We first used a multipathway reporter array to explore the potential signaling pathway of miR-140-5p regulated. As shown in Fig. 4A, miR-140-5p expression attenuated the activity Osimertinib manufacturer of TGF-β and mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK/ERK) signaling, both of which are crucial for the regulation of cell migration.20-22 We therefore focused on these two pathways to search for potential targets based on those genes with oncogenic properties using the miRanda, TargetScan, and PicTar algorithms, and only those targets detected by all programs were considered. Interestingly,

TGFBR1 and FGF9 were found to be the direct downstream targets, and they are implicated in TGF-β and MAPK/ERK signaling, Midostaurin respectively. To demonstrate that miR-140-5p binds to the 3′-UTR of TGFBR1 and FGF9, we performed miR-140-5p-based luciferase assay using the constructs described in Fig. 4B. As expected, miR-140-5p directly bound to TGFBR1 and FGF9 3′-UTR, and by which it remarkably reduced luciferase activities, whereas cells

with mutant TGFBR1 and FGF9 3′-UTR displayed much higher luciferase activities (Fig. 4C). Moreover, western blot analysis and immunostaining further demonstrated that ectopic miR-140-5p dramatically suppressed the endogenous protein levels for TGFBR1 and FGF9 in HCCLM3 and MHCC97-H cells (Fig. 4D,E). Consistent with these results, attenuated expression for Smad3, p-ERK, and H-Ras were noted in miR-140-5p-transduced cells (Fig. 4D,E). Taken together, these results

indicated that TGFBR1 and FGF9 were direct downstream targets for miR-140-5p in HCC cells. The above results prompted us to examine whether miR-140-5p suppresses HCC growth and metastasis through repression of TGFBR1 and FGF9 signaling. For this purpose, we first examined whether blockage of TGFBR1 and FGF9 would mimic the effect of miR-140-5p expression. We introduced siRNA for TGFBR1, FGF9, and both TGFBR1 and FGF9 into HCCLM3 cells. Western blot analysis confirmed that the expression of TGFBR1 and FGF9 was inhibited (Supporting Fig. 2). As expected, compared to the control group, HCCLM3 cells transfected with TGFBR1 and MCE公司 FGF9 siRNA displayed poor wound healing (Fig. 5B) and suppressed invasive activity (Fig. 5C). Interestingly, cell proliferation assay (Fig. 5D), cell cycle analysis (Fig. 5E), and colony formation assay (Fig. 5F) confirmed that HCCLM3 cells treated with FGF9 siRNA resembled the effect of ectopic miR-140-5p expression on HCCLM3 cells, and importantly, this phenotype was not produced in cells transfected with TGFBR1 siRNA alone. Nevertheless, ectopic TGFBR1 and FGF9 expression in miR-140-5p-transduced cells attenuated the inhibitory effect of miR-140-5p on HCC growth and metastasis (Fig.