MicroRNAs Discriminate Familial from Sporadic Non-BRCA1/2 Breast Carcinoma Arising in Patients ≤35 Years

<div><p>The influence of genetic factors may contribute to the poor prognosis of breast cancer (BC) at a very young age. However <i>BRCA1/2</i> mutations could not explain the majority of cases arising in these patients. MicroRNAs (miRs) have been implicated in biological processes associated with BC. Therefore, we investigated differences in miRs expression between tumors from young patients (≤35 years) with sporadic or familial history and non-carriers of <i>BRCA1/2</i> mutations. Thirty-six young Brazilian patients were divided into 2 groups: sporadic (NF-BC) or familial breast cancer (F-BC). Most of the samples were classified as luminal A and B and the frequency of subtypes did not differ between familial or sporadic cases. Using real time qPCR and discriminant function analysis, we identified 9 miRs whose expression levels rather than miR identity can discriminate between both patient groups. Candidate predicted targets were determined by combining results from miRWalk algorithms with mRNA expression profiles (n = 91 differently expressed genes). MiR/mRNA integrated analysis identified 91 candidate genes showing positive or negative correlation to at least 1 of the 9 miRs. Co-expression analysis of these genes with 9 miRs indicated that 49 differentially co-expressed miR-gene interactions changes in F-BC tumors as compared to those of NF-BC tumors. Out of 49, 17 (34.6%) of predicted miR-gene interactions showed an inverse correlation suggesting that miRs act as post-transcriptional regulators, whereas 14 (28.6%) miR-gene pairs tended to be co-expressed in the same direction indicating that the effects exerted by these miRs pointed to a complex level of target regulation. The remaining 18 pairs were not predicted by our criteria suggesting involvement of other regulators. MiR–mRNA co-expression analysis allowed us to identify changes in the miR-mRNA regulation that were able to distinguish tumors from familial and sporadic young BC patients non-carriers of BRCA mutations.</p></div

Co-expression matrixes.

<p>Co-expression matrixes of the 91 differentially expressed genes vs 9 differentially expressed miRs for F-BC (A) and NF-BC (B), respectively. The colors represent the co-expression values reaching from 1 to −1 for red and green, respectively. F-BC, familial breast cancer; NF-BC, non-familial breast cancer.</p

Target prediction graph.

<p>Graph displaying the number of predicted targets from miRWalk for each miRNA differentially expressed between F-BC and NF-BC groups. F-BC, familial breast cancer; NF-BC, non-familial breast cancer.</p

Cross-validation analysis graphic.

<p>Representative <i>cross-validation analysis</i> graphic of 35 patients from the F-BC and NF-BC groups. Black spots indicate F-BC samples while plus signs indicate NF-BC samples. The line represents the limit discriminant function between groups. F-BC, familial breast cancer; NF-BC, non-familial breast cancer.</p

Frequency of clinical and histopathological characteristics according to the groups—familial breast cancer (F-BC) and sporadic breast cancer (NF-BC).

<p>Frequency of clinical and histopathological characteristics according to the groups—familial breast cancer (F-BC) and sporadic breast cancer (NF-BC).</p

Illustration of network signatures.

<p>Seventeen miR–mRNA predicted interactions whose co-expression are significantly different between F-BC (A) and NF-BC (B) groups. Color edges represent positive (green) or negative (red) Pearson correlation values. The edge thickness indicates the magnitude of Pearson correlation values. The node size is proportional to the fold change of genes (orange nodes) and of miRs (blue nodes) between F-BC to NF-BC groups. F-BC, familial breast cancer; NF-BC, non-familial breast cancer.</p

Morphological changes in <i>D. rerio</i> during the 48 hours of exposure to piplartine.

<p><b>A</b>) Leakage of the ocular pigment caused by 1.8 ppm piplartine, <b>B</b>) tissue alterations on the head and mouth caused by 1.6 µg/ml piplartine, <b>C</b>) exophthalmia and hemorrhage caused by 1.4 µg/ml piplartine and D) control group.</p

Patient and sample characteristics.

<p>POS = positive; NEG: negative; Trip. NEG: triple negative; GN: nuclear grade; GH: histological grade; ER: estrogen receptor; PR: progesterone receptor; HER-2: growth factor receptor type 2; NCCN: <i>National Comprehensive Cancer Network</i>. ER, PR, and Her-2 receptor status was defined according to IHC.TNM =  tumor classification based on stage according to TNM criteria suggested by WHO (World Health Organization): I (T1N0M0); IIA (T0N1M0, T1N1M0, T2N0M0); IIB (T2N1M0,T3N0M0); IIIA (T0N2M0, T1N2M0, T2N2M0, T3N1M0, T3N2M0); IIIB (T4N0M0,T4N1M0, T4N2M0); IV (every T, N, and M1).</p

Mortality of <i>B. glabrata</i> adults exposed to methanolic extracts of different organs of <i>Piper tuberculatum</i>.

<p>n = 30 adult snails.</p><p>Values were obtained at the end of the 7^th day of observation.</p

Calculated (squares) and predicted (circles) PLS data.

<p>These data were generated from ESIMS data, versus measured and autoscaled LC₅₀ values of <i>P. tuberculatum</i> extracts for <i>B. glabrata</i>.</p