Hepatocellular carcinoma (HCC) is generally accompanied by high mortality and low cure rate. CCAAT enhancer-binding proteins (CEBPs) are transcriptional regulators that play a key role in maintaining liver function. Altered expression of C/EBP alpha and C/EBP beta occurs in many tumours including HCC. saRNAs are small double-stranded RNAs that enhance target gene expression at the transcriptional level. In this report, we activate CEPBA with saRNAs and suppress CEBPB with siRNAs in cells that represent three different degrees of HCC. We performed functional assays to investigate the effects of enhancing C/EBP alpha and its downstream targets, p21 and albumin across these lines. We also used Mass-spectrometry (MS) subsequent to a ChIP pull-down assay to characterise the components of the protein complex involved in regulating saRNA function. Putative saRNA interacting protein candidates that were identified by MS were knocked-down with siRNAs to investigate its impact on saRNA activity. We confirmed CEBPA-saRNA decreased proliferation and migration in the differentiated lines (HepG3/Hep3B). The undifferentiated line (PLCPRF5) showed saRNA-induced increase in CEBPA but with no loss in proliferation. This effect was reversed when CEBPB was suppressed with CEBPB-siRNA. When interrogating saRNA mode of action; three saRNA interacting proteins, CTR9, HnRNPA2/B1 and DDX5 were identified by MS. Targeted knock-down of these two proteins (by siRNA) abrogated saRNA activity. This study provides insight into how different HCC lines are affected by CEBPA-saRNAs and that endogenous abundance of CEBPB and saRNA accessory proteins may dictate efficacy of CEBPA-saRNA when used in a therapeutic context.
[1]Imperial Coll London, Dept Surg & Canc, Hammersmith Hosp, HPB Surg Unit, London W12 0HS, England;
[2]Fudan Univ, Zhongshan Hosp, Dept Surg, Shanghai 200032, Peoples R China;
[3]Harbin Med Univ, Affiliated Hosp 1, Dept Gen Surg, Harbin 150001, Heilongjiang, Peoples R China;
[4]Imperial Coll London, Dept Life Sci, London SW7 2AZ, England;
[5]Norwegian Univ Sci & Technol, Dept Canc Res & Mol Med, NO-7489 Trondheim, Norway;
[6]Norwegian Univ Sci & Technol, Dept Comp Sci, NO-7489 Trondheim, Norway;
[7]City Hope Natl Med Ctr, Beckman Res Inst, Div Mol Biol, Duarte, CA 91010 USA;
[8]Natl Taiwan Univ Hosp, Dept Surg, Taipei, Taiwan;
[9]Natl Taiwan Univ Hosp, Hepatitis Res Ctr, Taipei, Taiwan;
[10]Natl Taiwan Univ, Grad Inst Clin Med, Taipei, Taiwan
(8.0+极速模式)