JCV miR-J1-5p detection in feces.

<p>(A) To test whether JCV miRNA is present in stool, we extracted total RNA from stool samples and performed TaqMan based miRNA expression analyses. Expression of miR-J1-5p was normalized to mean miR-16 and -26b levels and further adjusted to the sample with the lowest miR-J1-5p expression level (1*). (B) To test the reproducibility of miRNA detection, we performed independent RNA extraction from the same samples in the subset of fecal samples from healthy subjects (n = 5). The samples were normalized to mean miR-16 and -26b expression. (C) Concomitant expression analyses of miR-J1-5p and -3p showed no correlation with JCV miRNA expression, arguing for potential cross-reactivity with BKV microRNA. (D&E) To measure JCV miR-J1-5p expression in feces from CRC patients, miR-J1-5p was analyzed by TaqMan PCR in 29 FOBT specimens from patients without and with colorectal neoplasia. Fold-expression was calculated using the 2^−ΔCt method normalized to mean miR-16 and -26b expression. D Represents the single sample values and E the mean values ± SD.</p

Clinico-pathological characteristics of patients and healthy volunteers in the study.

<p>*Clinical characteristics of one patient was incomplete.</p

JCV miR-J1-5p detection in CRC patient tissues.

<p>(A) Six FFPE tissue specimens were stained for JCV T-Ag expression to ensure the active presence of JCV in CRC tissues. Figure A1 shows a representative image for strong, and A2 shows weak JCV-T-Ag protein expression. (B) JCV miR-J1-5p expression was evaluated in each of 3 samples with strong and weak JCV T-Ag expression. Normalization of miR-J1-5p expression in FFPE tissues was performed using miR-16, as previously validated. (C & D) miR-J1-5p expression was evaluated in paired normal colonic mucosa and CRC fresh frozen tissues from 21 patients with CRC. In C, miR-J1-5p expression is shown for paired normal colonic mucosa and CRC tissues. miRNA expression is shown as 2^−ΔCt normalized to RNU6b expression. (E) miR-J1-5p expression in CRC tissues is shown correlated with miR-J1-5p expression in normal colon mucosa. The results are presented as 2^−ΔΔCt normalized to RNU6b and matching normal colonic mucosa, and the values are sorted in descending order. From a total of 21 CRC tissues samples, 12 samples (below the line) showed lower, and 6 samples (above the line) higher miR-J1-5p expression in CRC tissues compared to normal mucosa.</p

JCV miRNA sequence and detection.

<p>(A) Schematic presentation of the JCV genome. The black circle marks the transcript location of the JCV miR-J1 stem loop. (B) JCV miR-J1-5p and -3p sequences are compared to the Merkel Cell Polyomavirus-, SV40- and BK virus-miRNA sequences. (C & D) CRC cells were transfected in vitro with a JCVT-Ag-E plasmid, and JCV T-Ag message and miRNA expression were analyzed. In C, GAPDH and β-actin were used as loading controls for mRNA and protein expression, respectively. (D) Vector transfected cells showed no detectable miR-J1-5p expression, while JCV miR-J1-5p expression was high in transfected cells. To measure the expression of miR-J1-5p, expression in the vector was set to a Ct-value of 40, and 2^−ΔΔCt values were calculated using RNU6b for normalization.</p

The Clinical Significance of MiR-148a as a Predictive Biomarker in Patients with Advanced Colorectal Cancer

<div><h3>Aim</h3><p>Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies.</p> <h3>Methods</h3><p>We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival.</p> <h3>Results</h3><p>Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93).</p> <h3>Discussion</h3><p>MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy.</p> </div

Univariate and multivariate analysis of miR-148a expression, methylation and overall survival in stage IV MSS CRC patients.

a<p>p<0.05.</p

Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

<div><p>Aim</p><p>Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells.</p> <p>Materials and Methods</p><p>To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR.</p> <p>Results</p><p>As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines.</p> <p>Conclusions</p><p>Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.</p> </div

Associations between miR-148a status and therapeutic response or survival in stage IV CRC patients treated with 5-FU and oxaliplatin.

<p><b>A</b>. Therapeutic response according to miR-148a expression. Complete response, CR; partial response, PR; stable disease, SD; progressive disease; PD. <b>B</b>. Kaplan-Meyer curves for progression-free survival (PFS, left panel) and OS (right panel) in stage IV patients according to miR-148a expression. <b>C</b>. Kaplan-Meyer curves for PFS (left panel) and OS (right panel) in stage IV patients according to miR-148a methylation.</p

Functional categories of curcumin mediated genes in HCT116 colon cancer cells.

<p>HCT116 CRC cells were treated with curcumin for 240 days. Infinium genome-wide methylation analyses and Illumina gene expression analyses were performed to access the curcumin-mediated changes. Matching genes with curcumin-mediated methylation changes of β-value ≥0.1 and genes with ≥1.5-fold difference in gene expression were used for Ingenuity Pathway Analyses.</p

Curcumin-mediated changes in DNA methylation differ among 5-aza-CdR treated cells.

<p>The validated CpG loci of the cell lines were ordered in ascending order. The figure represents the magnitude and the location of curcumin-affected CpG loci in relation to control cell lines. The gray curve represents the distribution of the CpG loci in ascending order. The black lines represent the Δβ-values_{(βcontrol–βtreatment)} matching the control cells. The treatment with curcumin was associated with both hyper- and hypomethylation changes, predominantly in partially-methylated CpG loci, while 5-azaCdR treatment was responsible for non-selective hypomethylation.</p