目的：探讨LINC00958/血管内皮生长因子 C（VEGF-C）信号通路在宫颈癌的淋巴管生成和淋巴转移中的作用。方法：从2020 年9月至2022 年9月期间在河南省人民医院接受手术的患者中收集了42 例宫颈癌组织标本，通过qPCR检测宫颈癌组织和宫颈癌细胞（Hela、C33A、SiHa、Caski）中LINC00958 的表达情况。将LINC00958 过表达载体（LINC00958 组）或对照载体（CMV 组）转染Caski 细胞，敲减LINC00958（shLINC00958 组）、VEGF-C（shVEGF-C 组）的shRNA 序列或阴性对照shRNA （shNC 组）转染SiHa 细胞。分别通过CCK-8法、Transwell 实验检测过表达或敲减LINC00958 对宫颈癌细胞增殖、迁移和侵袭的影响。观察转染后细胞的培养上清液对人淋巴管内皮细胞（HLEC）淋巴管形成能力的影响。建立小鼠腘淋巴结转移模型，观察过表达LINC00958 或同时敲减VEGF-C对宫颈癌淋巴结转移的影响。结果：LINC00958 在宫颈癌组织中呈高表达（P<0.001），高水平的LINC00958 与大肿瘤、晚期肿瘤分级、浸润深度和淋巴转移有关联（P<0.05 或P<0.01）。与正常人宫颈上皮细胞ende1617相比，宫颈癌细胞中LINC00958 水平均显著升高（P<0.01 或P<0.001）。shLINC00958 组SiHa 细胞的增殖、迁移、侵袭能力及其培养上清液的促HLEC淋巴管形成能力均显著低于shNC 组（P<0.05、P<0.01 或P<0.001），LINC00958 组Caski 细胞的增殖、迁移、侵袭能力及其培养上清液的促HLEC淋巴管形成能力显著高于CMV组（P<0.05、P<0.01或P<0.001）。通过RNA下拉、RNA免疫沉淀实验发现宫颈癌细胞中LINC00958 能够特异性结合VEGF-C。LINC00958+shVEGF-C 组Caski 细胞的增殖、迁移、侵袭能力及其培养上清液的促淋巴管形成能力显著低于LINC00958 组（P<0.01 或P<0.001）；在小鼠腘淋巴结转移模型中，LINC00958+ shVEGF-C 组中小鼠腘窝淋巴结的体积和VEGF-C 蛋白、N-cadherin 蛋白以及LYVE-1的阳性细胞比例均显著低于LINC00958 组（均P<0.001）。结论：LINC00958通过直接与VEGF-C蛋白相互作用增强宫颈癌细胞的增殖、侵袭、淋巴管生成能力，促进小鼠腘淋巴结转移模型的淋巴结转移。

Objective: To investigate the role of LINC00958/vascular endothelial growth factor C (VEGF-C) signaling pathway in lymphovascular formation and lymphatic metastasis in cervical cancer. Methods: 42 samples of cervical cancer tissues and corresponding adjacent tissues were collected from patients who underwent surgery at Henan Provincial People's Hospital between September 2020 and September 2022. The expressions of LINC00958 in cervical cancer specimens and cervical cancer cells (Hela, C33A, SiHa, Caski) were examined by qPCR. Caski cells were transfected with LINC00958 overexpression vector (LINC00958 group) or vehicle control (CMV group), and SiHa cells were transfected with shRNA sequences of knocked-down LINC00958 (shLINC00958 group) and VEGF-C (shVEGF-C group), or negative control shRNA (shNC group). The effects of overexpressed or knocked-down LINC00958 on the proliferation, migration and invasion of cervical cancer cells were detected by CCK-8 method and Transwell assay, respectively. The effect of the culture supernatant of transfected cells on the lymphovascular formation capacity of human lymphatic endothelial cells (HLEC) was observed. A mouse popliteal lymph node metastasis model was established to investigate the effects of overexpressed LINC00958 or simultaneous knockdown of VEGF-C on cervical cancer lymph node metastasis. Results: LINC00958 was highly expressed in cervical cancer tissues (P<0.001), and high levels of LINC00958 were associated with large tumors, advanced tumor grade, depth of invasion, and lymphatic metastasis (P<0.05 or P<0.01). Compared with that in the normal human cervical epithelial cells ende1617, the level of LINC00958 in cervical cancer cells was significantly increased (P<0.01 or P<0.001). The proliferation, migration and invasion abilities of SiHa cells in the shLINC00958 group and the HLEC lymph vessel pro-formation ability of the culture supernatant were significantly lower than those in the shNC group (P<0.05, P<0.01 or P<0.001), and the proliferation, migration and invasion abilities of Caski cells in the LINC00958 group and the HLEC lymph vessel pro-formation ability of the culture supernatant were significantly higher than those in the CMV group (P<0.05, P<0.01 or P<0.001). RNA pull-down and RNA immunoprecipitation assays showed that LINC00958 in cervical cancer cells could specifically bind to VEGF-C. The proliferation, migration and invasion abilities of Caski cells in the LINC00958+shVEGF-C group and the lymph vessel pro-formation ability of the culture supernatant were significantly lower than those in the LINC00958 group (P<0.01 or P<0.001). In the mouse popliteal lymph node metastasis model, the volume of popliteal lymph nodes in the LINC00958+shVEGF-C group and the proportion of VEGF-C protein, N-cadherin protein and LYVE-1 positive cells were significantly lower than those in the LINC00958 group (all P<0.001). Conclusion: LINC00958 directly interacts with VEGF-C protein to enhance the proliferation and invasion abilities and lymph vessel formation ability of cervical cancer cells, and promote lymph node metastasis in the mouse popliteal lymph node metastasis model.

河南省医学科技攻关计划联合共建项目（No. LHGJ20200024）