miR-140-5p Affects TCC8113 Cell Biological Activity and Matrix Metalloproteinases 9 (MMP-9) in Oral Squamous Cell Carcinoma (OSCC) Cell Line

Whether the miR-140-5p affects the biological activity of Tca8113 cells and MMP-9 in OSCC cell line was explored. Tca8113 cells were divided into Tc group (normal Tca8113 cells), Tm group (Tca8113 cell transfection+miR-140-5p mimic), and Nc group (Tca8113 cell transfection+miR-140-5p-negative control) followed by analysis of MMP-9 expression by western blot, cell migration by Transwell assay, cell viability by MTT method, and apoptosis by flow cytometry. Compared with TC group and NC group, mir-140-5p in TM group was significantly higher (P <0.05), however, the MMP-9 level of TM group was significantly lower (P <0.05).Western blot analysis showed that there was no significant difference (P <0.05) in the comparison of MMP-9 protein expression between TC group and NC group, and MMP-9 expression in the TM group was decreased significantly (P <0.05). MTT assay showed that the cell viability of TM group was increased slowly and lower than that of group (P <0.05). In conclusion, mir-140-5p can induce apoptosis of OSCC cell line Tca8113, which may be achieved by reducing the expression of MMP-9.

A Rapid Evolving microRNA Cluster Rewires Its Target Regulatory Networks in Drosophila

New miRNAs are evolutionarily important but their functional evolution remains unclear. Here we report that the evolution of a microRNA cluster, mir-972C rewires its downstream regulatory networks in Drosophila. Genomic analysis reveals that mir-972C originated in the common ancestor of Drosophila where it comprises six old miRNAs. It has subsequently recruited six new members in the melanogaster subgroup after evolving for at least 50 million years. Both the young and the old mir-972C members evolved rapidly in seed and non-seed regions. Combining target prediction and cell transfection experiments, we found that the seed and non-seed changes in individual mir-972C members cause extensive target divergence among D. melanogaster, D. simulans, and D. virilis, consistent with the functional evolution of mir-972C reported recently. Intriguingly, the target pool of the cluster as a whole remains relatively conserved. Our results suggest that clustering of young and old miRNAs broadens the target repertoires by acquiring new targets without losing many old ones. This may facilitate the establishment of new miRNAs in existing regulatory networks.

miR-27b-3p Targeting BDNF Inhibits the TrkB/CREB Signaling Pathway and Improves IL-1 β-Induced Chondrocyte Inflammation

Background: Explore the mechanism of "miR-27b-3p targeting BDNF inhibits the TrkB/CREB signaling pathway and improves IL-1 β-induced chondrocyte inflammation".Methods: The animal and cell models of arthritis were constructed, and various biochemical detection methods were used to detect the changes of apoptosis, inflammation and oxidative stress.Results: The results showed that the expression of miR-27b-3p was downregulated in IL-1β-treated chondrocytes and cartilage tissues isolated from a KOA rat model. miR-27b-3p overexpression notably reduced IL-1β-induced chondrocyte apoptosis and the expression levels of caspase-3 and caspase-9. In addition, cell transfection with miR-27b-3p mimics increased the mRNA and protein expression levels of inducible nitric oxide synthase and cyclooxygenase-2. The levels of nitric oxide, prostaglandin E2 (PGE2), TNF-α and IL-6 were also reduced following cell transfection with miR-27b-3p mimics. Furthermore, bioinformatics analysis predicted that miR-27b-3p could directly target brain-derived neurotrophic factor (BDNF). Additionally, the present study suggested that the tropomyosin receptor kinase B (TrkB)/cAMP response‑element binding protein (CREB) signaling axis could be a downstream pathway of the miR-27b-3p/BDNF axis. The percentage of apoptotic cells and the expression levels of nitric oxide, PGE2, TNF-α and IL-6 were enhanced in chondrocytes co-treated with BDNF + IL-1β. However, these effects were restored following transfection of chondrocytes with miR-27b-3p mimics. Staining of cartilage tissues with safranin O showed that miR-27b-3p overexpression Significantly attenuated KOA-induced cartilage degradation.Conclusions:miR-27b-3p targeting BDNF inhibits the TrkB/CREB signaling pathway and improves IL-1 β-induced chondrocyte inflammation.

