摘要
目的:通过转录组测序技术(RNA-seq)分析仙连解毒方干预"湿热瘀毒证"小鼠结直肠肿瘤的转录组学特征。方法:将90只C57BL/6(雄性)小鼠随机分为正常组、"湿热瘀毒证"结直肠肿瘤模型组及仙连解毒方组,每组30只。模型组和仙连解毒方组予以高脂饲料喂养,以经典氧化偶氮甲烷(AOM)/葡聚糖硫酸钠(DSS)方式构建结直肠癌模型,仙连解毒方组从造模当天开始给药(12.9 g·kg^(-1)),共给药112 d。末次给药结束4 h后,取各组小鼠结直肠组织,苏木素-伊红(HE)染色和亚甲基蓝染色观察小鼠结直肠的组织病理形态;提取结直肠组织总RNA,采用RNA-seq技术进行转录组学测序,进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析,并筛选、验证差异基因。结果:与模型组比较,仙连解毒方可显著改善小鼠结直肠充血水肿情况,减少小鼠结直肠肿瘤数量、体积;亚甲基蓝染色结果表明仙连解毒方可以显著抑制畸形隐窝灶(ACF)的发生(P<0.01);HE染色显示仙连解毒方可显著减轻结直肠组织的损伤、异型增生;RNA-seq测序结果显示,模型组与正常组差异表达的基因共615个,上调基因446个,下调基因169个。仙连解毒方组与模型组差异表达的基因共54个,上调基因29个,下调基因25个。RNA-seq测序分析发现仙连解毒方主要调控NIMA相关蛋白激酶7(Nek7,P<0.01),黏蛋白16(Muc16,P<0.01),Siah E3泛素蛋白连接酶家族成员3(Siah3,P<0.01),胰岛再生基因3g(Reg3g,P<0.01),RNA聚合酶2延长因子相关因子2(Eaf2,P<0.01),转化生长因子-α(TGF-α,P<0.05),分泌珠蛋白家族1A成员1(Scgb1a1,P<0.05),序列相似家族227成员B(Fam227B,P<0.05),细胞色素P450家族2亚家族c多肽40(Cyp2c40,P<0.01),锚蛋白重复及EF手结构域蛋白1(Ankef1,P<0.05)等基因的表达,并且肠上皮细胞增殖等生物过程、细胞色素P450酶对外源性药物的代谢作用,花生四烯酸代谢等信号通路被显著富集。结论:仙连解毒方具有干预"湿热瘀毒证"小鼠结直肠肿瘤发生、发展的作用,转录组学分析提示可能与干预代谢相关通路密切相关。
Objective:To analyze the transcriptome characteristics of Xianlian Jiedu prescription(XLJDP)in the intervention of colorectal carcinoma by high-throughput c DNA-sequencing(RNA-seq).Method:Ninety male C57BL/6 mice were randomly divided into the control group,colorectal carcinoma due to dampness,heat,stasis,and toxin model group,and XLJDP group,with 30 mice in each group.Mice in the model group and XLJDP group were fed a high-fat diet and provided with azoxymethane and dextran sodium sulfate(AOM/DSS)for inducing colorectal carcinoma.Those in the XLJDP group were further treated with intragastric administration of 12.9 g·kg^(-1)XLJDP since the day of modeling for 112 days.The colorectal tissues were collected from each group 4 h after the last drug treatment and stained with hematoxylin-eosin(HE)and methylene blue for observing the pathological changes.The total RNA was extracted from colorectal tissues for RNA-Seq-based transcriptome profiling,followed by gene oncology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis and the screening and verification of differentially expressed genes.Result:Compared with the model group,XLJDP significantly relieved the colorectal congestion and edema and decreased tumor number and volume in mouse colorectal tissues.The methylene blue staining results indicated that XLJDP significantly suppressed the development of aberrant crypt foci(ACF,P<0.01).As revealed by HE staining,XLJDP significantly alleviated the injury and dysplasia of colorectal tissues.Transcriptome analysis identified 615 differentially expressed genes(446 up-regulated and 169 down-regulated)between the model group and the blank group and 54 differentially expressed genes(29 up-regulated and 25down-regulated)between the XLJDP group and model group.XLJDP mainly affected the expression of NIMA-related protein kinase 7 gene(Nek7,P<0.01),Mucin 16(Muc16,P<0.01),Siah E3 ubiquitin protein ligase family member 3(Siah3,P<0.01),regenerating islet-derived protein 3-gamma(Reg3g,P<0.01),RNApolymeraseⅡelongation factor-associated factor 2(Eaf2,P<0.01),transforming growth factor‐alfa gene(TGF-α,P<0.05),secretoglobin family 1A member 1(Scgb1a1,P<0.05),family with sequence similarity 227member B(Fam227B,P<0.05),cytochrome P450 family 2 subfamily c polypeptide 40(Cyp2c40,P<0.01),and ankyrin repeat and EF-hand domain containing protein 1(Ankef1,P<0.05).Enrichment analysis showed that intestinal epithelial cell proliferation,metabolism of xenobiotics by cytochrome P450,and arachidonic acid metabolism signaling pathway were significantly enriched.Conclusion:XLJDP is able to interfere with colorectal tumorigenesis and development due to dampness,heat,stasis,and toxin in mice,which has been proved by transcriptome analysis to be related to the regulation of metabolism-related pathways.
作者
陈桐庆
余成涛
许惠琴
程海波
沈卫星
谭佳妮
徐长亮
赖岳阳
范旻旻
CHEN Tong-qing;YU Cheng-tao;XU Hui-qin;CHENG Hai-bo;SHEN Wei-xing;TAN Jia-ni;XU Chang-liang;LAI Yue-yang;FAN Min-min(Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor,Nanjing 210023,China;The First Clinical Medical College,Nanjing University of Chinese Medicine,Nanjing 210023,China;School of Medicine and Holistic Integrative Medicine,Nanjing University of Chinese Medicine,Nanjing 210023,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第8期63-71,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金重点项目(81930117)
江苏高校优势学科建设工程资助项目(PAPD)
江苏省自然科学基金青年项目(BK20190236)
张家港市卫健委青年科技项目(ZJGQNKJ201902)。
关键词
转录组学
仙连解毒方
结直肠肿瘤
细胞代谢
transcriptomics
Xianlian Jiedu prescription
colorectal carcinoma
cell metabolism
