| 摘要: |
| ［摘要］ 目的 分析剪接因子DDX3Y下调IL-6/JAK/STAT3信号通路促进男性胃癌细胞增殖和类器官生长的机制。方法 通过生物信息学方法分析DDX3Y在胃癌组织和癌旁组织中的表达水平以及DDX3Y表达情况与患者预后的关系。收集苏州大学附属第一医院收治的男性胃癌患者的胃癌组织和癌旁组织（距离胃癌组织>5 cm）进行免疫组化染色。对人胃永生化黏膜上皮细胞GES-1以及男性来源胃癌细胞SNU-1、NUGC-3、KATOIII、MKN74、HGC-27、NCI-N87进行培养。采用Western blot法检测DDX3Y在不同细胞系中的表达水平。通过慢病毒转染SNU-1细胞和HGC-27细胞，分别构建DDX3Y高表达/低表达的细胞（OE-DDX3Y/shDDX3Y），进行EdU细胞增殖实验。利用男性胃癌患者的胃癌组织构建类器官，并通过慢病毒转染进一步构建DDX3Y高表达/低表达的类器官，分析抑制IL-6/JAK/STAT3信号通路对类器官直径及活力的影响。使用RBPmap在线网站预测DDX3Y与IL-6前体mRNA结合的可能性。结果 癌旁组织中DDX3Y mRNA表达水平显著高于胃癌组织（P<0.05）。DDX3Y mRNA高表达患者的生存预后显著优于低表达患者（log-rank检验： χ2=7.856，P=0.005）。与GES-1细胞相比，DDX3Y蛋白在HGC-27细胞中显著高表达（P<0.05）；相较于HGC-27细胞，SNU-1细胞中DDX3Y蛋白表达量较低（P<0.05）。在SNU-1细胞中，OE-DDX3Y组的DDX3Y蛋白表达量显著高于高表达对照组（P<0.05），OE-DDX3Y组EdU染色阳性细胞比例显著低于高表达对照组（P<0.05）。在HGC-27细胞中，shDDX3Y-1组的DDX3Y蛋白表达量显著低于低表达对照组（P<0.05），shDDX3Y组EdU染色阳性细胞比例显著高于低表达对照组（P<0.05）。相较于高表达对照组，OE-DDX3Y组类器官直径显著减小（P<0.05），活力显著降低（P<0.05），抑制IL-6/JAK/STAT3信号通路后类器官直径进一步减小（P<0.05），活力进一步降低（P<0.05）。相较于低表达对照组，shDDX3Y组类器官直径显著增大（P<0.05），活力显著增加（P<0.05），抑制IL-6/JAK/STAT3信号通路后类器官直径显著减小（P<0.05），活力显著降低（P<0.05）。RBPmap在线网站预测结果提示DDX3Y很可能与IL-6的前体mRNA发生结合（Z-score>3，结合信号显著）。结论 剪接因子DDX3Y下调促进男性胃癌细胞增殖和类器官生长，其调控IL-6的可变剪接并可进一步通过影响IL-6/JAK/STAT3信号通路发挥促进肿瘤进展的作用。 |
| 关键词: DDX3Y 肿瘤细胞增殖 可变剪接 IL-6/JAK/STAT3信号通路 胃癌性别差异 |
| DOI：10.3969/j.issn.1674-3806.2025.06.11 |
| 分类号: |
| 基金项目:国家自然科学基金项目（编号：82372887，82403045）；江苏省自然科学基金项目（编号：BK20221242，BK20240372）；苏州市“科教兴卫”青年科技项目（编号：KJXW2023005）；苏州大学附属第一医院自然科学基金博习培育计划项目（编号：BXQN2023027） |
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| A study on mechanism of splicing factor DDX3Y promoting proliferation of gastric cancer cells and growth of organoids from males through down-regulating IL-6/JAK/STAT3 signaling pathway |
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ZHOU Yajing, LI Dongbao, WANG Pengbo, DUAN Kaipeng, DONG Chao, LI Weikang, SUN Xiaotong, XU Chengxiang, ZHOU Jin
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Department of General Surgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, China
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| Abstract: |
| ［Abstract］ Objective To analyze the mechanism of splicing factor DEAD-box helicase 3 Y-linked(DDX3Y) promoting the proliferation of gastric cancer cells and growth of organoids from males through down-regulating IL-6/JAK/STAT3 signaling pathway. Methods The expression levels of DDX3Y in gastric cancer tissues and their adjacent tissues, as well as the relationship between the expressions of DDX3Y and the patients′ prognosis were analyzed by using bioinformatics methods. The gastric cancer tissues and their adjacent tissues(>5 cm away from the gastric cancer tissues) of the male gastric cancer patients who were admitted to the First Affiliated Hospital of Soochow University were collected for immunohistochemical staining. Human immortalized gastric mucosal epithelial cells(GES-1) and male-derived gastric cancer cells(SNU-1, NUGC-3, KATOIII, MKN74, HGC-27 and NCI-N87) were cultured. The expression levels of DDX3Y in different cell lines were detected by using Western blot method. SNU-1 and HGC-27 cells were transfected with lentivirus to construct