DCPIB通过上调DDIT3抑制黑色素瘤细胞增殖的机制研究  

DCPIB inhibits melanoma cell proliferation by upregulating DDIT3

作　　者：栗梦雅 蔡梦迪 LI Mengya;CAI Mengdi(Key Laboratory of Preservation of Human Genetic Resources and Disease Control in China,Ministry of Education,Harbin 150081,China;Laboratory of Genetics,Harbin Medical University)

机构地区：[1]中国遗传资源保护与疾病防控教育部重点实验室,哈尔滨150081 [2]哈尔滨医科大学医学遗传学研究室

出　　处：《实用肿瘤学杂志》2025年第2期99-107,共9页Practical Oncology Journal

基　　金：中国遗传资源保护与疾病防控教育部重点实验室开放课题(编号:LPHGRDC2020-002);哈尔滨医科大学少帅揭榜项目(编号:HMUMIF-21007)。

摘　　要：目的 探讨选择性可逆体积调节性阴离子通道抑制剂DCPIB(4-[(2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-3H-inden-5-yl)oxy]butanoic acid)对黑色素瘤细胞增殖能力的影响及作用机制。方法 采用CCK-8法和EdU实验检测DCPIB对黑色素瘤细胞A375和RPMI-7951增殖能力的影响;通过转录组测序分析对照组(无水乙醇处理的A375细胞)和DCPIB处理组(10μM DCPIB处理的A375细胞)的差异表达基因,寻找显著变化的信号通路;qRT-PCR检测差异表达基因mRNA水平变化;Western blot检测相关信号通路蛋白水平变化。结果 DCPIB可显著抑制人黑色素瘤细胞A375和RPMI-7951的增殖(P<0.01)。转录组测序结果显示DCPIB处理组中DNA损伤诱导转录因子3(DNA damage-inducible transcript 3,DDIT3)显著上调(t=161.800,P<0.001),多种DNA损伤修复途径相关关键基因Ku70、Ku80、RAD51、MLH1、MSH6、PARP1、FANCD2 mRNA表达水平下调,且qRT-PCR验证了DCPIB处理A375细胞36 h后上述DNA损伤修复通路关键基因的mRNA表达水平显著降低(P<0.05)。GO功能富集和KEGG通路富集分析结果显示DCPIB处理后内质网应激相关基因HSPA5、DDIT3、ATF4、XBP1、ERN1、EIF2AK3 mRNA表达水平上调,下游PI3K-Akt信号通路表达异常,qRT-PCR验证DCPIB处理组A375细胞的DDIT3下游分子TRIB3表达上调(t=2.819,P=0.048),下游Akt-mTOR信号通路受到抑制,p-Akt(t=7.638,P=0.002)和p-mTOR(t=4.898,P=0.008)蛋白表达水平明显下降。结论 DCPIB可显著抑制黑色素瘤细胞的增殖,其机制可能与DDIT3-TRIB3-Akt-mTOR信号轴有关。Objective The objective of this study was to investigate the effect of DCPIB(4-[(2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-3H-inden-5-yl)oxy]butanoic acid),a selective reversible volume-regulated anion channel inhibitor,on the proliferation capacity of melanoma cells and its mechanism of action.Methods CCK-8 assay and EdU assay were used to detect the effect of DCPIB on the proliferation capacity of A375 cells and RPMI-7951 cells.Transcriptome sequencing was conducted to analyze the differentially expressed genes(DEGs)between the control group(A375 cells treated with absolute ethanol)and the DCPIB-treated group(A375 cells treated with 10μM of DCPIB)to find the significantly changed signaling pathways.qRT-PCR was used to detect the changes in the mRNA levels of DEGs.Western blot was used to detect the changes in the expression of proteins related to relevant signaling pathways.Results DCPIB significantly inhibited the proliferation of A375 cells and RPMI-7951 cells(P<0.01).The results of transcriptome sequencing revealed that DNA damage-inducible transcript 3(DDIT3)was significantly upregulated in the DCPIB-treated group(t=161.800,P<0.001),and the levels of multiple key genes mRNA related to DNA damage repair pathway,including Ku70,Ku80,RAD51,MLH1,MSH6,PARP1,FANCD2 were downregulated.qRT-PCR verified that the levels of key genes mRNA in the above DNA damage repair pathways were significantly reduced after DCPIB treatment in A375 cells for 36 h(P<0.05).GO functional enrichment and KEGG pathway analyses revealed that after DCPIB treatment,the mRNA levels of endoplasmic reticulum stress-related genes,including HSPA5,DDIT3,ATF4,XBP1,ERN1,EIF2AK3 were upregulated,and the downstream PI3K-Akt signaling pathway was abnormally expressed.qRT-PCR verified that the expression of TRIB3,a downstream molecule of DDIT3 was upregulated in A375 cells treated with DCPIB(t=2.819,P=0.048),and the downstream Akt-mTOR signaling pathway was inhibited,and the levels of p-Akt(t=7.638,P=0.002)and p-mTOR proteins(t=4.898,P=0.008)were signifi

关 键 词：黑色素瘤 A375细胞 内质网应激 DNA损伤修复 Akt-mTOR信号通路 

分 类 号：R739.5[医药卫生—肿瘤]