PKC Regulates YAP Expression through Alternative Splicing of YAP 3'UTR Pre-mRNA by hnRNP F.

The Yes-associated protein (YAP) is a transcriptional co-activator that plays critical roles in organ development and tumorigenesis, and is verified to be inhibited by the Hippo signaling pathway. In the present study, we show that the YAP 3'UTR is alternatively spliced to generate a novel 950 bp 3'UTR mRNA from the full length 3'UTR region (3483 bp) in human cancer cells. The ratio of full length 3'UTR YAP mRNA to alternatively spliced 3'UTR YAP mRNA is up-regulated by exposure of the cells to PKC inhibitor chelerythrine chloride. Further study using luciferase reporter assay showed that the expression of the alternatively spliced 3'UTR mRNA is much lower compared with the full length 3'UTR mRNA, suggesting that alternatively spliced 3'UTR YAP mRNA may have a shorter half-life than full length 3'UTR mRNA. Interestingly, PKC represses YAP 3'UTR-mediated mRNA stability is dependent on a splicing factor, hnRNP F. Activation of PKC induces nuclear translocation of cytosolic hnRNP F. Ectopic expression of hnRNP F enhances YAP 3'UTR splicing. Our results suggest that hnRNP F regulates YAP 3'UTR-mediated mRNA stability in an alternative splicing-dependent manner, and PKC regulated YAP expression is dependent on nuclear translocation of hnRNP F in human cancer cell lines.

MicroRNA 630 Represses NANOG Expression through Transcriptional and Post-Transcriptional Regulation in Human Embryonal Carcinoma Cells.

The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3'UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the NANOG 3'UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3'UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3'UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3'UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3'UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.

Helium/Argon-Generated Cold Atmospheric Plasma Facilitates Cutaneous Wound Healing.

Cold atmospheric plasma jet (CAPJ) or non-thermal plasma jet has been employed in various biomedical applications based on their functions in bactericidal activity and wound healing. However, the effect of CAPJ generated by a particular composition of gases on wound closure and the underlying mechanisms that regulate wound healing signals remain elusive. In the present study, we investigated the impact of helium (He)- or a gas mixture of He and argon (He/Ar)-generated CAPJ on cell proliferation, which is a pivotal step during the wound healing process. With careful treatment duration control, He/Ar-CAPJ effectively induced keratinocyte proliferation and migration mediated through the activation of epithelial-to-mesenchymal transition (EMT) and cell cycle progression, which was evidenced by a decrease in E-cadherin levels and increases in N-cadherin, cyclin D1, Ki-67, Cdk2, and p-ERK levels. Rat wound healing studies showed that He/Ar-CAPJ treatment facilitated granulation tissue formation and mitigated inflammation in cutaneous tissue, resulting in accelerated wound closure. These findings highlight the possibility that He/Ar-CAPJ can be developed as a therapeutic agent for enhancing wound healing.

Heterogeneous ribonucleoprotein F regulates YAP expression via a G-tract in 3'UTR.

The Yes-associated protein (YAP) is a transcription coactivator that plays crucial roles in organ size control and tumorigenesis, and was demonstrated to be inhibited by the Hippo signaling pathway. To date, the molecular mechanisms regulating the expression of YAP in human cells remain unknown. In the present study, we found that hnRNP F and hnRNP U negatively regulate YAP expression. We also showed that downregulation of YAP expression by hnRNP F and hnRNP U was not at the transcriptional level. Knockdown of hnRNP F or hnRNP U increased YAP mRNA stability, suggesting the downregulation of YAP expression was by a post-transcriptional mechanism. A putative hnRNP F binding site was identified in the YAP 3'UTR at 685 to 698, and deletion of this putative hnRNP F element abolished the down-regulation effect of YAP mRNA stability by hnRNP F. Binding of the hnRNP F to the YAP 3'UTR was demonstrated by Cross-linked RNA Immunoprecipitation. mRNA stability is a possible secondary effect of alternative splicing or other nuclear process. Understanding the regulation of YAP expression would provide insights into the mechanisms underlying the maintenance of tissue size homeostasis and tumorigenesis.

Parameters Affecting the Antimicrobial Properties of Cold Atmospheric Plasma Jet.

Using the Taguchi method to narrow experimental parameters, the antimicrobial efficiency of a cold atmospheric plasma jet (CAPJ) treatment was investigated. An L9 array with four parameters of CAPJ treatments, including the application voltage, CAPJ-sample distance, argon (Ar) gas flow rate, and CAPJ treatment time, were applied to examine the antimicrobial activity against Escherichia coli (E. coli). CAPJ treatment time was found to be the most influential parameter in its antimicrobial ability by evaluation of signal to noise ratios and analysis of variance. 100% bactericidal activity was achieved under the optimal bactericidal activity parameters including the application voltage of 8.5 kV, CAPJ-sample distance of 10 mm, Ar gas flow rate of 500 sccm, and CAPJ treatment time of 300 s, which confirms the efficacy of the Taguchi method in this design. In terms of the mechanism of CAPJ's antimicrobial ability, the intensity of hydroxyl radical produced by CAPJ positively correlated to its antimicrobial efficiency. The CAPJ antimicrobial efficiency was further evaluated by both DNA double-strand breaks analysis and scanning electron microscopy examination of CAPJ treated bacteria. CAPJ destroyed the cell wall of E. coli and further damaged its DNA structure, thus leading to successful killing of bacteria. This study suggests that optimal conditions of CPAJ can provide effective antimicrobial activity and may be grounds for a novel approach for eradicating bacterial infections.

Simultaneous quantification of trans-resveratrol and its sulfate and glucuronide metabolites in rat tissues by stable isotope-dilution UPLC-MS/MS analysis.

A rapid, sensitive, and selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in the multiple reaction monitoring (MRM) mode has been developed and validated to investigate the distribution of trans-Resveratrol (Resv) and its metabolites in rats following intravenous (IV) administration at 20mg/kg body weight (BW). Resv and Resv metabolites were analyzed in the negative electrospray ionization mode and eluted with retention times of about 0.69-2.22min with a runtime of 7min. Stable deuterium-labeled Resv and its metabolites were used as the internal standards to correct for matrix effects and to allow for accurate quantification of Resv and its metabolites in a complex biological system. The method was validated with respect to linearity, within- and between-day precision, limit of quantification, recovery, and accuracy for all analytes. Upon examination at 0.5, 1, 2, and 4h post-administration, concentrations of Resv and its metabolites were the highest in the kidney, followed by plasma; specifically, the glucuronidated forms were the most abundant. In the liver and the brain, the predominant forms were the sulfated derivatives. In contrast, heart tissue contained the highest concentration of unmodified Resv at 0.5h post IV administration. The combined use of UPLC-MS/MS and isotope-dilution analysis, proved to be accurate and reliable in identifying and quantifying Resv and its various metabolites in biological samples at the nanomolar range. This technology is potentially applicable for other pharmacokinetic studies.