靶向TRAIL死亡受体DR5降低胃肠道间质瘤细胞抗失巢凋亡及免疫抑制因子分泌水平的研究

Targeting of TRAIL death receptor DR5 reduces anti-anoikis and secretion of immunosuppressive factors in gastrointestinal stromal tumor cells

目的探讨靶向肿瘤坏死因子相关的凋亡诱导配体(TRAIL)死亡受体5(DR5)对胃肠道间质瘤(GIST)细胞失巢凋亡及分泌免疫抑制因子的影响。方法收集2021-09-01-2022-02-16于石河子大学第一附属医院行手术切除治疗的38例GIST患者的肿瘤组织及癌旁组织。免疫组化染色检测组织中TRAIL与DR5蛋白表达水平,实时荧光定量聚合酶链反应法(qRT-PCR)测定组织内TRAIL与DR5 mRNA表达水平。将人GIST细胞系GIST-T1随机分为对照组、sh-NC组和sh-DR5组,采用脂质体介导法分别将sh-NC、sh-DR5重组质粒转染至对应组细胞中,采用qRT-PCR和蛋白质印迹法检测转染效果。收集转染后GIST-T1细胞培养液上清,酶联免疫吸附法测定上清中血管内皮生长因子(VEGF)、转化生长因子β_(1)(TGF-β_(1))、白细胞介素6(IL-6)及IL-8水平,软琼脂集落形成实验检测转染后GIST-T1细胞的集落形成情况。利用多聚2-羟乙基甲基丙烯酸酯(Poly-HEMA)悬浮培养GIST-T1细胞以诱导建立失巢凋亡模型,倒置相差显微镜下观察细胞生长情况,流式细胞术检测各组细胞凋亡情况,Calcein AM/EthD-1荧光双染鉴别各组细胞中活细胞和死细胞。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果与对照组癌旁组织比较,GIST组织中TRAIL表达降低而DR5表达升高,相对表达量分别为0.34±0.03和1.95±0.17,差异有统计学意义,t值分别为26.517和19.886,均P<0.001。相较于对照组与sh-NC组,sh-DR5组GIST-T1细胞中DR5 mRNA与蛋白相对表达量均下调,分别为0.24±0.01和0.17±0.01,差异有统计学意义,F值分别为32.567和50.124,均P<0.001。与对照组和sh-NC组比较,sh-DR5组GIST-T1细胞上清中VEGF、TGF-β_(1)、IL-6及IL-8水平均下降(均P<0.05),细胞集落形成个数减少、集落变小,经Poly-HEMA悬浮培养的细胞团块较为分散、直径变小,细胞凋亡率增加至(33.63±3.19)%,差异有统计学意义,F=87.025,P<0.001;EthD-1标记的失巢凋亡细胞比例也增加至(42.58±4.15)%,差异有统计学意义,F=86.165,P<0.001。结论在GIST组织中TRAIL表达降低而DR5表达升高,靶向DR5能够促进GIST-T1失巢凋亡,并降低免疫抑制因子的分泌水平,从而发挥抗肿瘤作用。

Objective To investigate the effect of targeting tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)death receptor 5(DR5)on anoikis and secretion of immunosuppressive factors in gastrointestinal stromal tumor(GIST)cells.Methods The tumor tissues and paracancerous tissues of 38 GIST patients who underwent surgical resection in the First Affiliated Hospital of Shihezi University from 2021-09-01 to 2022-02-16 were collected.Immunohistochemical staining was used to detect the expression levels of TRAIL and DR5 proteins in tissues,quantitative real-time polymerase chain reaction(qRT-PCR)reaction was used to measure the expression levels of TRAIL and DR5 mRNA in tissues;The human GIST cell line GIST-T1 was randomly divided into control group,sh-NC group and sh-DR5 group,the sh-NC and sh-DR5 recombinant plasmids were transfected into the corresponding groups of cells by liposome-mediated method,respectively,and the transfection effect was detected by qRT-PCR and Western blot;The supernatant of GIST-T1 cell culture medium after transfection was collected,and enzyme-linked immunosorbent assay was used to determine the levels of vascular endothelial growth factor(VEGF),transforming growth factor-β_(1)(TGF-β_(1)),interleukin 6(IL-6)and IL-8 in the supernatant,soft agar colony formation assay was used to detect the colony formation of GIST-T1 cells after transfection;GIST-T1 cells were cultured in suspension with poly-2-hydroxyethyl methacrylate(Poly-HEMA)to induce the establishment of an anoikis model,the cell growth was observed under an inverted phase contrast microscope,flow cytometry was used to detect cell apoptosis in each group,Calcein AM/EthD-1 fluorescent double staining was used to identify live and dead cells in each group.One-way analysis of variance was used for comparison between groups,and LSD-t test was used for comparison between groups in pairs.Results Compared with the GIST of the control group,TRAIL expression decreased and DR5 expression increased in the GIST tissue,the relative expression levels were 0.34±0.03 and1.95±0.17,respectively,the difference were statistically significant(t=26.517,P<0.001;t=19.886,P<0.001).Compared with the control group and the sh-NC group,the relative expression level of DR5mRNA and protein in GIST T1 cells of sh-DR5 group were down-regulated,to 0.24±0.01and 0.17±0.01,respectively,the difference were statistically significant(F=32.567,P<0.001;F=50.124,P<0.001).Compared with the control group and sh-NC group,the levels of VEGF,TGF-β_(1),IL-6 and IL-8 in the supernatant of GIST-T1 cells in sh-DR5 group were decreased(all P<0.05),and the number of cell colonies were decreased and the colonies became smaller,the cell clusters in Poly-HEMA suspension culture were more dispersed and smaller in diameter,the apoptosis rate was increased to(33.63±3.19)%,the difference was statistically significant(F=87.025,P<0.001),the proportion of apoptotic cells labeled with EthD-1 also increased to(42.58±4.15)%,the difference was statistically significant(F=86.165,P<0.001).Conclusions The expression of TRAIL is decreased and the expression of DR5 is increased in GIST tissues.Targeting DR5 can promote GIST-T1 anoikis and reduce the secretion of immunosuppressive factors,thereby exerting anti-tumor effects.