MicroRNA-590-3p relieves hypoxia/reoxygenation induced cardiomyocytes apoptosis and autophagy by targeting HIF-1α.

Autophagy and apoptosis are key factors in myocardial ischemia/reperfusion (I/R) injury. MicroRNAs (miRNAs or miRs) participate in occurrence and development of myocardial I/R injury by regulating autophagy and apoptosis. The purpose of the present study was to investigate the role of miR-590-3p in the regulation of autophagy and apoptosis in hypoxia/reoxygenation (H/R)-treated cardiomyocytes. Following 6 h hypoxia and 6 h reoxygenation in primary rat cardiomyocytes, miR-590-3p was downregulated. Transfection of miR-590-3p mimic inhibited the increased autophagy and apoptosis following H/R treatment. Subsequent experiments demonstrated that miR-590-3p regulated induction of autophagy and apoptosis by targeting hypoxia inducible factor (HIF)-1α. Forced expression of HIF-1α rescued the protective effect of miR-590-3p on H/R-induced cardiomyocytes. In summary, the present study showed that miR-590-3p exhibited a protective effect on H/R-induced cardiomyocyte injury and may be a novel target for the treatment of myocardial ischemia disease.

Promoting roles of long non-coding RNA FAM83H-AS1 in bladder cancer growth, metastasis, and angiogenesis through the c-Myc-mediated ULK3 upregulation.

Long non-coding RNA (lncRNA) FAM83H-AS1 has been recently identified with oncogenic roles in many human cancers. But its role in bladder cancer (BCa) pathogenesis and the mechanisms are largely unstudied. This study aims to evaluate the roles of FAM83H-AS1 in the malignant behaviors and the angiogenesis of BCa cells and the mechanical molecules involved. High expression of FAM83H-AS1 was found in 82 BCa tissues and in BCa cell lines compared to the normal ones. FAM83H-AS1 downregulation in T24 and BK10 cells inhibited viability, colony formation, migration, invasion, and angiogenesis of BCa cells and increased cell apoptosis. FAM83H-AS1 was found to bind to the transcription factor c-Myc to activate ULK3 expression. Overexpression of ULK3 was further introduced into T24 and BK10 cells in the presence of FAM83H-AS1 silencing, which blocked the inhibitory effects of FAM83H-AS1 downregulation on BCa cell growth. The activity of the Hedgehog signaling pathway was suppressed by FAM83H-AS1 while recovered by ULK3. Suppression of the Hedgehog pathway reduced the malignant behaviors of BCa cells promoted by ULK3. The in vitro experiment results were reproduced in vivo. This study evidenced that FAM83H-AS1 upregulates ULK3 expression through the transcription factor c-Myc and promotes the progression of BCa.

microRNA-451a promoter methylation regulated by DNMT3B expedites bladder cancer development via the EPHA2/PI3K/AKT axis.

BACKGROUND: The downregulation of microRNA (miR)-451a has been reported in bladder cancer (BCa) tissues. Herein, we elucidated the role of miR-451a in BCa with the involvement of DNA methyltransferase 3B (DNMT3B). METHODS: We first screened the differentially expressed miRNAs from the serum of 12 BCa patients and 10 healthy controls in the BCa database GSE113486. Subsequently, we detected miR-451a expression and CpG island methylation of the promoter in BCa cells T24 and 5637 with DNMT3B knockdown. The downstream mRNAs of miR-451a were predicted by bioinformatics and KEGG enrichment analysis. Afterwards, the expression patterns of DNMT3B, miR-451a and erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EPHA2) were altered in BCa cells to test the ability of cell proliferation, apoptosis, migration as well as invasion. Finally, the effect of miR-451a and DNMT3B was evaluated in vivo. RESULTS: miR-451a was significantly reduced in serum of BCa patients and cell lines. Moreover, the expression of DNMT3B in BCa cells was significantly increased, thus promoting methylation of the miR-451a promoter, resulting in miR-451a inhibition. Additionally, we found that miR-451a targeted and negatively regulated EPHA2, while EPHA2 could activate the PI3K/AKT signaling, driving BCa cell growth and metastasis. CONCLUSIONS: Our study proposed and demonstrated that miR-451a downregulation mediated by DNMT3B is critical for proliferation, migration, and invasion of BCa, which may be beneficial for developing more effective therapies against BCa.

[Establishment of STO Cell Lines Expressing Green Fluorescent Protein and Mouse Leukemia Inhibitory Factor].

OBJECTIVE: To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer. METHODS: The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed. RESULTS: The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer. CONCLUSION: The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.

Inducible expression of endomorphins in murine dendritic cells.

Bone marrow precursor cells were extracted from C57BL/6J mice aged 7-8 weeks, and dendritic cells were purified using anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme immunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [(3)H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of µ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of µ-opioid receptors.