目的：探究SOX9通过上调长链非编码RNA LINC01503的表达对喉鳞状细胞癌（LSCC）细胞的增殖、迁移、侵袭及肿 瘤干细胞干性的影响。方法： 常规培养人LSCC细胞AMC-HN-8、TU177、TU212和TU686，用转染试剂将敲减序列及其对照核 酸（si-SOX9-NC、si-SOX9#1、 si-SOX9#2、si-LINC01503-NC、si-LINC01503#1、si-LINC01503#2）或过表达质粒及其对照核酸 （pcDNA3.1-SOX-NC、pcDNA3.1-SOX-oe、pcDNA3.1-LIN01503-NC和pcDNA3.1-LIN01503-oe）分别转染至TU177细胞或TU686 细胞，记为si-SOX9-NC组、si-SOX9#1组、si-SOX9#2组、si-LINC01503-NC组、si-LINC01503#1组、si-LINC01503#2组；pcDNA3.1- SOX9-NC 组 、pcDNA3.1-SOX9-oe 组 、pcDNA3.1-LINC01503-NC 组 、pcDNA3.1-LINC01503-oe 组 、si-SOX9-NC + pcDNA3.1- LINC01503-NC组和si-SOX9 + pcDNA3.1-LINC01503-oe组。qPCR法检测SOX9 mRNA和LINC01503 在各组细胞中的表达，生 物信息学分析 SOX9 与 LINC0503 启动子区的结合位点，双萤光素酶报告基因实验和染色质免疫共沉淀实验验证 SOX9 与 LINC01503启动子区是否直接结合，WB法检测SOX9的敲减效率及LINC01503对TU177和TU686细胞干性标志物表达的影响， MTS法检测各组细胞的增殖活力，划痕愈合和Transwell小室实验检测各组细胞的迁移能力，克隆形成实验检测各组细胞的克隆 形成能力。结果：SOX9在各种LSCC细胞中呈高表达（均P < 0.05），数据库数据分析显示，在头颈部鳞状细胞癌中，SOX9与 LINC01503表达呈正相关（R = 0.12，P = 0.005 9）；SOX9可与LINC01503启动子区直接结合并促进其转录表达（均P < 0.05）；敲减 LINC01503可明显抑制TU177细胞的增殖、迁移、侵袭（均P < 0.05），过表达LINC01503明显促进TU686细胞增殖、迁移、侵袭的 能力（均P < 0.05），提高TU686 细胞克隆形成能力和细胞干性标志物分子 CD133、OCT4、SOX2的mRNA和蛋白水平表达 （均P < 0.05），敲减LINC01503则均可抑制TU686细胞的克隆形成和细胞干性标志物的表达（均P < 0.05）；敲减SOX9均可明显 抑制TU177细胞的增殖、迁移和侵袭能力，降低其干性细胞标志物的表达（均P < 0.05），同时过表达LINC01503则可部分逆转敲 减SOX9对TU177细胞恶性生物学行为和干性标志物表达的抑制作用（均P < 0.05）。结论：SOX9和LINC01503在LSCC细胞中 呈高表达，SOX9可能通过上调LINC01503表达提高LSCC细胞增殖、转移和侵袭能力和肿瘤干细胞干性。

Objective: To investigate the influence of SOX9 on the proliferation, migration, invasion, and stemness of laryngeal squamous cell carcinoma (LSCC) cells by upregulating the expression of long non-coding RNA LINC01503. Methods: Human LSCC cells AMC-HN-8, TU177, TU212, and TU686 were cultured routinely. Control nucleic acids (NC) and knockdown sequences (si-SOX9- NC, si-SOX9#1, si-SOX9#2, si-LINC01503-NC, si-LINC01503#1, si-LINC01503#2) or overexpression plasmids (pcDNA3.1-SOXNC, pcDNA3.1-SOX-oe, pcDNA3.1-LIN01503-NC, and pcDNA3.1-LIN01503-oe) were transfected into TU177 cells or TU686 cells. These groups were named as si-SOX9-NC group, si-SOX9#1 group, si-SOX9#2 group, si-LINC01503-NC group, si-LINC01503#1 group, si-LINC01503#2 group; pcDNA3.1-SOX9-NC group, pcDNA3.1-SOX9-oe group, pcDNA3.1-LINC01503-NC group, pcDNA3.1-LINC01503-oe group, si-SOX9-NC + pcDNA3.1-LINC01503-NC group, and si-SOX9 + pcDNA3.1-LINC01503-oe group. The expression of SOX9 mRNA and LINC01503 in each group of cells was detected by qPCR. Bioinformatics tool was employed to identify the binding site of SOX9 to lncRNA LINC01503 promoter region. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene experiments were carried out to validate the binding between SOX9 and the LINC01503 promoter. SOX9 knockdown efficiency and the influence of LINC01503 on the expression of stem cell markers in TU177 and TU686 cells were detected by WB assay. MTS assay was used to detect cell proliferation, scratch wound healing, and Transwell assays assessed cell migration, while colony formation assays evaluated the ability of cells to form colonies. Results: SOX9 was highly expressed in all LSCC cells (R = 0.12, P < 0.05). Database analysis showed a positive correlation between SOX9 and LINC01503 expression in head and neck squamous cell carcinoma (P = 0.005 9). It was proved that SOX9 directly binds with the LINC01503 promoter and promotes its transcriptional expression (P < 0.05). Knockdown of LINC01503 significantly suppressed the proliferation, migration, and invasion of TU177 cells (all P<0.05). Overexpression of LINC01503 notably enhanced the proliferation, migration, invasion and colony formation of TU686 cells (all P<0.05), as well as the expression of cell stemness markers CD133, OCT4, and SOX2 at both mRNA and protein levels (all P<0.05). However, knockdown of LINC01503 inhibited colony formation and expression of stem cell markers in TU686 cells (P < 0.05). Knockdown of SOX9 significantly suppressed the proliferation, migration, and invasion ability of TU177 cells and reduced the expression of their stem cell markers (all P<0.05). Meanwhile, overexpression of LINC01503 partially reversed the inhibitory effect of SOX9 knockdown on the malignant biological behavior and stem cell marker expression in TU177 cells (all P < 0.05). Conclusion: Both SOX9 and LINC01503 are highly expressed in LSCC cells. SOX9 may promote the proliferation, migration, invasion and stemness of laryngeal squamous cell carcinoma by upregulating LINC01503 expression.

河北省重点研发计划项目（No. 20377709D，22377736D）；河北省自然科学基金项目（No. H2022206326）