HOXA11启动子高甲基化通过激活TGF-β/Smad-EMT轴促进肝细胞癌转移

Promoter hypermethylation of HOXA11 promotes hepatocellular carcinoma metastasis through activation of the TGF-β/Smad-EMT axis

摘要:

背景与目的 肝细胞癌（HCC）侵袭转移是导致患者预后不良的重要原因。同源框A11（HOXA11）在多种肿瘤中具有组织特异性的生物学功能，但其在HCC中的作用及分子机制尚未明确。本研究探讨HOXA11表达失调的表观遗传学机制及其对HCC侵袭转移的调控作用。方法 收集11对HCC及癌旁组织，采用Western blot、免疫组织化学及重亚硫酸盐测序（BSP）检测HOXA11表达及启动子甲基化水平。利用地西他滨（DAC）进行去甲基化干预，构建HOXA11稳定敲低和过表达细胞系，通过CCK-8和Transwell实验检测细胞增殖、迁移及侵袭能力。采用Western blot检测TGF-β/Smad通路及上皮-间充质转化（EMT）相关蛋白表达，并通过TGF-β1挽救实验验证其调控机制。建立裸小鼠肺转移模型评价HOXA11对体内转移能力的影响。结果 HCC组织中HOXA11表达显著低于癌旁组织，而其启动子甲基化水平明显升高（均P<0.05）。DAC处理可降低HOXA11启动子甲基化水平并恢复其表达。HOXA11敲低显著促进HCC细胞增殖、迁移和侵袭，而过表达则产生相反作用（均P<0.05）。机制研究显示，HOXA11负向调控TGF-β/Smad信号通路活性，抑制EMT进程，表现为E-cadherin表达升高以及Snail、N-cadherin、vimentin和fibronectin表达降低。TGF-β1挽救实验进一步证实，HOXA11过表达能够部分拮抗TGF-β1诱导的Smad磷酸化及EMT表型转化。临床样本分析显示，HOXA11低表达与TGF-β/Smad通路激活及EMT标志物异常表达显著相关。动物实验表明，HOXA11过表达可显著降低肺转移负荷（P<0.05）。结论 HOXA11在HCC中因启动子高甲基化而表达沉默，其通过负向调控TGF-β/Smad信号通路抑制EMT进程，从而抑制HCC侵袭和转移。HOXA11可能成为HCC表观遗传治疗及抗转移干预的潜在靶点。

Abstract:

Background and Aims Invasion and metastasis are major causes of poor prognosis in hepatocellular carcinoma (HCC). Although homeobox A11 (HOXA11) has been implicated in various malignancies, its biological role and underlying mechanisms in HCC remain unclear. This study investigated the epigenetic regulation of HOXA11 and its effects on HCC progression and metastasis.Methods Eleven paired HCC and adjacent non-tumor tissues were collected. HOXA11 expression and promoter methylation were examined using Western blotting, immunohistochemistry, and bisulfite sequencing PCR (BSP). Decitabine (DAC) was used for demethylation treatment. Stable HOXA11 knockdown and overexpression cell lines were established. Cell proliferation, migration, and invasion were assessed by CCK-8 and Transwell assays. Expression of the TGF-β/Smad pathway- and epithelial-mesenchymal transition (EMT)-related proteins was analyzed by Western blotting. TGF-β1 rescue experiments were performed to verify the underlying mechanism. A nude mouse lung metastasis model was established to evaluate the effect of HOXA11 on metastatic potential in vivo.Results HOXA11 expression was significantly decreased in HCC tissues, whereas promoter methylation was significantly increased compared with adjacent tissues (both P<0.05). DAC treatment reduced promoter methylation and restored HOXA11 expression. HOXA11 knockdown significantly enhanced HCC cell proliferation, migration, and invasion, whereas HOXA11 overexpression exerted the opposite effects (all P<0.05). Mechanistically, HOXA11 negatively regulated the TGF-β/Smad signaling pathway and suppressed EMT, as evidenced by increased E-cadherin expression and decreased expression of Snail, N-cadherin, vimentin, and fibronectin. TGF-β1 rescue experiments further demonstrated that HOXA11 overexpression partially antagonized TGF-β1-induced Smad phosphorylation and EMT progression. Clinical analyses confirmed significant associations between low HOXA11 expression, activation of the TGF-β/Smad pathway, and EMT-related alterations. In vivo, HOXA11 overexpression significantly reduced pulmonary metastatic burden (P<0.05).Conclusion HOXA11 is epigenetically silenced through promoter hypermethylation in HCC. By negatively regulating the TGF-β/Smad signaling pathway and suppressing EMT, HOXA11 inhibits HCC invasion and metastasis. These findings suggest that HOXA11 may serve as a potential therapeutic target for epigenetic and anti-metastatic intervention in HCC.