Experimental Studies Of MiR-34a Combined Palmitic Acid Promote Hepatic Cell Senescence

Objective: With the change of lifestyle and diet, the incidence of fatty liver disease isincreasing in recent years, becoming an important factor endangering public health.Saturated free fatty acids can induce hepatic steatosis, through the "two-hit" led to thedevelopment of fatty liver disease. Studies have found that non-coding RNA miR-34a highexpressed in fatty liver disease. Therefore, the present study intends to establish a liver cellinjury model by combining free fatty acids and miR-34a to explore their effects on thebiological activity of the liver cells, to provide an initial basis for further exploration of thepathogenesis of fatty liver disease and find effective prevention and treatment measures.Methods:(1) Plasmids pEGFP-C1and pEGFP-C1/miR-34a were transiently transfectedinto HepG2cells and green fluorescent protein (GFP) was observed after24hours andcells were collected for RT-PCR after36hours.(2) MTT was used to detect theproliferation of HepG2cells treated with PA and transiently transfected pEGFP-C1andpEGFP-C1/miR-34a.(3) The cells were divided into four groups, including normal controlgroup, cells treated PA group, cells transfected with miR-34a group, cells treated PA andmiR-34a. Morphology of Cell nucleus was observed after stained with DAPI; meanwhile,cell senescence was determined by a specific β-galactosidase kit.(4) Cellsenescence-associated genes P21/P53and cell cycle-related genes CDK6/CyclinE2/E2F1and inflammatory cytokines TNF-α/MCP were detected by RT-PCR after collecting cellsfrom four groups.(5) Western blot was used to measure the expression of cell cycle-relatedprotein CDK6and cell senescence-associated protein Sirt1.Results:(1) Green fluorescent is observed by microscope after transfected pEGFP-C1andpEGFP-C1/miR-34a24hours, RT-PCR show that cells transfected with miR-34a arehighly expressed the green fluorescent.(2) MTT indicates that PA can inhibit cellproliferation with the increasing of concentration, the cell senescence is clearly induced bycombining treated PA and miR-34a.(3) senescent cells nucleus are bigger than normalafter stained with DAPI and the application of test kit also found that the experimentalcells specific enzyme β-galactosidase dark blue (4) RT-PCR show that combining treatedPA and transfected with miR-34a group cellular senescence-associated genes P21/P53/p16and inflammatory cytokines TNF-α/MCP are highly expressed compared with other threegroups, but cell cycle-related gene CDK6is low expressed.(5) Result from western blotreveals that the expression of cell cycle-related protein CDK6and cell senescence-associated protein Sirt1are inhabited.Conclusion: The research clearly show that combining PA and miR-34a can promoteintracellular β-galactosidase and block cell cycle and also prompt the important role of fatand miR-34a in the liver cell senescence and the potential target for drug intervention.