Gene ontology mapping of putative miR-224 targets which were significantly down-regulated in miR-224 expressing cells.

<p>Gene ontology mapping of putative miR-224 targets which were significantly down-regulated in miR-224 expressing cells.</p

miR-224 promotes cell proliferation <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Relative transcript expression of miR-224 (left panel) and non-specific let-7d expression (middle panel) measured using RT-qPCR and normalized against RNU48. Right panel shows the corresponding changes in cell proliferation measured with BrdU cell proliferation assay in cells transfected with either 50 nM of Control Oligos, miR-224 precursor or miR-224 precursor+inhibitor. (<b>B</b>) Colony formation from two stable clones of control (1 & 2) and two stable clones miR-224 expressing cells (A & B) grown on soft agar. Inset shows miR-224 expression and representative colony formation from the stable clones. Data in (A) and (B) were presented as Mean±SE from at least three independent experiments. (<b>C</b>) <i>In vivo</i> tumor growth from control or miR-224 expressing stable clones from (B) subcutaneously inoculated into BALB\c nude mice and plotted as tumor size versus time post subcutaneous injection. (<b>D</b>) <i>Left:</i> Average weight of tumors harvested from 4 mice each from clones 1 (control) and A (miR-224) or 3 mice each from clones 2 (control) and B (miR-224) at Day 26 post injection. Inset shows miR-224 expression in tumors from control or miR-224 expressing cells. <i>Right:</i> Photograph of representative mice inoculated with control or miR-224 expressing cells in the left and right dorsal area respectively. Data shown in (C) and (D) were presented as Mean±SD from four/three sacrificed nude mice per group respectively. * denotes p<0.05, ** denotes p<0.01 and *** denotes p<0.001.</p

Potentially Functional SNPs (pfSNPs) as Novel Genomic Predictors of 5-FU Response in Metastatic Colorectal Cancer Patients

<div><p>5-Fluorouracil (5-FU) and its pro-drug Capecitabine have been widely used in treating colorectal cancer. However, not all patients will respond to the drug, hence there is a need to develop reliable early predictive biomarkers for 5-FU response. Here, we report a novel potentially functional Single Nucleotide Polymorphism (pfSNP) approach to identify SNPs that may serve as predictive biomarkers of response to 5-FU in Chinese metastatic colorectal cancer (CRC) patients. 1547 pfSNPs and one variable number tandem repeat (VNTR) in 139 genes in 5-FU drug (both PK and PD pathway) and colorectal cancer disease pathways were examined in 2 groups of CRC patients. Shrinkage of liver metastasis measured by RECIST criteria was used as the clinical end point. Four non-responder-specific pfSNPs were found to account for 37.5% of all non-responders (P<0.0003). Five additional pfSNPs were identified from a multivariate model (AUC under ROC = 0.875) that was applied for all other pfSNPs, excluding the non-responder-specific pfSNPs. These pfSNPs, which can differentiate the other non-responders from responders, mainly reside in tumor suppressor genes or genes implicated in colorectal cancer risk. Hence, a total of 9 novel SNPs with potential functional significance may be able to distinguish non-responders from responders to 5-FU. These pfSNPs may be useful biomarkers for predicting response to 5-FU.</p></div

SMAD4 is a direct miR-224 target.

<p>(<b>A</b>) <i>Left:</i> Schematic diagram of the 3′UTR reporter constructs utilized to validate that the miR-224 targets the 3′UTR region of SMAD4, with the putative miR-224 binding sites and the mutated form highlighted in the box below. <i>Right:</i> Inhibitory effect of miR-224 on SMAD4 3′UTR examined through normalized β-galactosidase activity in cells co-transfected with Control oligos or miR-224 precursor and wild type SMAD4 3′UTR (Black bar) or mutant SMAD4 3′UTR reporter construct (White bar). (<b>B</b>) Relative SMAD4 transcript expression measured with RT-qPCR and normalized against β-actin (left panel) and SMAD4 protein expression measured by Western blotting in cells transfected with 50 nM of Control Oligos, miR-224 precursor or miR-224 precursor+inhibitor. (<b>C</b>) Relative SMAD4 transcript expression measured in the <i>in vivo</i> tumors arose from control (1 & 2) and miR-224 (A & B) stable clones expressing miR-224 (from Fig. 1D). (<b>D</b>) Relative fold change of miR-224 (Leftmost panel) and SMAD4 transcript expression (middle panel) and corresponding changes in cell proliferation measured with BrdU cell proliferation assay (rightmost panel) in cells transfected with either 50 nM of Control Oligos (white bar), miR-224 precursor (grey bar) or siRNA against SMAD4 (black bar). Inset shows the inhibition of SMAD4 protein by miR-224 precursor and siRNA against SMAD4. Data presented as Mean ± SE from three independent experiments. * denotes p<0.05, ** denotes p<0.01 and *** denotes p<0.001.</p

Methylation Profiles Reveal Distinct Subgroup of Hepatocellular Carcinoma Patients with Poor Prognosis

<div><p>Hepatocellular Carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. However, the role of epigenetic changes such as aberrant DNA methylation in hepatocarcinogenesis remains largely unclear. In this study, we examined the methylation profiles of 59 HCC patients. Using consensus hierarchical clustering with feature selection, we identified three tumor subgroups based on their methylation profiles and correlated these subgroups with clinicopathological parameters. Interestingly, one tumor subgroup is different from the other 2 subgroups and the methylation profile of this subgroup is the most distinctly different from the non-tumorous liver tissues. Significantly, this subgroup of patients was found to be associated with poor overall as well as disease-free survival. To further understand the pathways modulated by the deregulation of methylation in HCC patients, we integrated data from both the methylation as well as the gene expression profiles of these 59 HCC patients. In these patients, while 4416 CpG sites were differentially methylated between the tumors compared to the adjacent non-tumorous tissues, only 536 of these CpG sites were associated with differences in the expression of their associated genes. Pathway analysis revealed that forty-four percent of the most significant upstream regulators of these 536 genes were involved in inflammation-related NFκB pathway. These data suggest that inflammation via the NFκB pathway play an important role in modulating gene expression of HCC patients through methylation. Overall, our analysis provides an understanding on aberrant methylation profile in HCC patients.</p></div

Small oligos, Taqman assays, primer sequences, antibodies.

<p>Small oligos, Taqman assays, primer sequences, antibodies.</p

The molecular functions of the three SNPs in UMPS gene with minor allele uniquely found in non-responders are all linked to disabling UMPS.

<p>The molecular functions of the three SNPs in UMPS gene with minor allele uniquely found in non-responders are all linked to disabling UMPS.</p

The list of less-common SNPs with non-responder specific allele and their predicted molecular functions.

<p>The list of less-common SNPs with non-responder specific allele and their predicted molecular functions.</p

The demographic characteristics of the patients recruited for each study.

<p>The demographic characteristics of the patients recruited for each study.</p

Clustering analysis of 59 HCC tumors reveals 3 subgroups.

<p>(A) Consensus matrix (B) 2D hierarchical clustering of 170 probes that overlapped between 4416 differentially methylated CpG sites (between tumors and adjacent non-tumorous tissues) and 199 CpG loci that divided tumors into three subgroups. Subgroups were labeled as Group-1 (red), Group-2 (blue), Group-3 (green) and adjacent non-tumorous tissues, NT (white). Group A represents both Group-1 and Group-3, while Group B is Group-2. (C) Survival curves for the original 3 subgroups identified by CHC-FS. (D) Survival curve for Group B versus Group A. P-value was calculated by generalised Wilcoxon method. OS, overall survival; DFS, disease free survival.</p