Dielectrophoretic Manipulation of Cell Transfection Efficiency During Electroporation Using a Center Needle Electrode

Long duration electric pulses are frequently used to facilitate DNA electrotransfer into cells and tissues, while electroporation pulses can be combined with electrophoresis to maximize the transfection efficiency. In this work, we present the dielectrophoresis (DEP)-assisted methodology for electrotransfer of plasmid DNA (3.5 kbp pmaxGFP) into mammalian cells (CHO-K1). A prototype of an electroporation cuvette with center needle electrode for DEP-assisted transfection is presented resulting in a 1.4-fold of transfection efficiency increase compared to the electroporation-only procedure (1.4 kV/cm × 100 µs × 8). The efficiency of transfection has been compared between three DEP frequencies of 1, 100, and 1 MHz. Lastly, the effects of exposure time (1, 3, and 5 min) during the DEP application step have been determined. It is concluded that the proposed methodology and exposure setup allow a significant improvement of transfection efficiency and could be used as an alternative to the currently popular electrotransfection techniques.

The Double Mutation DSG2-p.S363X and TBX20-p.D278X Is Associated with Left Ventricular Non-Compaction Cardiomyopathy: Case Report

Left ventricular non-compaction cardiomyopathy (LVNC) is a rare heart disease, with or without left ventricular dysfunction, which is characterized by a two-layer structure of the myocardium and an increased number of trabeculae. The study of familial forms of LVNC is helpful for risk prediction and genetic counseling of relatives. Here, we present a family consisting of three members with LVNC. Using a next-generation sequencing approach a combination of two (likely) pathogenic nonsense mutations DSG2-p.S363X and TBX20-p.D278X was identified in all three patients. TBX20 encodes the cardiac T-box transcription factor 20. DSG2 encodes desmoglein–2, which is part of the cardiac desmosomes and belongs to the cadherin family. Since the identified nonsense variant (DSG2-p.S363X) is localized in the extracellular domain of DSG2, we performed in vitro cell transfection experiments. These experiments revealed the absence of truncated DSG2 at the plasma membrane, supporting the pathogenic relevance of DSG2-p.S363X. In conclusion, we suggest that in the future, these findings might be helpful for genetic screening and counseling of patients with LVNC.

Overexpression of RIPK4 Predicts Poor Prognosis and Promotes Metastasis in Ovarian Cancer

This study was conducted to evaluate the prognostic value of receptor-interacting protein kinase 4 (RIPK4) in ovarian cancer (OC) and its role in tumorigenesis. RNA expression and the corresponding clinical data were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. The relationship between clinical-pathological characteristics and RIPK4 expression was analyzed using the Wilcoxon signed-rank test and logistic regression. The Cox regression and the Kaplan-Meier method were used to evaluate the relationship between clinicopathological features and overall survival (OS). Gene set enrichment analysis (GSEA) was performed using Molecular Signatures Database. Scratch assay, transwell assay, and cell transfection were used to verify the function of RIPK4. Overexpression of RIPK4 was associated with the stage of OC and distant metastasis. Survival analysis revealed that patients with OC and higher expression of RIPK4 had a poorer prognosis. Univariate and multivariate analyses indicated that high expression of RIPK4 was associated with poor OS, as well as age and stage of OC. The areas under the curve (AUC) at 1, 4, and 8 years were 0.737, 0.634, and 0.669, respectively, according to the established OS prediction model. GSEA revealed that adherens junction, cadherin binding, and Wnt signaling pathway were enriched in the high RIPK4 expression group. Cell transfection confirmed RIPK4 was involved in the Wnt signaling pathway. RIPK4 can act as a potential prognostic molecular marker for poor survival in OC. Moreover, RIPK4 is associated with tumor metastasis and implicated in the regulation of the Wnt signaling pathway.