Stability of selected microRNAs in human blood, semen and saliva samples exposed to different environmental conditions.

Body fluid identification is an essential aspect of forensic investigations. MicroRNAs (miRNAs), a type of non-coding RNA has a lot of potential in forensic studies as biomarkers. Using a reference gene - GAPDH, this study ascertained the stability of hsa-mir-451a, hsa-miR-10b, and hsa-miR-205 in blood, semen and saliva respectively, exposed to different environmental conditions over different periods of time. Twenty adult males aged 20-35 donated blood, sperm, and saliva. These fluids were separated into four groups and exposed to Outdoor, Indoor, Fridge, and Freezer conditions for 37 days. Total RNA was isolated using Trizol-Isopropanol extraction protocol. FIREScript RT cDNA synthesis kit and Luna Universal qPCR Master Mix were used for cDNA synthesis and qPCR respectively. Under different environmental conditions, miR-451a and miR-10b was detected in all blood and semen samples but not in all saliva samples. Pearson's correlation test for Cq values showed multiple strong correlations (p < 0.05) within and outside the groups compared to GADPH. Regardless of the body fluid sample, the freezer group was more stable compared to other groups, although correlations are not homogeneous in all samples. Expression of the targeted genes in the body fluids showed that hsa-miR-451a, hsa-miR-10b, hsa-miR-205 could be utilized as biomarkers in forensics for body fluids identification.

miR-10b expression in breast cancer stem cells supports self-renewal through negative PTEN regulation and sustained AKT activation.

Cancer stem cells (CSCs) are linked to metastasis. Moreover, a discrete group of miRNAs (metastamiRs) has been shown to promote metastasis. Accordingly, we propose that miRNAs that function as metastatic promoters may influence the CSC phenotype. To study this issue, we compared the expression of 353 miRNAs in CSCs enriched from breast cancer cell lines using qRT-PCR analysis. One of the most altered miRNAs was miR-10b, which is a reported promoter of metastasis and migration. Stable overexpression of miR-10b in MCF-7 cells (miR-10b-OE cells) promoted higher self-renewal and expression of stemness and epithelial-mesenchymal transition (EMT) markers. In agreement with these results, inhibiting miR-10b expression using synthetic antisense RNAs resulted in a decrease in CSCs self-renewal. Bioinformatics analyses identified several potential miR-10b mRNA targets, including phosphatase and tensin homolog (PTEN), a key regulator of the PI3K/AKT pathway involved in metastasis, cell survival, and self-renewal. The targeting of PTEN by miR-10b was confirmed using a luciferase reporter, qRT-PCR, and Western blot analyses. Lower PTEN levels were observed in CSCs, and miR-10b depletion not only increased PTEN mRNA and protein expression but also decreased the activity of AKT, a downstream PTEN target kinase. Correspondingly, PTEN knockdown increased stem cell markers, whereas AKT inhibitors compromised the self-renewal ability of CSCs and breast cancer cell lines overexpressing miR-10b. In conclusion, miR-10b regulates the self-renewal of the breast CSC phenotype by inhibiting PTEN and maintaining AKT pathway activation.