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. 2018 Jan 1;8(2):423–436. doi: 10.7150/thno.22377

Figure 2.

Figure 2

MiR-193a-3p targets not only GRB7, but other key factors along MAPK/ERK signaling pathway in ovarian cancer cells. (A) The Venn diagram displays 77 common putative targets of miR-193a-3p predicted from the three algorithms, miRDB, miRanda and TargetScan Human 5.2, including three putative targets, ERBB4, KRAS and SOS2, involved in the MAPK/ERK signaling pathway. (B) Functional annotation analysis using the DAVID functional annotation tool (http://david.abcc.ncifcrf.gov/) showing the main signaling pathways associated with these 78 common targets of miR-193a-3p. (C) Duolink proximity ligation assay (PLA) with fluorescence and confocal microscopy showing the interactions between GRB7 and SOS2 and between GRB7 and KRAS (red dots) by transient transfection of GRB7-expressing plasmid (Upper). Anti-Myc, anti-SOS2 and anti-KRAS were used to detect Myc-tagged GRB7 and endogenous SOS2 and KRAS. Transient transfection of HA-SOS2 also showing the interaction between SOS2 and KRAS (red dots) using PLA and anti-HA as well as anti-KRAS (Lower). Scale bar, 20 μm. n = 3 independent experiments. (D) Relative luciferase activity of luciferase reporters with wild-type ERBB4, SOS2 and KRAS 3'UTRs co-transfected with miR-193a-3p. (E) Western blot analysis showing that transient transfection of GFP/GRB7-expressing plasmid profoundly elevated ERK activity in HEK293, SKOV3 and OVCA433 cells. (F) Western blot analysis indicating that transient transfection of pmR-ZsGreen1-miR-193a-3p reduced the expression of GRB7, ERBB4, SOS2, KRAS and pERK1/2 in SKOV3 cells. Cell lysates of all the above cell transfectants were harvested 24 h after cell transfection.