Question:
Long non-coding RNAs (lncRNAs) represent a constantly growing class of > 200 nucleotide-long
RNA molecules. They operate on an epigenetic, transcriptional and post-transcriptional
level to regulate multiple cellular functions. LncRNAs influence mechanisms like proliferation,
pluripotency and apoptosis, thereby affecting hallmarks of cancer. However, most lncRNAs
have not been explored in detail, and the role of the majority of lncRNAs in HCC is
completely unknown. The aim of this study was to identify and functionally verify
novel long non-coding RNAs in HCC using a novel qRT-PCR-based array approach.
Methods:
A qRT-PCR-based lncRNA expression array (pilot study package from Bio-Rad) was used
to analyze 87 potentially cancer-associated long non-coding RNAs. LncRNA expression
profiles of two HCC (Hep3B, PLC) cell lines were compared to primary human hepatocytes
(PHH) and activated hepatic stellate cells (aHSC) to reveal cancer(-subtype)-specific
lncRNA-expression profiles. Sanger sequencing and DNA gel electrophoresis of PCR products
served for confirmation of identified differentially expressed lncRNAs. A novel approach
for silencing of lncRNAs using specifically pre-designed si-RNA-pools consisting of
30 si-RNAs was used to study the function of lncRNAs. Boyden chamber and anchorage-dependent
clonogenicity assays were performed to study the effects of lncRNAs on migration,
invasion and stem cell properties.
Results:
Eight out of 87 lncRNAs were identified to be differentially regulated in HCC cell
lines as compared to the non-cancer cell types (primary human hepatocytes or hepatic
stellate cells). However, only two lncRNAs (lncADM-2, lncNBPF3-9) were specifically
and strongly overexpressed in HCC cells as compared to both hepatocytes and activated
stellate cells, pointing to specific de-regulation. DNA gel electrophoresis and sanger
sequencing confirmed strong expression of the predicted lncADM-2 transcript and revealed
that a predicted intron of lncNBPF3-9 is not spliced in HCC cells. First functional
experiments exploring the function of lncADM-2 show decreased migration and clonogenicity
of melanoma cells after si-RNA-pool-mediated knockdown.
Conclusions:
Our study revealed overexpression of two novel lncRNAs (lncADM-2, lncNBPF3-9) in HCC.
We found that a predicted exon of lncNBPF3-9 is not spliced in HCC cell lines resulting
in a longer, potentially functional novel transcript variant that will be further
evaluated. Moreover, a specifically designed si-RNA-pool targeting the lncADM-2 transcript
revealed this lncRNA to functionally promote migration and clonogenicity in HCC, outlining
a potential novel therapeutic target.
