presence or risk of concomitant adenomas.
CONCLUSIONS
A prediction model was developed for the presence of colorectal adenomas based on gene methylation patterns in the normal rectum. The results indicated that these genes may be involved in early stages of adenoma formation. The observed epigenetic alterations in these markers may be caused in part by the effects of smoking and/or age. Normal rectal methylation may be useful as a biomarker for narrowing the population in need of screening colonoscopy.
doi:10.1002/cncr.25348
PMCID: PMC2988654  PMID: 20572039
colorectal adenoma; methylation; smoking; normal rectum; field defect
3.  The miR-106b-25 polycistron, activated by genomic amplification, functions as an oncogene by suppressing p21 and Bim 
Gastroenterology  2009;136(5):1689-1700.
Background & Aims
Barrett’s esophagus (BE) is a highly premalignant disease that predisposes to the development of esophageal adenocarcinoma (EAC); however, the involvement of microRNAs (miRs) in BE-EAC carcinogenic progression is not known.
Methods
Esophageal cultured cells (HEEpiC, QhTRT, ChTRT, GihTRT and OE-33) and esophageal tissues (22 normal epithelia, 24 BE and 22 EAC) were studied. MiR microarrays and quantitative RT-PCR were employed to explore and verify differentially expressed miRs. Quantitative genomic PCR was performed to study genomic copy number variation at the miR-106b-25 polycistron and MCM7 gene locus on chromosome 7q22.1. In vitro cell proliferation, cell cycle, and apoptosis assays and in vivo tumorigenesis experiments were performed to elucidate biological effects of the miR-106b-25 polycistron. Western blotting and luciferase assays were performed to confirm direct mRNA targeting by miR-106b-25 polycistron.
Results
The miR-106b-25 polycistron exerted potential proliferative, anti-apoptotic, cell cycle-promoting effects in vitro and tumorigenic activity in vivo. MiRs -93 and -106b targeted and inhibited p21, while miR-25 targeted and inhibited Bim. This polycistron was upregulated progressively at successive stages of neoplasia, in association with genomic amplification and overexpression of MCM7. In addition, miRs -93 and -106b decreased p21 mRNA, while miR-25 did not alter Bim mRNA, suggesting the following discrete miR effector mechanisms: 1) for p21, mRNA degradation; 2) for Bim, translational inhibition.
Conclusions
The miR-106b-25 polycistron is activated by genomic amplification and is potentially involved in esophageal neoplastic progression and proliferation via suppression of two target genes, p21 and Bim.
PMCID: PMC2887605  PMID: 19422085
4.  A multicenter, double-blinded validation study of methylation biomarkers for progression prediction in Barrett’s esophagus 
Cancer research  2009;69(10):4112-4115.
Esophageal adenocarcinoma risk in Barrett’s esophagus (BE) is increased 30- to 125-fold versus the general population. Among all BE patients, however, neoplastic progression occurs only once per 200 patient-years. Molecular biomarkers are therefore needed to risk-stratify patients for more efficient surveillance endoscopy and to improve the early detection of progression. We therefore performed a retrospective, multicenter, double-blinded validation study of 8 BE progression prediction methylation biomarkers. Progression or nonprogression were determined at 2 years (tier 1) and 4 years (tier 2). Methylation was assayed in 145 nonprogressors (NPs) and 50 progressors (Ps) using real-time quantitative methylation-specific PCR. Ps were significantly older than NPs (70.6 vs. 62.5 years, p < 0.001). We evaluated a linear combination of the 8 markers, using coefficients from a multivariate logistic regression analysis. Areas under the ROC curve (AUCs) were high in the 2-, 4-year and combined data models (0.843, 0.829 and 0.840; p<0.001, p<0.001 and p<0.001, respectively). In addition, even after rigorous overfitting correction, the incremental AUCs contributed by panels based on the 8 markers plus age vs. age alone were substantial (Δ-AUC = 0.152, 0.114 and 0.118, respectively) in all three models. A methylation biomarker-based panel to predict neoplastic progression in BE has potential clinical value in improving both the efficiency of surveillance endoscopy and the early detection of neoplasia.
doi:10.1158/0008-5472.CAN-09-0028
PMCID: PMC2752375  PMID: 19435894
5.  Rarity of Somatic Mutation and Frequency of Normal Sequence Variation Detected in Sporadic Colon Adenocarcinoma Using High-Throughput cDNA Sequencing 
We performed high-throughput cDNA sequencing in colorectal adenocarcinoma and matching normal colorectal epithelium. All six hundred three genes in the UCSC database that were expressed in colon cancers and contained open reading frames of 1000 nucleotides or less were selected for study (total basepairs/bp, 366,686). 304,350 of these 366,686 bp (83.0%) were amplified and sequenced successfully. Seventy-eight sequence variants present in germline (i.e. normal) as well as matching somatic (i.e. tumor) DNA were discovered, yielding a frequency of 1 variant per 3,902 bp. Fifty-one of these sequence variants were homozygous (26 synonymous, 25 non-synonymous), while 27 were heterozygous (11 synonymous, 16 non-synonymous). Cancer tissue contained only one sequence-altered allele of the gene ATP50, which was present heterozygously alongside the wild-type allele in matching normal epithelium. Despite this relatively large number of bp and genes sequenced, no somatic mutations unique to tumor were found. High-throughput cDNA sequencing is a practical approach for detecting novel sequence variations and alterations in human tumors, such as those of the colon.
