Fig. 5. miR92a-3p promotes EMT by regulating Akt/Snail pathway via targeting PTEN.

Western blot analysis showing the effect of upregulated miR-92a-3p on the protein level of ZO-1, E-cadherin, N-cadherin, β-catenin, and Snail in 97h and Huh7 (a). Representative immunofluorescent images of E-cadherin and N-cadherin in Huh7 was shown with a magnification of ×600 (Blue: DAPI, Green: E-cadherin or N-cadherin) (b). The downstream mRNA was predicted by four miRNA databases, and real-time PCR results showing a significant change of mRNA level (with PTEN being the most significant) in 97h cells after being transfected with miR92a-3p mimics or negative control mimics (c). Sequence alignment between miR-92a-3p and PTEN mRNA shows two mRNA segments of PTEN match with the seed sites of miR92a-3p, and nucleotide substitution mutation of the seed sites of PTEN constructed accordingly, and Luciferase reporter gene assay was performed in triplicate in 97H cells. Inhibitory luciferase activities were observed after transfection of miR92a-3p mimics, representative data were shown in the bar graphs, ***P < 0.001 (d). Western blot results show the changing protein level of p-GSK 3β, mTOR, Akt, PTEN, phosphorylated mTOR, and phosphorylated Akt in HCC after transfected with miR mimics (e). 97h-luciferase cells were used to construct lung metastasis models with different treatments. Representative bioluminescent imaging of mice was performed after 8 weeks, and luciferin was intraperitoneally injected via the IVIS spectrum. The image of the metastatic condition of mice with different treatments was shown, n = 4 mice per group (f). Bars: (b) 10 μm.