METHODS: We used transcriptional sequencing and TCGA database analysis to identify APOL1 as a novel therapeutic target for ccRCC. The oncogenic functions of APOL1 were investigated by cell proliferation, colony formation, migration and invasion assays in ccRCC cells in vitro and xenografts derived from ccRCC cells in vivo. Oil red O staining and quantification were used to detect lipid droplets. Chromatin immunoprecipitation (ChIP) assays and luciferase reporter assays were carried out to identify HIF-2α bound to the promoter of APOL1 and lncRNA LINC02609. RNA-FISH and luciferase reporter assays were performed to determine that LncRNA LINC02609 functions as a competing endogenous RNA to regulate APOL1 expression by sponging miR-149-5p.
RESULTS: RNA-seq data revealed that HIF2α can regulate APOL1 and lncRNA LINC02609 expression. We also found that HIF-2α can bind to the promoter of APOL1 and lncRNA LINC02609 and transcriptionally regulate their expression directly. We further demonstrated that LncRNA LINC02609 functions as a competing endogenous RNA to regulate APOL1 expression by sponging miR-149-5p in ccRCC. Mechanistically, APOL1-dependent lipid storage is required for endoplasmic reticulum (ER) homeostasis and cell viability and metastasis in ccRCC. We also showed that high APOL1 expression correlated with worse clinical outcomes, and knockdown of APOL1 inhibited tumor cell lipid droplet formation, proliferation, metastasis and xenograft tumor formation abilities. Together, our studies identify that HIF2α can regulate the expression of the lipid metabolism related gene APOL1 by direct and indirect means, which are essential for ccRCC tumorigenesis.
CONCLUSIONS: Based on the experimental data, in ccRCC, the HIF-2α/LINC02609/APOL1 axis can regulate the expression of APOL1, thus interfering with lipid storage, promoting endoplasmic reticulum homeostasis and regulating tumor progression in ccRCC. Together, our findings provide potential biomarkers and novel therapeutic targets for future studies in ccRCC.
方法:我们使用转录测序和TCGA数据库分析来鉴定APOL1作为ccRCC的新治疗靶标。通过细胞增殖研究APOL1的致癌功能,菌落形成,体外ccRCC细胞和体内ccRCC细胞异种移植物的迁移和侵袭测定。油红O染色和定量用于检测脂滴。进行染色质免疫沉淀(ChIP)测定和荧光素酶报告基因测定以鉴定与APOL1和lncRNALINC02609的启动子结合的HIF-2α。进行RNA-FISH和荧光素酶报告基因测定以确定LncRNALINC02609作为竞争性内源RNA起作用,以通过形成miR-149-5p来调节APOL1表达。
结果:RNA-seq数据显示HIF2α可以调节APOL1和lncRNALINC02609的表达。我们还发现HIF-2α可以与APOL1和lncRNALINC02609的启动子结合,并直接转录调节它们的表达。我们进一步证明LncRNALINC02609作为竞争性内源性RNA通过在ccRCC中形成miR-149-5p来调节APOL1表达。机械上,APOL1依赖性脂质储存是ccRCC中内质网(ER)稳态和细胞活力和转移所必需的。我们还表明,高APOL1表达与更差的临床结果相关,APOL1的敲除抑制肿瘤细胞脂滴的形成,扩散,转移和异种移植肿瘤形成能力。一起,我们的研究发现HIF2α可以通过直接和间接手段调节脂质代谢相关基因APOL1的表达,这对ccRCC肿瘤发生至关重要。
结论:根据实验数据,在ccRCC中,HIF-2α/LINC02609/APOL1轴可以调节APOL1的表达,从而干扰脂质的储存,在ccRCC中促进内质网稳态并调节肿瘤进展。一起,我们的研究结果为ccRCC的未来研究提供了潜在的生物标志物和新的治疗靶点.