RESUMO
Atherosclerosis (AS) is a dynamic and multi-stage process that involves various cells types, such as vascular smooth muscle cells (VSMCs) and molecules such as microRNAs. In this study, we investigated how miR-338-3p works in the process of AS. To determine how miR-338-3p was expressed in AS, an AS rat model was established and primary rat VSMCs were cultured. Real-time polymerase chain reaction was performed to detect miR-338-3p expression. Markers of different VSMC phenotypes were tested by Western blot. Immunofluorescent staining was employed to observe the morphologic changes of VSMCs transfected with miR-338-3p mimics. A dual luciferase reporter assay system was used to verify that desmin was a target of miR-338-3p. To further identify the role of miR-338-3p in the development of AS, VSMC proliferation and migration were evaluated by EdU incorporation assay, MTT assay, and wound healing assay. miR-338-3p expression was upregulated in the aortic tissues of an AS rat model and in primary rat VSMCs from a later passage. The transfection of miR-338-3p mimics in VSMCs promoted the synthetic cell phenotype. Bioinformatics analysis proposed desmin as a candidate target for miR-338-3p and the dual luciferase reporter assay confirmed in vivo that desmin was a direct target of miR-338-3p. The MTT and EdU incorporation assay revealed increased cell viability when miR-338-3p mimics were transfected. The increased expression of PCNA was a consistent observation, although a positive result was not obtained with respect to VSMC mobility. In AS, miR-338-3p expression was elevated. Elevated miR-338-3p inhibited the expression of desmin, thus promoting the contractile-to-synthetic VSMC phenotypic transition. In addition to morphologic changes, miR-338-3p enhanced the proliferative but not mobile ability of VSMCs. In summary, miR-338-3p promotes the development of AS.
Assuntos
Aorta/metabolismo , Apolipoproteínas E/genética , Aterosclerose/genética , Desmina/genética , MicroRNAs/genética , Músculo Liso Vascular/metabolismo , Animais , Aorta/patologia , Apolipoproteínas E/deficiência , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Pareamento de Bases , Sequência de Bases , Linhagem Celular , Movimento Celular , Proliferação de Células , Desmina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Células HEK293 , Humanos , MicroRNAs/agonistas , MicroRNAs/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Cultura Primária de Células , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Transdução de SinaisRESUMO
ZNF300 plays an important role in the regulation of HBV-related hepatocellular carcinoma. However, little is known about the role of ZNF300 in lipid metabolism and NAFLD. In the present study, we observed that ZNF300 expression was markedly decreased in free fatty acid (FFA)-induced fatty liver. Overexpressed ZNF300 alleviated hepatic lipid accumulation, whereas knockdown of ZNF300 enhanced the FFA-induced lipid accumulation. Investigations of the underlying mechanisms revealed that ZNF300 directly binds to and regulates the PPARα expression, thus promoting fatty acid oxidation. Furthermore, bisulfite pyrosequencing PCR (BSP) analysis identified the hypermethylation status of ZNF300 gene in FFA-treated hepatocytes. Importantly, the suppression of ZNF300 could be blocked by DNA methyltransferase inhibitor (5-azadC) or DNMT3a-siRNA. These results suggested that ZNF300 plays an important role in hepatic lipid metabolism via PPARα promoting fatty acid oxidation and this effect might be blocked by DNMT3a-mediated methylation of ZNF300. Therefore, in addition to ZNF300 expression levels, the methylation status of this gene also has a potential as a prognostic biomarker.