HIF-1α/ACLY信号轴促进携带VHL失活突变的透明细胞肾细胞癌发生发展的机制

Activation of HIF-1α/ACLY signaling axis promotes progression of clear cell renal cell carcinoma with VHL inactivation mutation

目的基于低氧诱导因子-1α(HIF-1α)/ATP柠檬酸裂合酶(ACLY)信号通路,探究透明细胞肾细胞癌(clear cell renal cell carcinoma,ccRCC)潜在发病机制,为ccRCC的治疗提供新思路。方法收集苏州大学附属第一医院诊断为ccRCC的78例标本,通过外显子测序明确VHL基因突变情况;通过免疫组织化学染色评估HIF-1α/ACLY在VHL基因突变的ccRCC中表达情况,通过Western blot实验在携带VHL基因突变的ccRCC细胞系(786-O、A498、UM-RC-2、SNU-333和Caki-2)中进行进一步验证。在ccRCC细胞系中过表达或者干扰HIF-1α,通过即时定量PCR(qPCR)和Western blot实验检测ACLY的mRNA和蛋白质水平。CoCl2和低氧(1%O2)处理HeLa细胞激活HIF-1α,检测ACLY的mRNA和蛋白水平。通过JASPAR数据库结合染色质免疫共沉淀实验(ChIP)及荧光素酶报告基因实验等探究HIF-1α调控ACLY潜在分子机制。通过BODIPY染色等细胞生物学技术检测HIF-1α/ACLY调控轴对脂质积累的作用。比较ccRCC患者与良性病变患者ACLY表达情况,通过生存期分析探究ACLY作为ccRCC预后指标可行性。结果外显子测序发现,78例ccRCC患者中有55例携带VHL失活突变,其中HIF-1α与ACLY蛋白水平呈正相关,携带VHL基因突变的ccRCC细胞系中ACLY和HIF-1α的蛋白水平同样呈现不同程度正相关;在A498细胞中过表达HIF-1α可以增加ACLY的mRNA和蛋白水平,在Caki-2细胞中敲低HIF-1α则可以抑制ACLY的mRNA和蛋白质水平(均P<0.001),CoCl2和低氧处理可以通过激活HIF-1α显著提高ACLY的mRNA和蛋白水平(均P<0.001)。荧光素酶报告基因转录活性量化及ChIP-qPCR结果提示HIF-1α可以直接与ACLY启动子区域结合从而转录激活ACLY表达,提高ACLY蛋白水平(均P<0.001)。BODIPY染色结果提示细胞系中游离脂肪酸的含量和细胞中HIF-1α及ACLY的水平呈正相关,敲低HIF-1α可以有效降低细胞中脂质积累,过表达ACLY则可以逆转这一过程。同时,细胞功能实验提示HIF-1α敲低的ccRCC细胞增殖速率显著下降,外源性过表达ACLY则可以恢复肿瘤细胞的增殖能力(P<0.001)。通过生存分析发现,相较于ACLY低表达组,ACLY高表达组ccRCC患者预后较差,中位生存期较短(P<0.001)。结论ccRCC中VHL失活突变介导的HIF-1α高表达通过上调ACLY促进脂质合成,促进肿瘤进程,靶向HIF-1α/ACLY信号轴可能为ccRCC临床诊疗提供理论依据。

Objective To explore the potential pathogenesis of clear cell renal cell carcinoma(ccRCC)based on the HIF-1α/ACLY signaling pathway,as well as to provide new ideas for the treatment of ccRCC.Methods Seventy-eight ccRCC cases diagnosed at the First Affiliated Hospital of Soochow University,Suzhou,China were collected.The VHL mutation was examined using exon sequencing.The expression of HIF-1α/ACLY in VHL-mutated ccRCC was evaluated using immunohistochemical staining and further validated in VHL-mutated ccRCC cell lines(786-O,A498,UM-RC-2,SNU-333,and Caki-2)using Western blot.The mRNA and protein levels of ACLY were detected using real-time quantitative PCR and Western blot after overexpression or interference with HIF-1αin ccRCC cell lines.HeLa cells were treated with CoCl2 and hypoxia(1%O2)to activate HIF-1αand then subject to the detection of the ACLY mRNA and protein levels.The potential molecular mechanism of HIF-1α-induced ACLY activation was explored through JASPAR database combined with chromatin immunoprecipitation assay(ChIP)and luciferase reporter gene assay.The effect of HIF-1α/ACLY regulation axis on lipid accumulation was detected using BODIPY staining and other cell biological techniques.The expression of ACLY was compared between patients with ccRCC and those with benign lesions,and the feasibility of ACLY as a prognostic indicator for ccRCC was explored through survival analysis.Results Exon sequencing revealed that 55(70.5%)of the 78 ccRCC patients harbored a VHL inactivation mutation,and HIF-1αexpression was associated with ACLY protein levels.The protein levels of ACLY and HIF-1αin ccRCC cell lines carrying VHL mutation were also correlated to various degrees.Overexpression of HIF-1αin A498 cells increased the mRNA and protein levels of ACLY,and knockdown of HIF-1αin Caki-2 cells inhibited the mRNA and protein levels of ACLY(P<0.001 for all).CoCl2 and hypoxia treatment significantly increased the mRNA and protein levels of ACLY by activating HIF-1α(P<0.001 for all).The quantification of transcriptional activity of luciferase reporter gene and ChIP-qPCR results suggested that HIF-1αcould directly bind to ACLY promoter region to transcriptionally activate ACLY expression and increase ACLY protein level(P<0.001 for all).The results of BODIPY staining suggested that the content of free fatty acids in cell lines was associated with the levels of HIF-1αand ACLY.The depletion of HIF-1αcould effectively reduce the accumulation of lipid in cells,while the overexpression of ACLY could reverse this process.At the same time,cell function experiments showed that the proliferation rate of ccRCC cells with HIF-1αknockdown was significantly decreased,and overexpression of ACLY could restore proliferation of these tumor cells(P<0.001).Survival analysis further showed that compared with the ccRCC patients with low ACLY expression,the ccRCC patients with high ACLY expression had a poorer prognosis and a shorter median survival(P<0.001).Conclusions VHL mutation-mediated HIF-1αoverexpression in ccRCC promotes lipid synthesis and tumor progression by activating ACLY.Targeting the HIF-1α/ACLY signaling axis may provide a theoretical basis for the clinical diagnosis and treatment of ccRCC.