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,,():344-354.[doi:10.3760/cma.j.cn101785-20260112-00019]
Effect of PPARα-regulated HADHA on biological behavior of human wilms tumor
- 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.
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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