Gold-Nanoparticle-Based Colorimetric Discrimination of Cancer-Related Point Mutations with Picomolar Sensitivity

Point mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene are being increasingly recognized as important diagnostic and prognostic markers in cancer. In this work, we describe a rapid and low-cost method for the naked-eye detection of cancer-related point mutations in KRAS based on gold nanoparticles. This simple colorimetric assay is sensitive (limit of detection in the low picomolar range), instrument-free, and employs nonstringent room temperature conditions due to a combination of DNA-conjugated gold nanoparticles, a probe design which exploits cooperative hybridization for increased binding affinity, and signal enhancement on the surface of magnetic beads. Additionally, the scheme is suitable for point-of-care applications, as it combines naked-eye detection, small sample volumes, and isothermal (PCR-free) amplification

miR-342 Regulates BRCA1 Expression through Modulation of ID4 in Breast Cancer

<div><p>A miRNAs profiling on a group of familial and sporadic breast cancers showed that miRNA-342 was significantly associated with estrogen receptor (ER) levels. To investigate at functional level the role of miR-342 in the pathogenesis of breast cancer, we focused our attention on its “<i>in silico</i>” predicted putative target gene ID4, a transcription factor of the helix-loop-helix protein family whose expression is inversely correlated with that of ER. ID4 is expressed in breast cancer and can negatively regulate BRCA1 expression. Our results showed an inverse correlation between ID4 and miR-342 as well as between ID4 and BRCA1 expression. We functionally validated the interaction between ID4 and miR-342 in a reporter Luciferase system. Based on these findings, we hypothesized that regulation of ID4 mediated by miR-342 could be involved in the pathogenesis of breast cancer by downregulating BRCA1 expression. We functionally demonstrated the interactions between miR-342, ID4 and BRCA1 in a model provided by ER-negative MDA-MB-231 breast cancer cell line that presented high levels of ID4. Overexpression of miR-342 in these cells reduced ID4 and increased BRCA1 expression, supporting a possible role of this mechanism in breast cancer. In the ER-positive MCF7 and in the BRCA1-mutant HCC1937 cell lines miR-342 over-expression only reduced ID4. In the cohort of patients we studied, a correlation between miR-342 and BRCA1 expression was found in the ER-negative cases. As ER-negative cases were mainly BRCA1-mutant, we speculate that the mechanism we demonstrated could be involved in the decreased expression of BRCA1 frequently observed in non BRCA1-mutant breast cancers and could be implicated as a causal factor in part of the familial cases grouped in the heterogeneous class of non BRCA1 or BRCA2-mutant cases (BRCAx). To validate this hypothesis, the study should be extended to a larger cohort of ER-negative cases, including those belonging to the BRCAx class.</p></div

Effects of Warm Ischemic Time on Gene Expression Profiling in Colorectal Cancer Tissues and Normal Mucosa

<div><h3>Background</h3><p>Genome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures.</p> <h3>Methods</h3><p>RNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method.</p> <h3>Results</h3><p>Thirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation.</p> <h3>Conclusions</h3><p>Our results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.</p> </div

Expression of ID4 and BRCA1 in patients.

<p>(A) Negative correlation of ID4 expression and BRCA1 levels both measured by qRT-PCR analysis. The dashed line was obtained using a linear regression model. (B) Differences between delta Ct values of ID4 and BRCA1 in patients divided into hereditary (BRCA1- or BRCA2-mutant), familial (BRCAx) or sporadic groups.</p

Estimated association between BRCA1 and ID4, PGR, ERBB2, ESR1 and miR-342.

<p>The associations were estimated from regression models where BRCA1 was the outcome (y-axis) while the other genes (ID4, PGR, ERBB2, ESR1) and miR-342 were the covariates (x-axis). In the multivariate model (adjusted analysis, showed in solid lines) all the covariates and the interaction terms between ER status and miR-342 were included. The univariate models (showed in dashed lines) included only the specified gene (ID4, PGR, ERBB2, ESR1) or miR-342 as covariates. The analysis of the association of BRCA1 with ER and miR-342 was done dividing patients according to their ER status.</p

Top candidates identified in the Tumor dataset (Bonferroni P<0.05).

<p>Top candidates identified in the Tumor dataset (Bonferroni P<0.05).</p

RIN analysis.

<p>RIN distributions at each time point for both tumor (A) and normal (B) samples. A higher RIN indicate a higher RNA quality.</p

Expression of miR-342 and ID4 in patients.

<p>Negative correlation of ID4 expression measured by qRT-PCR and miR-342 levels detected by miRNA arrays. Samples are represented according to their positive/negative estrogen receptor status. The dashed line was obtained using a linear regression model.</p

Down-regulation of ID4 protein through miR-342 increases BRCA1 expression in MDA-MB-231 cells.

<p>(A) Expression of ID4 (left) and miR-342 (right) measured by qRT-PCR in five breast cancer cell lines. (B) qRT-PCR evaluation of miR-342 expression in MDA-MB-231, HCC1937 and MCF7 cells after transfection of pre-miR-342 (grey) or of a scramble oligonucleotide (black). (C) Western blotting analysis of ID4 and BRCA1 proteins on the same cells. qRT-PCR data are expressed as two elevated to –ΔCt (delta cycle threshold) value which is directly related to the expression levels. β-Actin and Vinculin were used as loading control.</p

Validation of gene expression deregulation through quantitative Real-Time PCR in NIH-3T3 transfectants.

<p>Significant up- (↑) or down- (↓) regulation obtained by microarray analysis is shown alongside quantitative Real-Time PCR fold change values for a direct comparison. n = no change.</p