Wang Zhi,Tu Lei,Ning Feng,et al.Effect of PPARα-regulated HADHA on biological behavior of human wilms tumor[J].Journal of Clinical Pediatric Surgery,2026,(04):344-354.[doi:10.3760/cma.j.cn101785-20260112-00019]
PPARα调控HADHA对人肾母细胞瘤细胞生物学行为的影响
- Title:
- Effect of PPARα-regulated HADHA on biological behavior of human wilms tumor
- 关键词:
- 基因; 肾母细胞瘤; 过氧化物酶体增殖物激活受体; 脂类代谢
- 摘要:
- 目的 探讨PPARα及HADHA对人肾母细胞瘤细胞增殖、凋亡、迁移等生物学行为的影响与调控机制。方法 下载GEO数据库肾母细胞瘤测序数据集GSE138869,筛选差异基因并进行富集分析;收集来自湖南省儿童医院的临床肾母细胞瘤及瘤旁组织样本,采用Western blot和qRT-PCR检测PPARα、HADHA及脂质代谢相关酶ACAT1、ACLY、FASN的表达。培养肾母细胞瘤细胞系WiT49和HFWT,构建PPARα过表达载体及HADHA小分子干扰RNA并转染细胞,采用CCK-8实验、划痕愈合实验、Transwell实验、流式细胞术,分别检测细胞增殖、迁移、侵袭及凋亡情况;采用油红O染色检测细胞脂质蓄积;采用双荧光素酶报告实验验证PPARα对HADHA的转录调控作用;建立裸鼠皮下成瘤模型,检测瘤块体积,采用免疫组化和WB检测相关蛋白表达。结果 GSE138869数据集共筛选出4 433个差异基因,富集于脂肪酸代谢及PPAR通路;PPARα和HADHA在肾母细胞瘤组织及细胞系中均呈低表达。与对照组相比,PPARα过表达组CCK-8吸光值降低(WiT49:1.57±0.04比2.28±0.02;HFWT:1.41±0.02比2.10±0.04),细胞凋亡率升高[WiT49:(15.93±0.86)%比(1.83±0.08)%;HFWT:(20.05±0.57)%比(3.17±0.52)%],划痕愈合率降低[WiT49:(45.62±2.58)%比(77.53±0.76)%;HFWT:(43.64±4.88)%比(83.02±0.78)%],侵袭细胞数减少(WiT49:51.33±5.73比193.67±5.91;HFWT:18.33±3.68比122.33±5.25),细胞内红色脂滴显著减少,且ACAT1表达上调、ACLY及FASN表达下调(P<0.05)。在PPARα过表达细胞中敲减HADHA后,细胞活力升高(WiT49:2.02±0.04比1.84±0.03;HFWT:1.59±0.01比1.41±0.01),凋亡率降低[WiT49:(7.87±0.55)%比(16.55±1.01)%;HFWT:(11.21±0.75)%比(20.19±1.83)%],迁移及侵袭能力增强,细胞内脂质颗粒增加,ACAT1表达减弱,ACLY及FASN表达上调(P<0.05)。双荧光素酶报告实验显示,HADHA-WT与OE-PPARα共转染细胞的荧光信号强度显著升高(P<0.05),而HADHA-MUT与OE-PPARα共转染细胞无显著差异。体内实验中,对照组裸鼠肿瘤体积[(983.15±279.11)mm3]及质量[(0.87±0.24)g]均显著高于OE-PPARα组[(257.74±45.45)mm3、(0.16±0.05)g],且OE-PPARα组肿瘤组织中PPARα、HADHA及ACAT1表达上调,ACLY、FASN、Ki-67及PCNA表达下调,脂质沉积减少,细胞凋亡增加。结论 PPARα可通过转录调控HADHA表达,抑制肾母细胞瘤细胞增殖、迁移及侵袭,促进细胞凋亡,并减少细胞脂质沉积,为肾母细胞瘤的靶向治疗提供新的分子靶点。
- Abstract:
- Objective To explore the effects and regulatory mechanisms of PPAR α and HADHA on biological behaviors of human nephroblastoma cells,including proliferation,apoptosis and migration.Methods The authors downloaded the GEO database nephroblastoma sequencing dataset GSE138869,screened for differentially expressed genes and performed enrichment analysis; Collected clinical samples of nephroblastoma and adjacent tissues and employed Western Blot and qRT PCR to detect the expressions of PPAR α,HADHA and lipid metabolism related enzymes ACAT1,ACLY and FASN.Also we cultivated nephroblastoma cell lines WiT49 and HFWT,constructed PPAR α overexpression vector (OE-PPAR α) and HADHA small interfering RNA (siHADHA) and transfected the cells.Cell proliferation,migration,invasion and apoptosis were detected by CCK-8 assay,scratch healing assay,Transwell assay and flow cytometry,respectively; Oil red O staining was used for detecting cellular lipid accumulation; Dual luciferase reporter assay was utilized for verifying the transcriptional regulatory effect of PPAR α on HADHA; With a subcutaneous tumor model in nude