RAB31促进肾透明细胞癌增殖、侵袭及转移的机制研究

董宇豪; 陈欣然; 高石亮; 邹南鑫; 张旭

doi:10.3969/j.issn.2095-5227.24091203

摘要: 背景　肾透明细胞癌是肾癌最常见的病理类型，探究肾透明细胞癌的发生发展机制、找寻新的治疗靶点有重要意义。目的　探索RAB31对肾透明细胞癌的增殖、迁移、侵袭能力以及线粒体分裂融合方面的影响。方法　采用荧光定量PCR与Western blot检测配对肾透明细胞癌组织和癌旁组织中RAB31的表达情况；分析RAB31的表达与肾透明细胞癌患者分期分级的关系；采用 Western blot 检测NC-RAB31组、shRAB31-1组、shRAB31-2组、NC-786-O组、OE-RAB31组细胞的RAB31及MMP2、SLUG等EMT相关蛋白，BAX、BCL-2等凋亡相关蛋白，DRP1、FIS1等线粒体分裂相关基因在肾透明细胞癌细胞中的表达情况；采用CCK8实验、划痕实验和Transwell实验分别检测RAB31对肾透明细胞癌细胞增殖和迁移能力的影响；利用RNA-seq技术对稳定敲低RAB31的肾透明细胞癌细胞进行测序分析。结果 RAB31在肾透明细胞癌组织中的表达高于在癌旁组织中的表达。Western blot结果显示，与NC-RAB31组比较，sh-RAB31-1组与shRAB31-2组肾透明细胞癌细胞EMT相关通路被抑制，凋亡通路被激活，线粒体融合增多，过表达组则相反。CCK8实验结果显示，稳定敲低RAB31 的肿瘤细胞增殖能力低于正常肿瘤细胞，结果具有统计学差异(P＜0.001)；Transwell 实验结果显示，稳定敲低RAB31 的肿瘤细胞侵袭和迁移能力低于正常肿瘤细胞，结果具有统计学差异(P＜0.001)。划痕实验结果显示，稳定敲低RAB31的肿瘤细胞迁移能力低于正常肿瘤细胞，结果具有统计学差异(P＜0.001)。结论 RAB31在肾透明细胞癌组织中高表达并可能通过增强肿瘤细胞能量代谢提升其增殖和迁移能力。

Abstract: Background Kidney renal clear cell carcinoma (KIRC) represents the predominant histological subtype of renal neoplasms. Elucidating the pathogenesis of ccRCC and identifying novel therapeutic targets are crucial for advancing treatment strategies.Objective This study aimed to investigate the role of RAB31 in the proliferation, migration, and invasiveness of ccRCC,as well as its impact on mitochondrial dynamics.Methods The expression levels of RAB31 in ccRCC and adjacent non-tumorous tissues were assessed using quantitative real-time PCR (qRT-PCR) and Western blot analysis. The correlation between RAB31expression and patient prognosis was statistically analyzed. Expression profiles of epithelial-mesenchymal transition (EMT) markers
(MMP2, SLUG), apoptosis-related proteins (BAX, BCL-2), and mitochondrial fission regulators (DRP1, FIS1) in ccRCC cells were evaluated by Western blot. The effects of RAB31 on ccRCC cell proliferation and migration were determined using CCK-8 and Transwell assays, respectively. RNA sequencing (RNA-seq) was employed to analyze the transcriptomic alterations in ccRCC cells following RAB31 knockdown. Results The expression of RAB31 in renal clear cell carcinoma tissues was higher than that in adjacent tissues. Western blot results showed that compared with the NC-RAB31 group, the EMT related pathway was inhibited, the apoptosis pathway was activated, and mitochondrial fusion was increased in the sh-RAB31-1 and shRAB31-2 groups, which was opposite in the overexpression group. CCK8 assay showed that the proliferation ability of tumor cells with stable knockdown of RAB31 was lower than that of normal tumor cells, and the results were statistically significant (P＜0.001). The results of Transwell assay showed that tumor cells with stable knockdown of RAB31 had lower invasion and migration abilities than normal tumor cells,and the results were statistically significant (P＜0.001). Wound healing assay showed that the migration ability of tumor cells with stable RAB31 knockdown was lower than that of normal tumor cells, and the results were statistically significant (P＜0.001).Conclusion RAB31 is highly expressed in renal clear cell carcinoma tissues and may promote tumor cell proliferation and migration by enhancing energy metabolism.