Effect of MIPU1 gene interference on biological behavior of U251 cell line. / shRNA干扰MIPU1åŸºå› å¯¹è„‘æ˜Ÿå½¢ç»†èƒžç˜¤U251细胞生物学行为的影响.

OBJECTIVES: To investigate effect of MIPU1 silence on proliferation, apoptosis, migration and invasion in U251 cells. METHODS: The shRNA recombinant plasmids targeting MIPU1 gene was transfected into U251 cells. Western blotting was used to identify the inhibitory efficiency at 72 h after transfection. The cell viability was measured by MTT colorimetric assay. Hoechest staining and caspase-3 activity were used to detect apoptosis. Then wound healing assay and transwell migration assay were applied to detect the migration and invasion of cells. RESULTS: The expression of MIPU1 protein was effectively knocked down in transfected cells (P<0.05). The cellular proliferation was obviously inhibited and apoptosis was increased in shRNA-transfected MIPU1 cells (all P<0.05). The migration and invasion ability of cells transfected with positive plasmid was lower than that in the control group (P<0.05). CONCLUSIONS: Down-regulation of MIPU1 can promote apoptosis while inhibit the proliferation, invasion, and migration of U251 cells.

Transplantation of bone marrow stromal stem cells overexpressing tropomyosin receptor kinase A for peripheral nerve repair.

BACKGROUND AIMS: Previously we reported that overexpression of tropomyosin receptor kinase A (TrkA) could improve the survival and Schwann-like cell differentiation of bone marrow stromal stem cells (BMSCs) in nerve grafts for bridging rat sciatic nerve defects. The aim of this study was to investigate how TrkA affects the efficacy of BMSCs transplantation on peripheral nerve regeneration and functional recovery. METHODS: Rat BMSCs were infected with recombinant lentiviruses to construct TrkA-overexpressing BMSCs and TrkA-shRNA-expressing BMSCs, which were then seeded in acellular nerve allografts for bridging 10-mm rat sciatic nerve defects. RESULTS: At 8 weeks post-transplantation, compared with Vector and Control BMSCs-laden groups, TrkA-overexpressing BMSCs-laden group demonstrated obviously improved axon growth, such as significantly higher expression of myelin basic protein and superior results of myelinated fiber density, axon diameter and myelin sheaths thickness. In accordance with this increased nerve regeneration, the animals of TrkA-overexpressing BMSCs-laden group showed significantly better restoration of sciatic nerve function, manifested as greater sciatic function index value and superior electrophysiological parameters including shorter onset latency and higher peak amplitude of compound motor action potentials and faster nerve conduction velocity. However, these beneficial effects could be reversed in TrkA-shRNA-expressing BMSCs-laden group, which showed much fewer and smaller axons with thinner myelin sheaths and correspondingly poor functional recovery. CONCLUSIONS: These results demonstrated that TrkA may regulate the regenerative potential of BMSCs in nerve grafts, and TrkA overexpression can enhance the efficacy of BMSCs on peripheral nerve regeneration and functional recovery, which may help establish novel strategies for repairing peripheral nerve injuries.

Cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): therapeutic and pathophysiological implications.

The hepatic endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) are mixed-function oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic and environmental relevance. P450 ER-content and hence function is regulated by their coordinated hemoprotein syntheses and proteolytic turnover. Such P450 proteolytic turnover occurs through a process known as ER-associated degradation (ERAD) that involves ubiquitin-dependent proteasomal degradation (UPD) and/or autophagic-lysosomal degradation (ALD). Herein, on the basis of available literature reports and our own recent findings of in vitro as well as in vivo experimental studies, we discuss the therapeutic and pathophysiological implications of altered P450 ERAD and its plausible clinical relevance. We specifically (i) describe the P450 ERAD-machinery and how it may be repurposed for the generation of antigenic P450 peptides involved in P450 autoantibody pathogenesis in drug-induced acute hypersensitivity reactions and liver injury, or viral hepatitis; (ii) discuss the relevance of accelerated or disrupted P450-ERAD to the pharmacological and/or toxicological effects of clinically relevant P450 drug substrates; and (iii) detail the pathophysiological consequences of disrupted P450 ERAD, contributing to non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) under certain synergistic cellular conditions.