mice,we measured tumor volume and detected related protein expression through immunohistochemistry and Western blot.Results A total of 4433 differentially expressed genes were screened from the GSE138869 dataset,enriched in fatty acid metabolism and PPAR pathway; PPAR α and HADHA showed low expression in both nephroblastoma tissues and cell lines.As compared with control group,PPAR α overexpression group showed a decrease in CCK-8 absorbance values (WiT49:1.57±0.04 vs. 2.28±0.02; HFWT:1.41±0.02 vs.2.10±0.04) and apoptotic rate spiked[WiT49:(15.93±0.86)% vs.(1.83±0.08)%; HFWT:(20.05±0.57)% vs.(3.17±0.52)%,scratch healing rate declined[WiT49:(45.62±2.58)% vs.(77.53±0.76)%]; HFWT:(43.64±4.88)% vs.(83.02 ± 0.78)%,with a drop in the number of invasive cells (WiT49:51.33±5.73 vs.193.67±5.91; HFWT:18.33±3.68 vs.122.33±5.25) showed a significant reduction in intracellular red lipid droplets with up-regulation of ACAT1 expression and down-regulation of ACLY and FASN expression (P<0.05).After knocking down HADHA in PPAR α overexpressing cells,cell viability rose (WiT49:2.02±0.04 vs.1.84±0.03; HFWT:1.59±0.01 vs.1.41±0.01),apoptosis rate dropped[WiT49:(7.87±0.55)% vs.(16.55±1.01)%; HFWT:(11.21±0.75)% vs.(20.19±1.83)%,showed enhanced migration and invasion capability,increased intracellular lipid particles,decreased ACAT1 expression and up-regulated ACLY and FASN expression (P<0.05).Dual luciferase reporter assay indicated that fluorescent signal intensity of co-transfected cells with HADHA-WT and OE-PPAR α spiked significantly (P<0.05) while there was no significant difference between co-transfected cells with HADHA-MUT and OE-PPAR α.In in vivo experiments,tumor volume[(983.15±279.11) mm3]and mass[(0.87±0.24) g]of control group were significantly higher than those of OE-PPAR α group[(257.74±45.45) mm3,(0.16±0.05) g].The expressions of PPAR α,HADHA and ACAT1 became up-regulated in tumor tissue of OE-PPAR α group while the expressions of ACLY,FASN,Ki-67 and PCNA were down-regulated.Lipid deposition declined and cell apoptosis accelerated.Conclusions PPAR α may regulate HADHA expression through transcription,inhibit proliferation,migration,invasion of nephroblastoma cells,promote apoptosis and reduce lipid deposition.Thus it provides a novel molecular candidate for targeted therapy of nephroblastoma.
参考文献/References:
[1] Cunningham ME, Klug TD, Nuchtern JG, et al.Global disparities in Wilms tumor[J].J Surg Res, 2020, 247:34-51.DOI:10.1016/j.jss.2019.10.044.
[2] van den Heuvel-Eibrink MM, Hol JA, Pritchard-Jones K, et al.Position paper:rationale for the treatment of Wilms tumour in the UMBRELLA SIOP-RTSG 2016 protocol[J].Nat Rev Urol, 2017, 14(12):743-752.DOI:10.1038/nrurol.2017.163.
[3] Spreafico F, Fernandez CV, Brok J, et al.Wilms tumour[J].Nat Rev Dis Primers, 2021, 7(1):75.DOI:10.1038/s41572-021-00308-8.
