Phospholipid scramblase Xkr8 is required for developmental axon pruning via phosphatidylserine exposure.

The mature mammalian brain connectome emerges during development via the extension and pruning of neuronal connections. Glial cells have been identified as key players in the phagocytic elimination of neuronal synapses and projections. Recently, phosphatidylserine has been identified as neuronal "eat-me" signal that guides elimination of unnecessary input sources, but the associated transduction systems involved in such pruning are yet to be described. Here, we identified Xk-related protein 8 (Xkr8), a phospholipid scramblase, as a key factor for the pruning of axons in the developing mammalian brain. We found that mouse Xkr8 is highly expressed immediately after birth and required for phosphatidylserine exposure in the hippocampus. Mice lacking Xkr8 showed excess excitatory nerve terminals, increased density of cortico-cortical and cortico-spinal projections, aberrant electrophysiological profiles of hippocampal neurons, and global brain hyperconnectivity. These data identify phospholipid scrambling by Xkr8 as a central process in the labeling and discrimination of developing neuronal projections for pruning in the mammalian brain.

Papillary Thyroid Carcinoma Tissue miR-146b, -21, -221, -222, -181b Expression in Relation with Clinicopathological Features.

We analyzed miR-146b, miR-21, miR-221, miR-21, and miR-181b in formalin fixed paraffin-embedded papillary thyroid carcinoma (PTC) tissue samples of 312 individuals and evaluated their expression relationship with clinicopathological parameters. A higher expression of miR-21 was related to unifocal lesions (p < 0.011) and autoimmune thyroiditis (0.007). miR-221, miR-222 expression was higher in the PTC tissue samples with extrathyroidal extension (p = 0.049, 0.003, respectively). In a group of PTC patients with pT1a and pT1b sized tumors, the expression of miR-146b, miR-21, miR-221, and miR-222 in PTC tissue samples was lower than in patients with pT2, pT3, and pT4 (p = 0.032; 0.0044; 0.003; 0.001; 0.001, respectively). Patients with lymph node metastases had higher expression of miR-21, -221, -222, and -181b (p < 0.05). A high expression of miR-146b, miR-21, miR-221 panel was associated with decreased overall survival (OS) (Log rank p = 0.019). Univariate analysis revealed that presence of metastatic lymph nodes and high expression of miR-146b, miR-21, and miR-221 panels were associated with increased hazard of shorter OS. After multivariate analysis, only sex (male) and age (≥55 years) emerged as independent prognostic factors associated with shorter OS (HR 0.28 (95% CI 0.09-0.86) and HR 0.05 (95% CI 0.01-0.22), respectively). In conclusion, 5 analyzed miRs expression have significant relations to clinicopathologic parameters so further investigations of these molecules are expedient while searching for prognostic PTC biomarkers.

Different stability of miRNAs and endogenous control genes in archival specimens of papillary thyroid carcinoma.

BACKGROUND: The most popular miRNA quantitation technique is RQ-PCR with relative gene expression method that requires an endogenous control (EC) gene for data normalization. However, there are insufficient data and selection criteria on the most suitable ECs for miRNA expression studies in many cancer types including papillary thyroid carcinoma (PTC). Therefore, in this study we evaluated the impact of chosen EC and archival formalin-fixed, paraffin-embedded (FFPE) PTC tissue age on estimated target miRNA expression. METHODS: RQ-PCR was used to determine expression levels of five miRNAs (miR-146b, miR-222, miR-21, miR-221 and miR-181b) and three different endogenous controls (RNU48, let-7a, miR-16), which were used to normalize the data. In total, 400 FFPE PTC tissues were analyzed that have been stored from 1 to 15 years. RESULTS: The stability of commonly used ECs RNU48 and let-7a significantly differs from the stability of target miRNA in archival FFPE PTC tissues. Moreover, these differences have a great impact on miRNA expression results when FFPE tissue samples have been stored for a different period of time. CONCLUSIONS: It is important to select an ECs not only stable in the tissue of interest but also with similar stability to target miRNA, especially when working with samples of different age.

Plasma-Derived miRNA-222 as a Candidate Marker for Papillary Thyroid Cancer.

We analyzed five miRNA molecules (miR-221; miR-222; miR-146b; miR-21; miR-181b) in the plasma of patients with papillary thyroid cancer (PTC), nodular goiter (NG) and healthy controls (HC) and evaluated their diagnostic value for differentiation of PTC from NG and HC. Preoperative PTC plasma miRNA expression (n = 49) was compared with plasma miRNA in the HC group (n = 57) and patients with NG (n = 23). It was demonstrated that miR-221; miR-222; miR-146b; miR-21 and miR-181b were overexpressed in preoperative PTC plasma samples compared to HC (p < 0.0001; p < 0.0001; p < 0.0001; p < 0.0001; p < 0.002; respectively). The upregulation in tumor tissue of these miRNAs was consistent with The Cancer Genome Atlas Thyroid Carcinoma dataset. A significant decrease in miR-21; miR-221; miR-146b and miR-181b expression was observed in the plasma of PTC patients after total thyroidectomy (p = 0.004; p = 0.001; p = 0.03; p = 0.036; respectively). The levels of miR-222 were significantly higher in the preoperative PTC compared to the NG group (p = 0.004). ROC curve (receiver operating characteristic curve) analysis revealed miR-222 as a potential marker in distinguishing PTC from NG (AUC 0.711; p = 0.004). In conclusion; circulating miR-222 profiles might be useful in discriminating PTC from NG.

Association of MicroRNA Expression and BRAFV600E Mutation with Recurrence of Thyroid Cancer.

Many miRNAs and cancer-related mutations have been proposed as promising molecular markers of papillary thyroid carcinoma (PTC). However, there are limited data on the correlation between miRNA expression, BRAFV600E mutation, and PTC recurrence. Therefore, to evaluate the potential of BRAFV600E mutation and five selected miRNAs (-146b, -222, -21, -221, -181b) in predicting PTC recurrence, these molecular markers were analyzed in 400 formalin-fixed, paraffin-embedded PTC tissue specimens. The expression levels of miRNAs were measured using qRT-PCR. It was demonstrated that expression levels of all analyzed miRNAs are significantly higher in recurrent PTC than in non-recurrent PTC (p < 0.05). Moreover, higher expression levels of miR-146b, miR-222, miR-21, and miR-221 were associated with other clinicopathologic features of PTC, such as tumor size and lymph node metastases at initial surgery (p < 0.05). No significant differences in the frequency of BRAFV600E mutation in recurrent PTC and non-recurrent PTC were determined. Our results suggest that miRNA expression profile differs in PTC that is prone to recurrence when compared to PTC that does not reoccur after the initial surgery while BRAFV600E mutation frequency does not reflect the PTC recurrence status. However, the prognostic value of the analyzed miRNAs is rather limited in individual cases as the pattern of miRNA expression is highly overlapping between recurrent and non-recurrent PTC.