背景:含三方基序26(TRIM26),TRIM蛋白家族的成员,在几种类型的癌症中发挥双重功能。然而,TRIM26在透明细胞肾细胞癌(ccRCC)中的确切作用尚未得到研究.
方法:通过公共资源和实验验证,检测TRIM26在ccRCC组织和细胞系中的表达。TRIM26对细胞增殖的影响,迁移,入侵,和上皮-间质转化(EMT)过程通过CCK-8,集落形成,EdU成立,伤口愈合,Transwell入侵,蛋白质印迹,和免疫荧光分析。使用RNA-seq和随后的生物信息学分析来鉴定TRIM26的下游途径。通过免疫共沉淀评估TRIM26和ETK之间的相互作用,qRT-PCR,蛋白质印迹,环己酰亚胺(CHX)追逐,和体内泛素化试验。
结果:我们已经表明TRIM26在ccRCC组织和细胞系中均表现出下调。此外,这种TRIM26表达下降与ccRCC患者的不良总生存期和无病生存期结局密切相关.增益和功能丧失实验表明,增加TRIM26的表达抑制了增殖,迁移,入侵,ccRCC细胞的EMT过程。相反,降低TRIM26的表达具有相反的效果。RNA测序,结合生物信息学分析,与TRIM26过表达组相比,对照组中mTOR信号通路显著富集。然后通过蛋白质印迹测定证实了这一发现。随后的检查显示,TRMI26与ETK有直接相互作用,非受体酪氨酸激酶。这种相互作用促进了ETK的泛素化和降解,导致ccRCC中AKT/mTOR信号通路失活。ETK过表达抵消了TRIM26过表达对细胞增殖的抑制作用,迁移,和入侵。
结论:我们的研究结果表明了一种新的机制,TRIM26通过与ETK结合并使其不稳定来阻碍ccRCC的发展,从而导致AKT/mTOR信号的失活。TRIM26显示出有望作为ccRCC患者的治疗靶标和预后生物标志物。
BACKGROUND: Tripartite motif-containing 26 (
TRIM26), a member of the TRIM protein family, exerts dual function in several types of cancer. Nevertheless, the precise role of TRIM26 in clear cell renal cell carcinoma (ccRCC) has not been investigated.
METHODS: The expression of
TRIM26 in ccRCC tissues and cell lines were examined through the use of public resources and experimental validation. The impacts of TRIM26 on cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) process were determined via CCK-8, colony formation, EdU incorporation, wound healing, Transwell invasion, Western blot, and Immunofluorescence assays. RNA-seq followed by bioinformatic analyses were used to identify the downstream pathway of
TRIM26. The interaction between TRIM26 and ETK was assessed by co-immunoprecipitation, qRT-PCR, Western blot, cycloheximide (CHX) chase, and in vivo ubiquitination assays.
RESULTS: We have shown that TRIM26 exhibits a downregulation in both ccRCC tissues and cell lines. Furthermore, this decreased expression of TRIM26 is closely linked to unfavorable overall survival and diseases-free survival outcomes among ccRCC patients. Gain- and loss-of-function experiments demonstrated that increasing the expression of TRIM26 suppressed the proliferation, migration, invasion, and EMT process of ccRCC cells. Conversely, reducing the expression of
TRIM26 had the opposite effects. RNA sequencing, coupled with bioinformatic analysis, revealed a significant enrichment of the mTOR signaling pathway in the control group compared to the group with TRIM26 overexpression. This finding was then confirmed by a western blot assay. Subsequent examination revealed that TRMI26 had a direct interaction with ETK, a non-receptor tyrosine kinase. This interaction facilitated the ubiquitination and degradation of ETK, resulting in the deactivation of the AKT/mTOR signaling pathway in ccRCC. ETK overexpression counteracted the inhibitory effects of TRIM26 overexpression on cell proliferation, migration, and invasion.
CONCLUSIONS: Our results have shown a novel mechanism by which TRIM26 hinders the advancement of ccRCC by binding to and destabilizing ETK, thus leading to the deactivation of AKT/mTOR signaling.
TRIM26 shows promise as both a therapeutic target and prognostic biomarker for ccRCC patients.