DNAJA1 Stabilizes EF1A1 to Promote Cell Proliferation and Metastasis of Liver Cancer Mediated by miR-205-5p

Figure 5

DNAJA1 was directly targeted by miR-205-5p and interacted with EF1A1. (a) Predicted microRNAs which targeted to DNAJA1 were predicted by three common bioinformatic algorithms. (b) Luciferase assay analyses of the indicated cells transfected with the indicated reporters with miR-205-5p, miR335-5p, miR579-3p, and miR-217. (c) Western blot analysis of DNAJA1 in HCC-M3 cells transfected with miR-205-5p, miR335-5p, miR579-3p, and miR-217 mimics. (d) Luciferase assay analyses of HCC-M3 cells simultaneously transfected with the 3-UTRs of WT and mutant DNAJA1 and miR-205-5p mimics. (e) Western blot analysis of DNAJA1 in HCC-M3 cells transfected with miR-205-5p with or without DNAJA1. (f) Mutual combination between DNAJA1 and EF1A1 in Huh 7 cells by Co-IP assay. (g) Colocalization of DNAJA1 and EF1A1 by immunofluorescence (blue: DAPI, green: DNAJA1, and red: EF1A1). (h) The protein level of DNAJA1 and EF1A1 in DNAJA1 overexpressing or inhibiting cells by western blot. (i) Effect of time-course inhibition of DNAJA1 on the expression of EF1A1 and the status of its ubiquitination by western blot. (j) Effect of miR-205-5p on the expression of EF1A1 and DNAJA1 and ubiquitin in HCC-M3 cells by western blot. GAPDH was used as internal control.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)