DDX3Y-high-expression/DDX3Y-low-expression cells(OE-DDX3Y/shDDX3Y), and EdU cell proliferation assays were performed. The organoids were established by using tumor tissues of the male gastric cancer patients, and lentiviral transfection was used to further generate organoids with DDX3Y-high-expression/DDX3Y-low-expression. The effects of inhibiting IL-6/JAK/STAT3 signaling pathway on organoid diameter and viability was analyzed. The possibility of DDX3Y binding to IL-6 precursor mRNA was predicted by using the RBPmap online website. Results DDX3Y mRNA expression level in the adjacent cancer tissues was significantly higher than that in the gastric cancer tissues(P<0.05). The survival prognosis of the patients with high expression of DDX3Y mRNA was significantly better than that of the patients with low expression of DDX3Y mRNA(log-rank test: χ2=7.856, P=0.005). Compared with that in GES-1 cells, the expression of DDX3Y protein in HGC-27 cells was significantly higher(P<0.05). Compared with that in HGC-27 cells, expression of DDX3Y protein in SNU-1 cells was significantly lower(P<0.05). In the SNU-1 cells, the expression level of DDX3Y protein in the OE-DDX3Y group was significantly higher than that in the high expression control group(P<0.05), and the proportion of EdU staining positive cells in the OE-DDX3Y group was significantly lower than that in the high expression control group(P<0.05). In the HGC-27 cells, the expression level of DDX3Y protein in the shDDX3Y-1 group was significantly lower than that in the low expression control group(P<0.05), and the proportion of EdU staining positive cells in the shDDX3Y group was significantly higher than that in the low expression control group(P<0.05). Compared with that in the high expression control group, the organoid diameter in the OE-DDX3Y group was significantly decreased(P<0.05), and the vitality in the OE-DDX3Y group was significantly reduced(P<0.05). After inhibiting the IL-6/JAK/STAT3 signaling pathway, the organoid diameter in the OE-DDX3Y group was further decreased(P<0.05), and the vitality in the OE-DDX3Y group was further reduced(P<0.05). Compared with that in the low expression control group, the organoid diameter in the shDDX3Y group was significantly increased(P<0.05), and the vitality in the shDDX3Y group was significantly increased(P<0.05). After inhibiting the IL-6/JAK/STAT3 signaling pathway, the organoid diameter in the shDDX3Y group was significantly decreased(P<0.05), and the viability in the shDDX3Y group was significantly reduced(P<0.05). The prediction results of the RBPmap online website suggested that DDX3Y was very likely to bind to the IL-6 precursor mRNA(Z-score>3, with a significant binding signal). Conclusion Downregulation of splicing factor DDX3Y promotes male gastric cancer proliferation and organoid growth. The alternative splicing of IL-6 regulated by the splicing factor DDX3Y can further promote tumor progression by influencing the IL-6/JAK/STAT3 signaling pathway. |
| Key words: DEAD-box helicase 3 Y-linked(DDX3Y) Tumor cell proliferation Alternative splicing IL-6/JAK/STAT3 signaling pathway Sexual dimorphism in gastric cancer |
