Fig. 5. LPIN1 is a target of miR-451a. A, Flow chart of miR-451a target screening. B, 293T cells were co-transfected with miR-451a mimics and wild-type promoter luciferase reporter constructs of LPIN1. Luciferase activities were determined 36 h after transfection. Luciferase values were normalized to Renilla activities. C, miR-451a binding site in the predicted target sequences of LPIN1 3′-UTR is indicated. D, 293T cells were transfected with wild-type or mutant LPIN1 promoter luciferase reporter constructs in the presence or absence of miR-451a mimics. Luciferase activities were determined 36 h after transfection. Luciferase values were normalized to Renilla activities. E and F, Different concentrations of miR-451a mimics were transfected into SMMC-7721 cells (E) and human umbilical vein endothelial cells (HUVECs) (F) for 48 h. The expression of LPIN1 was detected by western blotting. G, The expression of LPIN1 was determined by immunohistochemistry (IHC) in tumor and adjacent non-tumor tissues from hepatocellular carcinoma (HCC) patients. H, Spearman's correlation analysis was performed to determine the relationship between miR-451a and LPIN1 expression in HCC patients. miR-451a levels were obtained from quantitative PCR and LPIN1 expression was based on IHC scores. NC, negative control; *P<0.05, ***P<0.001.