Abstract
BackgroundBone metastasis (BM) has long been recognized as a major threat to the quality of life of hepatocellular cancer (HCC) patients. While LncRNA34a (Lnc34a) has been shown to regulate colon cancer stem cell asymmetric division, its effect on HCC BM remains unknown.MethodsIn situ hybridization and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of Lnc34a in HCC tissues and cell lines. Ventricle injection model was constructed to explore the effect of Lnc34a on BM in vivo. The methylation of miR-34a promoter and histones deacetylation were examined by using bisulfate-sequencing PCR and chromatin immunoprecipitation assays. RNA pull down and RNA immunoprecipitation were performed to investigated the interaction between Lnc34a and epigenetic regulators. Dual-luciferase reporter assay was conducted to find miR-34a target. The involvement of TGF-β pathway in the BM from HCC was determined by qRT-PCR, western, and elisa assays.ResultsWe found that Lnc34a was significantly overexpressed in HCC tissues and associated with BM. Both in vitro and in vivo experiments indicate that the restoration or knockdown of Lnc34a expression in HCC cells had a marked effect on cellular migration, invasion, and metastasis. Mechanistic analyses suggested that Lnc34a epigenetically suppresses miR-34a expression through recruiting DNMT3a via PHB2 to methylate miR-34a promoter and HDAC1 to promote histones deacetylation. On the other hand, miR-34a targets Smad4 via the TGF-β pathway, followed by altering the transcription of the downstream genes (i.e., CTGF and IL-11) that are associated with BM.ConclusionsOur study is the first to document the pro-bone metastatic role of Lnc34a in BM of HCC and reveal a novel mechanism for the activation of the TGF-β signaling pathway in HCC BM, providing evidence of a potential therapeutic strategy in HCC BM.
Highlights
Bone metastasis (BM) has long been recognized as a major threat to the quality of life of hepatocellular cancer (HCC) patients
Aberrant expression of Lnc34a in human HCC To examine the relative expression of Lnc34a in the immortalized normal human hepatocyte cell line L02 and a serious of HCC cells with different metastatic potential, quantitative real-time polymerase chain reaction (qRT-PCR) was performed
Subcellular fractionation assays showed that Lnc34a was mainly in the nuclear of HCC-LM3 cell (Fig. 1b) and SMMC-7721 cell (Fig. 1c), and RNA fluorescence in situ hybridization (RNA fluorescence in Situ Hybridization (FISH)) suggest that Lnc34a was primarily located in the nucleus (Fig. 1d); Lnc34a may function via epigenetic regulation
Summary
Bone metastasis (BM) has long been recognized as a major threat to the quality of life of hepatocellular cancer (HCC) patients. While LncRNA34a (Lnc34a) has been shown to regulate colon cancer stem cell asymmetric division, its effect on HCC BM remains unknown. The majority of lncRNAs are found in the cell nucleus [16] where they regulate gene expression via epigenetic modifications, on both a transcriptional and posttranscriptional level [17]. Lnc34a is a newly characterized lncRNA consisting of 693 bp that has no protein coding potential and epigenetically silences miR-34a expression, thereby initiating the asymmetric division of colon cancer stem cells [18]. The role of Lnc34a in BM in the context of HCC remains unknown
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