[摘 要] 目的：探讨同源异型盒蛋白D11（HOXD11）对Ki-67的转录调控作用及其对喉鳞状细胞癌（LSCC）细胞恶性生物学行为的影响和机制。方法：结合高通量测序数据，通过GEO、UALCAN数据库分析HOXD11在LSCC中差异表达。收集2022年1月至2025年1月联勤保障部队第九八〇医院手术切除的60例LSCC患者的癌及癌旁组织标本，以及人LSCC细胞系AMC-HN-8、TU-177、TU-686和人正常喉上皮细胞（HNLC），构建敲低或过表达HOXD11的细胞系，将细胞分为对照组、HOXD11敲低组及HOXD11过表达组。通过RT-qPCR法检测HOXD11和Ki-67基因在LSCC组织和细胞中mRNA表达水平，免疫组织化学（IHC）法分析两者在LSCC组织中的蛋白表达及分布，WB法进一步验证蛋白差异表达。采用MTS、克隆形成实验及Transwell实验分别检测敲低或过表达HOXD11对LSCC细胞增殖、迁移与侵袭的影响。通过双萤光素酶报告基因实验和染色质免疫沉淀（ChIP）实验验证HOXD11对Ki-67启动子活性的调控作用。结果：GEO和UALCAN数据库分析表明，HOXD11在LSCC中高表达（P < 0.01）。在LSCC组织中HOXD11和Ki-67 mRNA和蛋白表达均显著高于癌旁组织（均P < 0.01），同时，两者表达水平之间存在正相关（r = 0.26, P < 0.05）；LSCC细胞系中HOXD11 mRNA表达显著高于HNLC（均P < 0.01）。敲低HOXD11显著抑制LSCC细胞的增殖、迁移和侵袭能力（均P < 0.01），而过表达HOXD11则促进细胞的这些恶性生物学行为（均P < 0.01）。双萤光素酶报告基因实验及ChIP实验均证实，HOXD11可直接结合到Ki-67启动子区上，调控其表达（P < 0.01）。回复实验显示，过表达Ki-67可部分逆转敲低HOXD11对LSCC细胞增殖、迁移与侵袭的抑制作用（均P < 0.01）。结论：HOXD11在LSCC组织及细胞系中均呈高表达，其机制在于通过直接调控Ki-67的转录活性，从而增强LSCC细胞的增殖、迁移及侵袭能力。

[Abstract] Objective: To investigate the transcriptional regulation of HOXD11 (homeobox D11) on Ki-67, its effects on the malignant biological behavior of laryngeal squamous cell carcinoma (LSCC) cells and the underlying mechanisms. Methods: High-throughput sequencing data from GEO and UALCAN databases were used to analyze the differential expression of HOXD11 in LSCC. Tumor and adjacent tissue specimens from 60 patients with LSCC surgically resected at the 980 Hospital of the Joint Logistics Support Force between January 2022 and January 2025, along with human LSCC cell lines AMC-HN-8, TU-177, TU-686, and human normal laryngeal epithelial cells (HNLC) were collected. Cell lines with stable HOXD11 knockdown or overexpression were established, and the cells were divided into the control group, the HOXD11 knockdown group, and the HOXD11 overexpression group. The mRNA expression levels of HOXD11 and Ki-67 genes in LSCC tissues and cells were detected by RT-qPCR. The protein expression and distribution of HOXD11 and Ki-67 in LSCC tissues were analyzed by immunohistochemistry (IHC). Western blot (WB) further verified protein differential expression. MTS, clonogenic assay and Transwell assay were used to detect the effects of HOXD11 knockdown or overexpression on the proliferation, migration and invasion of LSCC cells. Dual luciferase reporter gene experiment and ChIP experiment were used to confirm the regulatory effect of HOXD11 on Ki-67 promoter activity. Results: GEO and UALCAN database analyses showed that HOXD11 was highly expressed in LSCC (P < 0.01). The expressions of HOXD11 and Ki-67 mRNA in LSCC tissues were significantly higher than those in adjacent tissues (both P < 0.01). At the same time, there was a positive correlation between the expressions of HOXD11 and Ki-67 mRNA (r = 0.26, P < 0.05). The expressions of HOXD11 mRNA in LSCC cell lines were significantly higher than those in HNLC (all P < 0.01). HOXD11 knockdown significantly inhibited the proliferation, migration and invasion of LSCC cells (all P < 0.01), while HOXD11 overexpression promoted these malignant biological behaviors of LSCC cells (all P < 0.01). Both dual-luciferase reporter gene assay and ChIP assay confirmed that HOXD11 could directly bind to the Ki-67 promoter region to regulate its expression (P < 0.01). The rescue experiment showed that overexpression of Ki-67 partially reversed the inhibitory effect of HOXD11 knockdown on the proliferation, migration and invasion of LSCC cells (all P < 0.01). Conclusion: HOXD11 is highly expressed in LSCC tissues and cell lines. It directly regulates Ki-67 transcriptional activity and promotes the proliferation, migration and invasion of LSCC cells.

河北省医学科学研究课题计划（20240854）