[4] Brok J, Treger TD, Gooskens SL, et al.Biology and treatment of renal tumours in childhood[J].Eur J Cancer, 2016, 68:179-195.DOI:10.1016/j.ejca.2016.09.005.
[5] Dome JS, Graf N, Geller JI, et al.Advances in Wilms tumor treatment and biology:progress through international collaboration[J].J Clin Oncol, 2015, 33(27):2999-3007.DOI:10.1200/jco.2015.62.1888.
[6] Ma?odobra-Mazur M, O?dakowska M, Dobosz T.Exploring PPAR gamma and PPAR alpha’s regulation role in metabolism via epigenetics mechanism[J].Biomolecules, 2024, 14(11):1445.DOI:10.3390/biom14111445.
[7] 朱金澳, 陈沙沙, 胡朗, 等.HADHA的乙酰化修饰在心血管疾病中的作用研究进展[J].心血管病学进展, 2025, 46(10):906-911.DOI:10.16806/j.cnki.issn.1004-3934.2025.10.010. Zhu JA, Chen SS, Hu L, et al.Role of HADHA acetylation modification in cardiovascular diseases[J].Adv Cardiovasc Dis, 2025, 46(10):906-911.DOI:10.16806/j.cnki.issn.1004-3934.2025.10.010.
[8] Ma ML, Zhang CH, Cao R, et al.UBE2O promotes lipid metabolic reprogramming and liver cancer progression by mediating HADHA ubiquitination[J].Oncogene, 2022, 41(48):5199-5213.DOI:10.1038/s41388-022-02509-1.
[9] Wu XY, Feng R, Wang XQ, et al.Roles of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, a lipid metabolism enzyme, in Wilms tumor patients[J].J Cancer Res Ther, 2021, 17(5):1281-1285.DOI:10.4103/jcrt.jcrt_1388_21.
[10] 覃善璐, 董岿然.肾母细胞瘤胚胎学起源的研究进展[J].临床小儿外科杂志, 2024, 23(7):697-700.DOI:10.3760/cma.j.cn101785-202207041-018. Qin SL, Dong KR.Research advances in embryological origin of Wilms tumor[J].DOI:10.3760/cma.j.cn101785-202207041-018.
[11] Treger TD, Chowdhury T, Pritchard-Jones K, et al.The genetic changes of Wilms tumour[J].Nat Rev Nephrol, 2019, 15(4):240-251.DOI:10.1038/s41581-019-0112-0.
[12] Wagner N, Wagner KD.Peroxisome proliferator-activated receptors and the hallmarks of cancer[J].Cells, 2022, 11(15):2432.DOI:10.3390/cells11152432.
[13] Luo YH, Xie C, Brocker CN, et al.Intestinal PPARα protects against colon carcinogenesis via regulation of methyltransferases DNMT1 and PRMT6[J].Gastroenterology, 2019, 157(3):744-759.e4.DOI:10.1053/j.gastro.2019.05.057.
[14] You ML, Jin JH, Liu Q, et al.PPARα promotes cancer cell Glut1 transcription repression[J].J Cell Biochem, 2017, 118(6):1556-1562.DOI:10.1002/jcb.25817.
[15] Liu S, Liu XL, Wu F, et al.HADHA overexpression disrupts lipid metabolism and inhibits tumor growth in clear cell renal cell carcinoma[J].Exp Cell Res, 2019, 384(1):111558.DOI:10.1016/j.yexcr.2019.111558.
[16] He W, Li YF, Liu SB, et al.From mitochondria to tumor suppression:ACAT1’s crucial role in gastric cancer[J].Front Immunol, 2024, 15:1449525.DOI:10.3389/fimmu.2024.1449525.
[17] Xiao YC, Yang YH, Xiong HB, et al.The implications of FASN in immune cell biology and related diseases[J].Cell Death Dis, 2024, 15(1):88.DOI:10.1038/s41419-024-06463-6.
[18] Du F, Zhang HJ, Shao W, et al.Adenosine diphosphate-ribosylation factor-like 15 can regulate glycolysis and lipogenesis related genes in colon cancer[J].J Physiol Pharmacol, 2022, 73(3):403-411.DOI:10.26402/jpp.2022.3.08.
[19] Gu L, Zhu YH, Lin X, et al.The IKKβ-USP30-ACLY axis controls lipogenesis and tumorigenesis[J].Hepatology, 2021, 73(1):160-174.DOI:10.1002/hep.31249.
