ABSTRACT
RNA sequencing (RNA-seq) is emerging in genetic diagnoses as it provides functional support for the interpretation of variants of uncertain significance. However, the use of amniotic fluid (AF) cells for RNA-seq has not yet been explored. Here, we examined the expression of clinically relevant genes in AF cells (n = 48) compared with whole blood and fibroblasts. The number of well-expressed genes in AF cells was comparable to that in fibroblasts and much higher than that in blood across different disease categories. We found AF cells RNA-seq feasible and beneficial in prenatal diagnosis (n = 4) as transcriptomic data elucidated the molecular consequence leading to the pathogenicity upgrade of variants in CHD7 and COL1A2 and revising the in silico prediction of a variant in MYRF. AF cells RNA-seq could become a reasonable choice for postnatal patients with advantages over fibroblasts and blood as it prevents invasive procedures.
ABSTRACT
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common human cancers. Recently, emerging evidence has suggested the role of long non-coding RNAs (lncRNAs) in human carcinogenesis. In this study, we aimed to investigate the expression and functional implications of lncRNAs in human HCC. METHODS: Eighty-eight well-annotated lncRNAs were profiled in primary HCC by quantitative RT-PCR. Functional relevance of lncRNAs was elucidated in HCC cell lines and nude mice models. The regulatory relationship between miRNA and lncRNA was predicted in silico and further validated by luciferase reporter assay and expression analysis. RESULTS: In our profiling study, HOTTIP was identified as the most significantly up-regulated lncRNA in human HCCs, even in early stage of HCC formation. Functionally, knock-down of HOTTIP attenuated HCC cell proliferation in vitro and markedly abrogated tumourigenicity in vivo. In addition, knock-down of HOTTIP also inhibited migratory ability of HCC cells and significantly abrogated lung metastasis in orthotopic implantation model in nude mice. HOTTIP is an antisense lncRNA mapped to the distal end of the HOXA gene cluster. Knock-down of HOTTIP significantly suppressed the expression of a number of HOXA genes. Furthermore, we identified miR-125b as a post-transcriptional regulator of HOTTIP. Ectopic expression of miR-125b reduced HOTTIP-coupled luciferase activity and suppressed the endogenous level of HOTTIP. Moreover, in human HCCs, HOTTIP expression negatively correlated with that of miR-125b. CONCLUSIONS: HOTTIP is a novel oncogenic lncRNA, which negatively regulated by miR-125b. Overexpression of HOTTIP contributes to hepatocarcinogenesis by regulating the expression of its neighbouring protein-coding genes.
