Supplementary Material for: Nesprin1 deficiency is associated with poor prognosis of renal cell carcinoma and resistance to sunitinib treatment

Introduction: Nuclear envelope spectrin repeat protein (Nesprin) 1 encoded by SYNE1, crucially regulates the morphology and functions of the cell. Mutations in the SYNE1 gene are associated with various diseases; however, their significance in renal cell carcinoma (RCC) remains unknown. In this study, we have investigated the association of SYNE1/Nesprin1 with the progression and prognosis of clear cell RCC (ccRCC). Methods: In silico analyses of publicly available datasets of patients with RCC were performed. Based on the cohort data, Nesprin1 expression in nephrectomized tissue samples acquired from patients with ccRCC was analyzed using immunohistochemical staining. The invasion, migration, and proliferation of the SYNE1-knockdown human RCC cell lines were analyzed in vitro; moreover, RNA sequencing and Gene Set Enrichment Analysis were conducted to study the molecular mechanism underlying the association of SYNE1/Nesprin1 with prognosis of RCC. Results: Patients with RCC-associated SYNE1 gene mutations exhibited significantly worse overall and progression-free survivals. Patients with Nesprin1-negative ccRCC tumors exhibit significantly poorer overall, cancer-specific, and recurrence-free survival rates than those recorded in the Nesprin1-positive group. SYNE1 knockdown enhanced the invasion and migration of RCC cells, however, it did not influence the proliferation of cells. RNA sequencing and Gene Set Enrichment Analysis revealed that SYNE1 knockdown significantly altered the expression of genes associated with oxidative phosphorylation. Consistently, patients with RCC exhibiting low SYNE1 expression, who were treated with the vascular endothelial growth factor receptor inhibitor sunitinib, had worse progression-free survival. Conclusions: The results indicate that the expression of SYNE1/Nesprin1 and SYNE1 mutations in patients with RCC are closely linked to their prognosis and responsiveness to sunitinib treatment.

引言：由SYNE1基因编码的核膜血影蛋白重复蛋白1（Nesprin 1）对细胞形态与功能具有关键调控作用。SYNE1基因的突变与多种疾病相关，但其在肾细胞癌（renal cell carcinoma, RCC）中的临床意义仍不明确。本研究旨在探讨SYNE1/Nesprin 1与透明细胞肾细胞癌（clear cell RCC, ccRCC）的进展及预后的关联。
方法：本研究对公开可用的肾细胞癌患者数据集进行了生物信息学分析。基于队列数据，采用免疫组化染色法分析了透明细胞肾细胞癌患者肾切除组织样本中的Nesprin 1表达水平。体外实验分析了SYNE1基因敲低的人肾细胞癌细胞系的侵袭、迁移与增殖能力；此外，通过RNA测序及基因集富集分析（Gene Set Enrichment Analysis），探究了SYNE1/Nesprin 1与肾细胞癌预后相关的分子机制。
结果：携带SYNE1基因突变的肾细胞癌患者的总生存期与无进展生存期均显著更差。Nesprin 1阴性的透明细胞肾细胞癌患者的总生存期、肿瘤特异性生存期及无复发生存率均显著低于Nesprin 1阳性组患者。SYNE1基因敲低可增强肾细胞癌细胞的侵袭与迁移能力，但对细胞增殖无显著影响。RNA测序及基因集富集分析结果显示，SYNE1基因敲低显著改变了与氧化磷酸化相关的基因表达。与此一致的是，接受血管内皮生长因子受体抑制剂舒尼替尼（sunitinib）治疗且SYNE1低表达的肾细胞癌患者，其无进展生存期更短。
结论：本研究结果表明，肾细胞癌患者的SYNE1/Nesprin 1表达水平及SYNE1基因突变情况，与其预后及舒尼替尼治疗响应度密切相关。