Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo.
Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation.


CONCLUSIONS
A novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model.
Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, UK.
Vikash
V
Reebye
Pål
P
Sætrom
Paul J
PJ
Mintz
Kai-Wen
KW
Huang
Piotr
P
Swiderski
Ling
L
Peng
Cheng
C
Liu
Xiaoxuan
X
Liu
Steen
S
Lindkaer-Jensen
Dimitris
D
Zacharoulis
Nikolaos
N
Kostomitsopoulos
Noriyuki
N
Kasahara
Joanna P
JP
Nicholls
Long R
LR
Jiao
Madhava
M
Pai
Duncan R
DR
Spalding
Malkhaz
M
Mizandari
Tinatin
T
Chikovani
Mohamed M
MM
Emara
Abdelali
A
Haoudi
Donald A
DA
Tomalia
John J
JJ
Rossi
Nagy A
NA
Habib
D000418:Albumins / Q000378:metabolism; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000150:complications / Q000188:drug therapy* / Q000473:pathology; D004353:Drug Evaluation, Preclinical; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D056945:Hep G2 Cells; D006801:Humans; D007275:Injections, Intravenous; D008099:Liver / Q000473:pathology; D008103:Liver Cirrhosis / Q000150:complications; D008111:Liver Function Tests; D008114:Liver Neoplasms, Experimental / Q000150:complications / Q000188:drug therapy* / Q000473:pathology; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D012313:RNA / Q000627:therapeutic use*; D051381:Rats; D017208:Rats, Wistar; D019947:Receptors, Interleukin-6 / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism
D000418:Albumins; D022763:CCAAT-Enhancer-Binding Protein-alpha; C508603:IL6R protein, human; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D019947:Receptors, Interleukin-6; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D012313:RNA