Department of Radiation Oncology, Zhongshan Hospital, Fudan University, 180 Feng Lin Road, Shanghai, 200032, China.
Department of Radiation Oncology, Shanghai East Hospital, Tongji University School of Medicine, 150 Jimo Road, Shanghai, 200120, China.
Mol Cancer. 2019 Jul 26;18(1):120. doi: 10.1186/s12943-019-1044-9.
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.
In 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.
We 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.
Our 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.
骨转移（BM）一直被认为是肝癌（HCC）患者生活质量的主要威胁。虽然 LncRNA34a（Lnc34a）已被证明可调节结肠癌干细胞的不对称分裂，但它对 HCC BM 的影响尚不清楚。
原位杂交和实时定量聚合酶链反应（qRT-PCR）用于检测 HCC 组织和细胞系中 Lnc34a 的表达。构建脑室注射模型，探讨 Lnc34a 对体内 BM 的影响。通过 bisulfate-sequencing PCR 和染色质免疫沉淀测定检测 miR-34a 启动子的甲基化和组蛋白去乙酰化。进行 RNA 下拉和 RNA 免疫沉淀实验，以研究 Lnc34a 与表观遗传调控因子之间的相互作用。双荧光素酶报告基因检测法寻找 miR-34a 靶标。通过 qRT-PCR、western blot 和 ELISA 测定法确定 HCC BM 中 TGF-β 途径的参与情况。
我们发现 Lnc34a 在 HCC 组织中显著过表达，并与 BM 相关。体外和体内实验均表明，在 HCC 细胞中恢复或敲低 Lnc34a 表达对细胞迁移、侵袭和转移有明显影响。机制分析表明，Lnc34a 通过 PHB2 招募 DNMT3a 对 miR-34a 启动子进行甲基化，通过 HDAC1 促进组蛋白去乙酰化，从而在表观遗传学上抑制 miR-34a 的表达。另一方面，miR-34a 通过 TGF-β 途径靶向 Smad4，随后改变与 BM 相关的下游基因（即 CTGF 和 IL-11）的转录。
本研究首次证明了 Lnc34a 在 HCC BM 中的促骨转移作用，并揭示了 HCC BM 中 TGF-β 信号通路激活的新机制，为 HCC BM 的潜在治疗策略提供了证据。
