目的 探讨内质网膜蛋白4B(RTN 4B)对胃癌血管生成的影响。方法 选取2023年1月至3月空军军医大学第一附属医院消化外科手术切除的胃癌组织及配对的癌旁组织,各15例。免疫组织化学染色法检测组织RTN 4B表达,检测CD34标记的血管微密度(MVD)。选取胃癌细胞系(AGS、NCI-N87、MGC803、BGC8231)和正常胃黏膜细胞系(GES-1),PCR检测RTN 4B mRNA表达;Western-blot检测RTN 4B蛋白。用Lipofectamine2000转染RTN 4B siRNA质粒(RTN 4B-siRNA组),阴性对照质粒转入AGS细胞(CON组),分别将RTN 4B过表达质粒(pcDNA3.1-RTN 4B组)和阴性对照质粒(pcDNA3.1-CON组)转入NCI-N87细胞,检测各组细胞血管生成能力及HIF-1α、VEGF蛋白表达水平。结果 癌组织RTN 4B表达率高于癌旁组织[73.33%(11/15)vs. 13.33%(2/15),P<0.05]。癌组织MVD高于癌旁组织[(28.06±3.05)vs.(21.70±5.26),P<0.05)]。在癌组织中,高表达RTN 4B组织的MVD高于低表达组织[(30.25±6.16)vs.(25.71±9.25);P<0.05)。胃癌细胞系(AGS、NCI-N87、MGC803、BGC8231)RTN 4B基因及蛋白表达水平均高于正常胃黏膜细胞系(GES-1),差异有统计学意义(P<0.05)。血管生成实验结果显示,pcDNA3.1-RTN 4B组管长度和节点均高于pcDNA3.1-CON组,RTN 4B-siRNA组管长度和节点均低于CON组,差异有统计学意义(P<0.05)。pcDNA3.1-RTN 4B组HIF-1α、VEGF蛋白表达水平均高于pcDNA3.1-CON组,RTN 4B-siRNA组HIF-1α、VEGF蛋白表达水高于CON组,差异均有统计学意义(P<0.05)。结论 RTN 4B在胃癌组织中高表达,并且能够促进胃癌血管生成。
Abstract
Objective To investigate the effect of reticulon 4B (RTN 4B) on angiogenesis in gastric cancer. Methods From January to March 2023, 15 gastric cancer tissues and matched adjacent tissues were selected from the First Affiliated Hospital of Air Force Military Medical University. Immunohistochemical staining was used to detect the expression of RTN 4B in tissues, and the microvascular density (MVD) labeled with CD34. RTN 4B mRNA expression in gastric cancer cell lines (AGS, NCI-N87, MGC803, BGC8231) and normal gastric mucosa cell lines (GES-1) were detected by real-time quantitative PCR. Western blot was used to detect the expression of RTN 4B protein. RTN 4B siRNA plasmids (RTN 4B-siRNA group) and negative control plasmids (CON group) were transfected into AGS cells using Lipofectamine2000 transfection reagent.RTN 4B overexpression plasmids (pcDNA3.1-RTN 4B group) and negative control plasmids (pcDNA3.1-CON group) were transfected into NCI-N87 cells, respectively. The angiogenesis ability of cells and the expression levels of HIF-1α and VEGF proteins were detected in each group. Results The expression rate of RTN 4B in cancer tissue was higher than in adjacent tissues [73.33% (11/15) vs. 13.33% (2/15), P<0.05]. The MVD of cancer tissue was higher than that of adjacent tissues, and the difference was statistically significant[(28.06±3.05) vs. (21.70±5.26), P<0.05]. The high expression rate of RTN 4B and MVD in cancer tissues were higher than those in adjacent tissues[(28.06±3.05) vs. (21.70±5.26); P<0.05]. In cancer tissues, the MVD of high expression RTN 4B tissues was higher than that of low expression tissues, and the difference was statistically significant (P<0.05). The expression levels of RTN 4B gene and protein in gastric cancer cell lines (AGS, NCI-N87, MGC803, BGC8231) were significantly higher than those in normal gastric mucosa cell lines (GES-1) (P<0.05). The results of the angiogenesis experiment showed that the length and nodes of the pcDNA3.1-RTN 4B group were higher than those of the pcDNA3.1-CON group, with statistically significant differences (P<0.05). The expression level of HIF-1 α and VEGF protein in PCDNA3.1-RTN 4B group were higher than those of the pcDNA3.1-CON group, and the difference was statistically significant (P<0.05). The results of angiogenesis experiments showed that the RTN 4B-siRNA group had lower tube length and nodes compared to the CON group, and those in RTN 4B-siRNA group were lower than those of the CON group, with statistically significant differences (P<0.05). Conclusions RTN 4B is highly expressed in gastric cancer tissues and can promote angiogenesis in gastric cancer.
关键词
内质网膜蛋白4B /
胃癌 /
血管微密度 /
血管生成
Key words
reticulon 4B /
gastric cancer /
microdensity of blood vessel /
angiogenesis
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基金
陕西省自然科学基础计划项目(2020JM-332)
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