EPHB2介导胃肠道间质瘤伊马替尼继发性耐药

Secondary resistance to imatinib in gastrointestinal stromal tumors mediated by EPHB2

目的研究胃肠道间质瘤(GIST)治疗中伊马替尼继发性耐药机制。方法选取2018年12月至2021年4月南京医科大学第一附属医院收治的GIST患者伊马替尼耐药10例和伊马替尼敏感10例,经手术或穿刺活检获取其肿瘤组织。以外购的敏感亲代细胞GIST-T1S,梯度加药构建GIST伊马替尼耐药株GIST-T1R,运用RT-PCR及western blot、免疫组化法,分别在RNA及蛋白水平检测酪氨酸蛋白激酶受体家族中的促红素人肝细胞(erythropoietin-producing human hepatocellular,EPH)B2在敏感或耐药细胞、组织中的表达量;GIST细胞系敲低或过表达EPHB2后,CCK-8法检测细胞存活率确定药物对细胞的半数抑制浓度(IC_(50)),克隆形成实验检测EPHB2对细胞增殖能力的影响。结果EPHB2在继发性伊马替尼耐药患者的GIST组织中显著高表达(P<0.01)。相比于GIST-T1S细胞,GIST-T1R细胞中EPHB2 RNA及蛋白水平均明显上调(P<0.01)。在GIST-T1S细胞中,过表达EPHB2显著升高伊马替尼对细胞的IC_(50),敲低EPHB2则显著降低伊马替尼对细胞的IC_(50)(P<0.01)。结论EPHB2介导GIST细胞对伊马替尼的耐药并促进其恶性增殖。

Objective To explore the mechanism of secondary resistance to imatinib(IM)in gastrointestinal stromal tumors(GIST).Methods Ten IM-resistant and ten IM-sensitive GIST patients admitted to the First Affiliated Hospital with Nanjing Medical University from December 2018 to April 2021 were selected,and their tumor tissues were obtained by surgery or needle biopsy.The IM-resistant strain GIST-T1R of GIST was constructed by gradient addition using purchased IM-sensitive parental cells GIST-T1S.Real-time quantitative polymerase chain reaction,immunohistochemistry and western blot were used to detect the expression of erythropoietin-producing human hepatocellular(EPH)B2 in IM-sensitive or IM-resistant cells and tissues at RNA and protein levels,respectively.After knockdown or overexpression of EPHB2 in GIST cell lines,CCK-8 assay was used to detect cell viability to determine the half inhibitory concentration(IC_(50))of IM on cells,and clone formation assay was used to detect the effect of EPHB2 on cell proliferation ability.Results EPHB2 was significantly overexpressed in GIST tissues of patients with secondary IM-resistance(P<0.01).Compared with GIST-T1S cells,EPHB2 RNA and protein levels were significantly up-regulated in GIST-T1R cells(P<0.01).In GIST-T1S cells,overexpressing EPHB2 significantly increased the IC_(50)of IM on cells,and knocking down of EPHB2 significantly decreased the IC_(50)of IM on cells(P<0.01).Conclusion EPHB2 mediates IM-resistance of GIST cells and promotes their malignant proliferation.