维A酸衍生物ECPIRM对皮肤T细胞淋巴瘤HH细胞增殖、凋亡的影响及其机制初探

Effect of a retinoid derivative ECPIRM on proliferation and apoptosis of a cutaneous T-cell lymphoma cell line HH and its mechanisms:a preliminary investigation

目的探讨维A酸衍生物ECPIRM对人皮肤T细胞淋巴瘤HH细胞的增殖抑制作用及潜在机制。方法以0(对照组)、5、10、20μmol/L ECPIRM处理HH细胞72 h,采用CCK8法检测ECPIRM对HH细胞增殖活力的影响,采用流式细胞仪检测ECPIRM对HH细胞凋亡的影响。以10μmol/L ECPIRM处理HH细胞72 h,采用转录组学测序分析ECPIRM对HH细胞基因表达的调控作用,结合KEGG通路分析和GO功能富集分析比较ECPIRM诱导的差异表达的相关基因。采用反转录qPCR验证相关通路中关键基因的表达变化。组间差异采用单因素方差分析,采用LSD-t检验进行两两比较。结果CCK8结果显示,ECPIRM对HH细胞IC50为(4.91±2.48)μmol/L,对照组与5、10、20μmol/L ECPIRM组HH细胞活力分别为100.00%±2.87%、49.58%±4.53%、48.36%±2.88%、31.44%±2.46%,4组细胞活力差异有统计学意义(F=162.86,P<0.001),5、10、20μmol/L组细胞活力均低于对照组(t值分别为15.36、15.73和20.89,均P<0.001)。流式细胞仪检测结果显示,4组细胞凋亡率差异有统计学意义(11.51%±1.84%、23.83%±5.72%、36.19%±8.33%、49.75%±4.10%,F=17.62,P<0.001),10、20μmol/L组细胞凋亡率均高于对照组(t值分别为4.46、6.92,均P<0.01)。转录组学分析发现,ECPIRM诱导的增殖抑制作用可能与类固醇的代谢调控有关。反转录qPCR验证发现,10μmol/L ECPIRM组L-氨基酸氧化酶(IL4I1)、乙酰辅酶A乙酰转移酶2(ACAT2)、3-羟基-3-甲基戊二酰辅酶A合酶1(HMGCS1)、甲羟戊酸二磷酸脱羧酶(MVD)、3-β-羟基类固醇-8,7-异构酶(EBP)、极低密度脂蛋白受体(VLDLR)、3-羟基3-甲基戊二酰辅酶A还原酶(HMGCR)mRNA相对表达与对照组比较明显受到抑制(均P<0.05)。结论维A酸衍生物ECPIRM对HH细胞可发挥显著的抗增殖及凋亡诱导作用,其机制可能与类固醇代谢关键基因的表达抑制有关。

Objective To evaluate the inhibitory effect of a retinoid derivative ECPIRM on proliferation of a cutaneous T-cell lymphoma(CTCL)cell line HH,and to explore its potential mechanisms.Methods Cultured HH cells were treated with ECPIRM at different concentrations of 0(control group),5,10 and 20μmol/L separately for 72 hours,cell counting kit-8(CCK8)assay was performed to evaluate the effect of ECPIRM on the proliferative activity of HH cells,and flow cytometry to investigate the effect of ECPIRM on apoptosis of HH cells.Some HH cells were treated with 10μmol/L ECPIRM for 72 hours,transcriptome sequencing was performed to investigate gene expression changes triggered by ECPIRM in HH cells,and Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis and gene ontology(GO)enrichment analysis were then performed to analyze differentially expressed genes in HH cells induced by ECPIRM.Reverse transcription-qPCR was subsequently conducted to verify changes in key gene expression in related pathways.Intergroup differences were analyzed by using one-way analysis of variance,and least significant difference(LSD)-t test was used for multiple comparisons.Results CCK8 assay showed that the 50%inhibitory concentration(IC50)of ECPIRM on HH cells was 4.91±2.48μmol/L,the viability of HH cells significantly differed among the control group,and 5-,10-and 20-μmol/L ECPIRM groups(100.00%±2.87%,49.58%±4.53%,48.36%±2.88%,31.44%±2.46%,respectively,F=162.86,P<0.001),and was significantly lower in the 5-,10-and 20-μmol/L ECPIRM groups than in the control group(t=15.36,15.73,20.89,respectively,all P<0.001).Flow cytometry showed that there was a significant difference in the apoptosis rate among the 4 groups(11.51%±1.84%,23.83%±5.72%,36.19%±8.33%,49.75%±4.10%,respectively,F=17.62,P<0.001),and the 10-and 20-μmol/L groups showed significantly increased apoptosis rates compared with the control group(t=4.46,6.92 respectively,both P<0.01).Transcriptomics analysis revealed that the inhibitory effect of ECPIRM on the cellular proliferative activity may be related to the metabolic regulation of steroids.As reverse transcription-qPCR revealed,the 10-μmol/L ECPIRM group showed significantly decreased mRNA expression of L-amino acid oxidase(IL4I1),acetyl-coenzyme A acetyltransferase 2(ACAT2),3-hydroxy-3-methylglutaryl-coenzyme A synthase 1(HMGCS1),mevalonate diphosphate decarboxylase(MVD),3-β-hydroxysteroid-8,7-isomerase(EBP),very low-density lipoprotein receptor(VLDLR),3-hydroxy 3-methylglutaryl-CoA reductase(HMGCR)compared with the control group(all P<0.05).Conclusion The retinoid derivative ECPIRM exerted marked anti-proliferative and apoptosis-inducing effects on HH cells,which may be related to the decreased expression of key genes involved in steroid metabolism.