PMCID: PMC2287164  PMID: 18389087
6.  Screening for Microsatellite Instability Identifies Frequent 3′-Untranslated Region Mutation of the RB1-Inducible Coiled-Coil 1 Gene in Colon Tumors 
PLoS ONE  2009;4(11):e7715.
Background
Coding region microsatellite instability (MSI) results in loss of gene products and promotion of microsatellite-unstable (MSI-H) carcinogenesis. Recent studies have indicated that MSI within 3′-untranslated regions (3′UTRs) may post-transcriptionally dysregulate gene products. Within this context, we conducted a broad mutational survey of 42 short 3′UTR microsatellites (MSs) in 45 MSI-H colorectal tumors and their corresponding normal colonic mucosae.
Methodology/Principal Findings
In order to estimate the overall susceptibility of MSs to MSI in MSI-H tumors, the observed MSI frequency of each MS was correlated with its length, interspecies sequence conservation level, and distance from some genetic elements (i.e., stop codon, polyA signal, and microRNA binding sites). All MSs were stable in normal colonic mucosae. The MSI frequency at each MS in MSI-H tumors was independent of sequence conservation level and distance from other genetic elements. In contrast, MS length correlated significantly with MSI frequency in MSI-H tumors (r = 0.86, p = 7.2×10−13). 3′UTR MSs demonstrated MSI frequencies in MSI-H tumors higher than the 99% upper limit predicted by MS length for RB1-inducible coiled-coil 1(RB1CC1, mutation frequency 68.4%), NUAK family SNF1-like kinase 1(NUAK1, 31.0%), and Rtf1, Paf1/RNA polymerase II complex component, homolog (RTF1, 25.0%). An in silico prediction of RNA structure alterations was conducted for these MSI events to gauge their likelihood of affecting post-transcriptional regulation. RB1CC1 mutant was predicted to lose a microRNA-accessible loop structure at a putative binding site for the tumor-suppressive microRNA, miR-138. In contrast, the predicted 3′UTR structural change was minimal for NUAK1- and RTF1 mutants. Notably, real-time quantitative RT-PCR analysis revealed significant RB1CC1 mRNA overexpression vs. normal colonic mucosae in MSI-H cancers manifesting RB1CC1 3′UTR MSI (9.0-fold; p = 3.6×10−4).
Conclusions
This mutational survey of well-characterized short 3′UTR MSs confirms that MSI incidence in MSI-H colorectal tumors correlates with MS length, but not with sequence conservation level or distance from other genetic elements. This study also identifies RB1CC1 as a novel target of frequent mutation and aberrant upregulation in MSI-H colorectal tumors. The predicted loss of a microRNA-accessible structure in mutant RB1CC1 RNA fits the hypothesis that 3′UTR MSI involves in aberrant RB1CC1 posttranscriptional upregulation. Further direct assessments are indicated to investigate this possibility.
doi:10.1371/journal.pone.0007715
PMCID: PMC2766054  PMID: 19888451
7.  Rarity of Somatic Mutation and Frequency of Normal Sequence Variation Detected in Sporadic Colon Adenocarcinoma Using High-Throughput cDNA Sequencing 
We performed high-throughput cDNA sequencing in colorectal adenocarcinoma and matching normal colorectal epithelium. All six hundred three genes in the UCSC database that were expressed in colon cancers and contained open reading frames of 1000 nucleotides or less were selected for study (total basepairs/bp, 366,686). 304,350 of these 366,686 bp (83.0%) were amplified and sequenced successfully. Seventy-eight sequence variants present in germline (i.e. normal) as well as matching somatic (i.e. tumor) DNA were discovered, yielding a frequency of 1 variant per 3,902 bp. Fifty-one of these sequence variants were homozygous (26 synonymous, 25 non-synonymous), while 27 were heterozygous (11 synonymous, 16 non-synonymous). Cancer tissue contained only one sequence-altered allele of the gene ATP50, which was present heterozygously alongside the wild-type allele in matching normal epithelium. Despite this relatively large number of bp and genes sequenced, no somatic mutations unique to tumor were found. High-throughput cDNA sequencing is a practical approach for detecting novel sequence variations and alterations in human tumors, such as those of the colon.
PMCID: PMC2287164  PMID: 18389087

Results 1-7 (7)