Investigation of the miR-637 and miR-523-5p as candidate biomarkers in breast cancer.

OBJECTIVES: The distinction of benign lesions from malign tumors is crucial for the diagnosis and treatment of breast cancers. BACKGROUND: The aim of this study was to investigate the use of miRNAs as plasma biomarkers for the discrimination of malign and benign breast tumors. METHODS: Whole blood samples obtained from 40 individuals in 3 groups designated as invasive ductal carcinoma group, fibroadenoma group and healthy controls were included in this study. The expression levels of 372 miRNAs were determined using RT-PCR.  Results: The comparison of fibroadenoma group with healthy controls revealed an upregulation of thirty miRNAs and downregulation of twenty-nine miRNAs. The comparison of invasive ductal carcinoma (IDC) group with controls has shown that eight miRNAs were upregulated while eleven miRNAs were downregulated. When comparing IDC and fibroadenoma groups, 15 miRNAs were found to be upregulated, while 10 miRNAs were downregulated. Further analysis of these miRNAs aimed to determine their power in distinguishing  IDCs from fibroadenomas. Among the miRNAs analyzed, seven miRNAs have shown sufficient discriminative power, of which three miRNAs, namely miR-637, miR-523-5p and miR-490-3p, have shown a significantly high discriminative power. CONCLUSIONS: Circulating miR-637 and miR-523-5p combination maybe used to discriminate between invasive ductal carcinomas and fibroadenomas. (Tab. 9, Fig. 4, Ref. 30).

Androgen Stimulation of PCA3 and miR-141 and Their Release from Prostate Cancer Cells.

OBJECTIVE: Prostate cancer antigen 3 (PCA3) and microRNA-141 (miR-141) are emerging molecules in prostate cancer (PCa) pathogenesis and have been shown to be involved in androgen signaling. In this original research, we designed an experimental cell model with androgen-sensitive LNCaP cells to comparatively assess the extent of androgen responsiveness of PCA3-mRNA and miR-141 along with prostate specific antigen (PSA)mRNA and their release into culture medium. These molecules were also measured in the plasma of the patients with early PCa which is considered to be analogous to androgenresponsive cells. MATERIALS AND METHODS: In this experimental study, LNCaP cells were exposed to androgen ablation for 48 hours and treated then with dihydrotestosterone (DHT) for 24 hours. Expression of all three RNA molecules in cells, culture medium or plasma was measured by quantitative polymerase chain reaction (qPCR). RESULTS: Our results show that DHT differentially affects the expression of these molecules. PCA3 was the most evidently induced molecule (up to 400-fold, p<0.001), while the effect was moderate for PSA-mRNA (up to 30-fold, p<0.001). In contrast, the stimulation of miR-141 was much weaker (up to 1.5-fold, p>0.05). With regard to the release into culture medium, a similar picture was observed except for PCA3. PCA3 was below the detection level despite its high stimulation. DHT treatment led to a significant release of PSA-mRNA (up to 12-fold). Similar to its induction pattern in LNCaP cells, miR-141 was released at a limited quantity into the medium (up to 1.7- fold, p=0.07). In plasma, only PCA3 differed significantly between the patients and healthy subjects (p=0.001). CONCLUSION: Our findings indicate that PCa-related RNA molecules respond differentially to androgen stimulation suggesting differential regulation by androgens.

Abundant circulating microRNAs in breast cancer patients fluctuate considerably during neoadjuvant chemotherapy.

Previous studies have revealed the aberrant expression of a number of microRNAs (miRNA/miRs) in the blood circulation of patients with breast cancer (BC). The aim of the present study was to assess the effect of neoadjuvant chemotherapy on the levels of a panel of BC-associated miRNAs, which are at relatively low (let-7, miR-10b, miR-34, miR-155, miR-200c and miR-205) or abundant (miR-21, miR-195 and miR-221) levels in the circulation. Patients with primary operable or locally advanced BC were enrolled in the study. The plasma levels of the miRNAs at baseline and at the fourth cycle of treatment were compared. Patients with stage II disease exhibited higher basal miRNAs levels than those with higher stages. The difference was most evident for miR-155 and miR-21 (P=0.05). From the initial to the fourth cycle of chemotherapy, the miRNA levels changed substantially. In samples in which the miRNA levels generally declined, a marked decrease (≤15,500-fold) was evident for the abundant miRNAs. Notably, the occurrence of a decrease in miRNA levels was more frequent in patients with smaller tumor sizes (P<0.05 for miR-21 and miR-195). This proof-of-concept study provides evidence that highly expressed miRNAs are affected most frequently by chemotherapy, particularly in patients with early stage tumors. This information may be valuable in assessing the response of the patients to therapy.

miR-141 and miR-375 induction and release are different from PSA mRNA and PCA3 upon androgen stimulation of LNCaP cells.

Recent studies have demonstrated the differential expression of miR-141 and miR-375 in circulation of patients with advanced/metastatic prostate cancer (PCa). The aim of this study was to investigate the regulation of miR-141 and miR-375 by androgens and their release into the incubation medium in relation to prostate-specific antigen (PSA) mRNA and prostate cancer antigen 3 (PCA3). Plasma levels of these molecules were measured in a small cohort of patients with localized PCa. As an in vitro cell model we used androgen-sensitive LNCaP cells exposed to an androgen ablation of 48 h, and then treated with dihydrotestosterone (DHT) for 24 h. Expression of the four RNA molecules was measured by quantitative polymerase chain reaction (qPCR). miR-141 and miR-375 were induced in a dose-dependent manner where the median stimulation reached only 1.5-fold at maximum. The effect of DHT on PSA mRNA (up to 30-fold) and PCA3 (up to 195-fold) was much more evident. With regard to the release into the incubation medium, similar results were obtained with the exception of PCA3. At the highest DHT dose (100 nM), median miR-141 and miR-375 release was increased 1.7- and 1.4-fold (P=0.07), respectively. DHT treatment led to a significant release of PSA mRNA (up to 12-fold) into the medium while PCA3 could not be amplified from the incubation medium. In plasma only PCA3 differed significantly between localized PCa patients and healthy subjects. In conclusion, our study provides evidence that miR-141 and miR-375 are increasingly released into incubation medium from androgen-stimulated cells. However, the extent of their induction was weaker than PSA mRNA or PCA3, suggesting differential regulation by androgens.

Induction of p53-inducible microRNA miR-34 by gamma radiation and bleomycin are different.

microRNAs (miRNAs) are small molecules in their mature form and master regulators of gene expression. Recent work has shown that miRNAs are involved in the p53 network. Of the various miRNAs, miR-34 is regulated by the p53 protein. miR-34 can be induced by ionizing radiation (IR) in vitro and in vivo. However, there is no data in the literature for induction of miR-34 by a chemical agent inducing DNA damage. Here we studied the expression of miR-34 in HeLa and MCF-7 cells exposed to genotoxic stress-induced by bleomycin (BLM) or γ-radiation. We first analyzed p53 accumulation upon DNA damage induction. The basal level of p53 in MCF-7 cells was higher (approx. 6-fold) than in HeLa cells, and its accumulation was similar for both DNA-damaging agents in both cell lines. We have shown that miR-34 is significantly induced by γ-radiation in HeLa cells, but not in MCF-7 cells. BLM did not significantly affect miR-34 expression in both cell types. In conclusion, our findings reveal that miR-34 induction by genotoxic stress may be cell-type specific.