论文部分内容阅读
目的:运用分子生物学方法观察小泛素样修饰蛋白(small ubiquitin-related modifier,SUMO)通路成员在骨肉瘤中的表达特点,为基于蛋白质SUMO化修饰的靶向治疗提供理论依据。方法:收集2017年1月至2019年6月于我院就诊并手术的新鲜骨肉瘤组织样本18例,经Western blot及免疫组织化学方法检测癌灶及癌旁SUMO通路核心成员SUMO1、SAE1、Ubc9、SENP1的蛋白表达水平。分别干涉SUMO1、干涉UBC9及过表达SENP1,进行如下分组:空白对照组、对照组、siR-SUMO1组、siR-Ubc9组和SENP1组。Western blot验证基因转染效率,细胞增殖检测试剂盒检测EdU的阳性表达率,划痕实验及Transwell侵袭实验检测细胞迁移和侵袭能力,流式细胞术检测细胞凋亡率。以骨髓间充质干细胞(BMSCs)为研究对象,评估三种治疗方案的副作用。结果:Western blot及免疫组织化学结果均显示,骨肉瘤组织中SUMO1、SAE1、Ubc9的蛋白表达水平均明显高于癌旁组织(n P<0.05),但SENP1明显低于癌旁组织。基于143B骨肉瘤细胞的实验结果表明,siR-SUMO1组、siR-Ubc9组、SENP1组均能够明显抑制骨肉瘤细胞中SUMO通路的活化,表现为较对照组和无义组更低的肿瘤细胞增殖率(n P<0.05),更低的细胞迁移和侵袭能力(n P<0.05),以及更高的细胞凋亡率(n P0.05)。n 结论:SUMO通路在骨肉瘤中被异常激活,外源性补充或内源性激活SENP1是靶向治疗的备选方案之一。“,”Objective:To observe the expression characteristics of small ubiquitin-like modified protein (SUMO) pathway members in osteosarcomaby using molecular biology methods, and provide theoretical basis for targeted therapy based on protein SUMO modification.Methods:Eighteen fresh osteosarcoma tissue samples surgically resected at Tianjin Cancer Hospital from January 2017 to June 2019 were collected. Western blot and immunohistochemical methods were used to detect the protein expressions of SUMO1, SAE1, Ubc9, and SENP1, which are core members of SUMO pathway, in the cancerous and adjacent tissue. Taking osteosarcoma cell line 143B as the research object, three targeted treatment regimens were designed and grouped as follows: control group, nonsense group, siR-SUMO1 group, siR-Ubc9 group, and SENP1 group. Western blot was used to verify the efficiency of gene transfection, the positive expression rate of EdU was detected by cell proliferation detection kit, the ability of cell migration and invasion was measured by scratch test and Transwell invasion test, and the apoptosis rate was detected by flow cytometry. Bone marrow mesenchymal stem cells (BMSCs) were used to evaluate the side effects of three treatment regimens.Results:Results from Western blot and immunohistochemistry showed that the protein expression levels of SUMO1, SAE1, andUbc9 in osteosarcoma tissues were significantly higher than those in adjacent tissues (n P<0.05), but SENP1 was significantly lower than in adjacent tissues. The experimental results based on 143B osteosarcoma cells showed that the siR-SUMO1 group, siR-Ubc9 group, and SENP1 group can significantly inhibit the activation of SUMO pathway in osteosarcoma cells, showing lower tumor cell proliferation (n P<0.05), slower cell migration and invasion capacity (n P<0.05), and higher apoptotic rate (n P0.05).n Conclusion:The SUMO pathway is abnormally activated in osteosarcoma. Exogenous supplementation or endogenous activation of SENP1 is one of the alternatives for targeted osteosarcoma treatment.