Deregulated miRNAs in Hereditary Breast Cancer Revealed a Role for miR-30c in Regulating KRAS Oncogene

<div><p>Aberrant miRNA expression has been previously established in breast cancer and has clinical relevance. However, no studies so far have defined miRNAs deregulated in hereditary breast tumors. In this study we investigated the role of miRNAs in hereditary breast tumors comparing with normal breast tissue. Global miRNA expression profiling using Exiqon microarrays was performed on 22 hereditary breast tumors and 15 non-tumoral breast tissues. We identified 19 miRNAs differentially expressed, most of them down-regulated in tumors. An important proportion of deregulated miRNAs in hereditary tumors were previously identified commonly deregulated in sporadic breast tumors. Under-expression of these miRNAs was validated by qRT-PCR in additional 18 sporadic breast tumors and their normal breast tissue counterparts. Pathway enrichment analysis revealed that deregulated miRNAs collectively targeted a number of genes belonging to signaling pathways such as MAPK, ErbB, mTOR, and those regulating cell motility or adhesion. <em>In silico</em> prediction detected KRAS oncogene as target of several deregulated miRNAs. In particular, we experimentally validated KRAS as a miR-30c target. Luciferase assays confirmed that miR-30c binds the 3′UTR of KRAS transcripts and expression of pre-miR-30c down-regulated KRAS mRNA and protein. Furthermore, miR-30c overexpression inhibited proliferation of breast cancer cells. Our results identify miRNAs associated to hereditary breast cancer, as well as miRNAs commonly miss-expressed in hereditary and sporadic tumors, suggesting common underlying mechanisms of tumor progression. In addition, we provide evidence that KRAS is a target of miR-30c, and that this miRNA suppresses breast cancer cell growth potentially through inhibition of KRAS signaling.</p> </div

Differentially expressed miRNAs between normal breast tissue and hereditary breast tumors.

<p>Heat map of the expression of the 19 miRNAs differentially expressed between normal samples and tumors, overexpression in red, lower expression in green. Expression of these miRNAs in Human Mammary Epithelial Cells (HMEC) cells is also represented.</p

Shared differentially expressed miRNAs in sporadic breast tumors and hereditary tumors.

<p>Venn diagrams representing commonly deregulated miRNAs in two different studies carried out in sporadic breast cancer samples <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038847#pone.0038847-Iorio1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038847#pone.0038847-Volinia1" target="_blank">[12]</a> and in the present study on hereditary breast tumors. Regardless of the genetic background or histopathological features of the tumors, there are miRNAs consistently altered in breast tumor samples.</p

miRNAs differentially expressed between normal breast and familial tumor tissue.

1<p>FDR: False discovery rate adjusted p value.</p

Significantly enriched signaling pathways associated to the differentially expressed. miRNAs.

<p>Significantly enriched signaling pathways associated to the differentially expressed. miRNAs.</p

miR-30c effects on KRAS expression and cell proliferation.

<p>(A) Schematic representation of miR-30c binding site within the KRAS 3′UTR region. (B) Luciferase activity of a reporter construct carrying the KRAS 3′UTR downstream of the luciferase gene. The construct was co-transfected with pre-miR-30c or scramble control in MDA-MB-436 cells. (C) KRAS expression at transcription level. Significant reduced level of KRAS mRNA expression was detected by qRT-PCR after pre-miR-30c transfection, comparing with scramble control. (D) Regulation of KRAS protein level by miR-30c. MDA-MB-436 cells were transfected with pre-miR-30c or pre-miR-scramble oligonucleotides. After 48 hours KRAS protein was evaluated by western blot. GAPDH was used as loading control. The signal in each line was quantified and the ratio of KRAS to GAPDH was determined. (E) Effect of miR-30c expression on proliferation of MDA-MB-436 cells. MTT cell viability assay was performed at 48, 72, 96, 120 or 144 hours after transfection of MDA-MB-436 cells with pre-miR-30c or pre-miR-scramble oligonucleotides.</p

Correlation of expression of miR-30c and KRAS in two breast cancer cell lines.

<p>Inverse correlation between the expression level of miR-30 determine by qRT-PCR (A) and detection of KRAS protein in MCF7 and MDA-MB-436 cells (B).</p

Validation of miR-30c expression by qRT-PCR in hereditary and sporadic tumors.

<p>Expression levels of miR-30c in an independent set of 12 familial (FamBC) and 8 sporadic tumors (SpoBC) comparing to normal breast tissue expression. Differences were estimated by t-test and <i>p</i> values are shown for each comparison.</p

DataSheet1_Association between missense variants of uncertain significance in the CHEK2 gene and hereditary breast cancer: a cosegregation and bioinformatics analysis.PDF

Inherited mutations in the CHEK2 gene have been associated with an increased lifetime risk of developing breast cancer (BC). We aim to identify in the study population the prevalence of mutations in the CHEK2 gene in diagnosed BC patients, evaluate the phenotypic characteristics of the tumor and family history, and predict the deleteriousness of the variants of uncertain significance (VUS). A genetic study was performed, from May 2016 to April 2020, in 396 patients diagnosed with BC at the University Hospital Lozano Blesa of Zaragoza, Spain. Patients with a genetic variant in the CHEK2 gene were selected for the study. We performed a descriptive analysis of the clinical variables, a bibliographic review of the variants, and a cosegregation study when possible. Moreover, an in-depth bioinformatics analysis of CHEK2 VUS was carried out. We identified nine genetic variants in the CHEK2 gene in 10 patients (two pathogenic variants and seven VUS). This supposes a prevalence of 0.75% and 1.77%, respectively. In all cases, there was a family history of BC in first- and/or second-degree relatives. We carried out a cosegregation study in two families, being positive in one of them. The bioinformatics analyses predicted the pathogenicity of six of the VUS. In conclusion, CHEK2 mutations have been associated with an increased risk for BC. This risk is well-established for foundation variants. However, the risk assessment for other variants is unclear. The incorporation of bioinformatics analysis provided supporting evidence of the pathogenicity of VUS.</p

Description of the TNM, histological subtype and grade of the evaluable patients, including TN patients.

<p>Only pathological T and N were considered.</p