[20] Zheng M, Zhang SH, Zhou JJ, et al.ACAT1 suppresses clear cell renal cell carcinoma progression by AMPK mediated fatty acid metabolism[J].Transl Oncol, 2024, 47:102043.DOI:10.1016/j.tranon.2024.102043.
[21] Du QQ, Liu P, Zhang CY, et al.FASN promotes lymph node metastasis in cervical cancer via cholesterol reprogramming and lymphangiogenesis[J].Cell Death Dis, 2022, 13(5):488.DOI:10.1038/s41419-022-04926-2.
相似文献/References:
[1]王跃 吴晔明. 6例Currarino综合征HLXB9基因突变的探讨[J].临床小儿外科杂志,2011,10(02):119.
[J].Journal of Clinical Pediatric Surgery,2011,10(04):119.
[2]邹华新,刘铭.Rb和P21基因在婴幼儿皮肤血管瘤中的表达及意义[J].临床小儿外科杂志,2008,7(04):0.
[3]钱晨 林厚维 孙平 徐国峰 徐卯升 杨涛 耿红全. 尿道下裂的MAMLD1基因研究[J].临床小儿外科杂志,2012,11(02):106.
[4]杨恩 刘铭 彭强. 血管瘤血管内皮生长因子受体KDR基因异常甲基化的研究[J].临床小儿外科杂志,2012,11(04):268.
[J].Journal of Clinical Pediatric Surgery,2012,11(04):268.
[5]张 媛 王常林.肾母细胞瘤黑色素抗原基因 — 1 mRNA的表达及临床意义[J].临床小儿外科杂志,2013,12(02):90.[doi:10.3969/j.issn.1671— 6353.2013.02.004]
ZHANG Yuan,WANG Chang-lin.Expression of melanoma antigen-1 mRNA in nephroblastoma and its clinical significance[J].Journal of Clinical Pediatric Surgery,2013,12(04):90.[doi:10.3969/j.issn.1671— 6353.2013.02.004]
[6]何军,肖海波,王淑华,等. p53免疫组化判定肾母细胞瘤预后的研究[J].临床小儿外科杂志,2016,15(02):163.
[7]何炜婧,柳龚堡,董岿然..肾母细胞瘤伴静脉瘤栓的治疗探讨[J].临床小儿外科杂志,2017,16(05):426.
[8]卫园园,范晋楠,李亚蕊.ADD3基因rs17095355位点多态性与胆道闭锁相关性研究的Meta分析[J].临床小儿外科杂志,2020,19(06):496.[doi:10.3969/j.issn.1671-6353.2020.06.007]
Wei Yuanyuan,Fan Jinnan,Li Yarui.Meta-analysis of the relationship between single nucleotide polymorphism of rs17095355 and biliary atresia[J].Journal of Clinical Pediatric Surgery,2020,19(04):496.[doi:10.3969/j.issn.1671-6353.2020.06.007]
[9]王金湖,蔡嘉斌,李民驹,等.儿童肾母细胞瘤国际及国内诊治方案解读[J].临床小儿外科杂志,2020,19(09):765.[doi:10.3969/j.issn.1671-6353.2020.09.002]
Wang Jinhu,Cai Jiabin,Li Minju,et al.Recent international and domestic advances in the diagnosis and treatment of Wilms tumor[J].Journal of Clinical Pediatric Surgery,2020,19(04):765.[doi:10.3969/j.issn.1671-6353.2020.09.002]
[10]卢冠锦,李盛华,周明旺,等.先天性马蹄内翻足致病基因及遗传因素的研究进展[J].临床小儿外科杂志,2020,19(09):789.[doi:10.3969/j.issn.1671-6353.2020.09.006]
Lu Guanjin,Li Shenghua,Zhou Mingwang,et al.Research advances in pathogenic genes and hereditary factors of congenital talipes equinovarus[J].Journal of Clinical Pediatric Surgery,2020,19(04):789.[doi:10.3969/j.issn.1671-6353.2020.09.006]
备注/Memo
收稿日期:2026-1-12。
基金项目:湖南省省级科技计划(2023JJ40347); 国家自然科学基金青年基金(82203414); 湖南省卫生健康委员会科研计划(202204055028)
通讯作者:何军,Email:hejunhnetyy@163.com