After treatment with TGF-β (10 ng/ml, 96 hours) the methylation of E-cadherin promoter was induced (35.5 ± 1.96% in PLC/PRF/5), which was abrogated by pre-treatment with a methylation inhibitor 5-aza-2′-deoxycytidine (5-Aza) indicating the involvement of DNA methylation in this process. Treatment of

HCC cells with TGF-β (10 ng/ml, 72 hours) increased protein expression of DNMT3B and DNMT1, which are reported as targets of miR-29a. After treatment of cells with TGF-β (10 ng/ml), expression of miR-29a decreased by 27.8% in PLC/RF/5 cells and by 26.7% in HepG2 cells by 72 hours. Transfection of precursor miR-29a in PLC/PRF/5 cells partially blocked the suppression of E-cadherin protein expression (control, 48%; miR-29a, 72%) and methylation level of the promoter CpG islands (control, 27.3 ± 9.15%; miR-29a, 13.7 ± 2.85%) induced by TGF-β. [Conclusion] We show that the involvement

PD0325901 of miR-29a in TGF-β-induced EMT via epigenetic regulation of E-cadherin in HCC cells. These observations identify miR-29a as a unique mechanism of the regulation of EMT in HCC. Disclosures: The following people have nothing to disclose: Takayuki Kogure, Yasuteru Kondo, Eiji Kakazu, Masashi Ninomiya, Osamu Kimura, Tomoaki Iwata, Tatsuki Morosawa, Yasuyuki Fujisaka, Tooru Shimosegawa Background: selleck screening library Protein kinases MST1 & MST2 are the core of the Hippo pathway, which inactivate the transcriptional co-activator YAP through LATS phosphorylation. Inhibition of MST1 & MST2 leads to the activation of YAP where it translocates to the nucleus and promotes the medchemexpress transcription of pro proliferative genes. We hypothesize that knockdown

(KD) of MST1 & MST2 will push hepatocytes into cell cycle through activation of YAP. Methods:We are exploring a gene therapy approach using siRNAs coupled with liposomes to target the Hippo pathway to promote hepatocyte proliferation in non-regenerating livers. Results: We identified siRNA sequences that lead to 92 and 89% KD of MST1 and MST2 in a mouse liver hepatoma cell line in vitro. siRNA:liposome complexes injected i.v. resulted in 80% KD of expression in the liver using FVII as a control gene target. Using siRNAs targeting MST1 & MST2 with liposomes reduced expression to 66 and 40%, respectively in liver after 72 hours. The KD was confirmed by RT-qPCR and immunoblot. KD of MST1 and MST2 in mouse liver resulted in an increase of nuclear Yap localization and hepatocyte proliferation measured by incorporation of EdU and Ki67 immunostaining. After MST1 and MST2 KD there was a 3 and 5-fold increase of BIRC5/survivin and Foxm 1, respectively -both YAP target genes normally up-regulated in a regenerating liver. Conclusion: The KD of MST1 & MST2 provokes nuclear YAP translocation and hepatocyte proliferation in wild-type mice.