基于转录组测序技术挖掘游泳运动抗肝细胞癌作用的潜在干预机制

Potential Intervention Mechanisms of Swimming Exercise Against Hepatocellular Carcinoma Cancer Based on Transcriptome Sequencing Technology

目的基于转录组测序技术挖掘游泳运动对肝细胞癌(hepatocellular carcinoma,HCC)裸鼠肿瘤生长发挥抑制作用的潜在机制。方法将12只4周龄BALB/C雄性裸鼠随机区组分为运动组、对照组,每组各6只。运动组裸鼠游泳适应1周后与对照组均进行皮下瘤接种,之后运动组裸鼠进行游泳训练(0%体重负荷,30 min/d,每周5天,连续4周)。训练结束后处死裸鼠收集血清并测量瘤重,同时使用细胞计数试剂盒(cell counting kit 8,CCK-8)试验验证游泳运动血清对于HCC细胞增殖的抑制能力,并进一步运用转录组测序探究游泳运动干预HCC的潜在机制。结果运动组裸鼠瘤重较对照组显著下降[(0.136±0.020)g vs.(0.290±0.099)g,t=26.000,P<0.05];CCK-8试验结果显示,运动组36 h[(0.418±0.001)g vs.(0.431±0.002)g,t=6.000,P<0.05]以及72 h[(0.402±0.019)g vs.(0.452±0.022)g,t=2.955,P<0.05]的细胞光密度(optical density,OD)值较对照组均显著降低。转录组测序分析结果显示,运动组与对照组相比,测序共筛选差异基因1263个,与HCC靶标基因交集后共获得89个交集靶标基因。蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络分析结果显示,信号转导与转录激活因子1(signal transducerand activator of transcription 1,STAT1)、抗原加工相关转运物1(transporters associated with antigen processing 1,TAP1)、多功能肽酶(recombinant proteasome subunit beta type 9,PSMB9)、人类白细胞抗原B(human leukocyte antigen B,HLA-B)、干扰素调节因子1(interferon regulatory factor 1,IRF1)以及β2-微球蛋白(beta-2 microglobulin,β2M)等基因各算法得分靠前。基因本体(gene ontology,GO)富集分析结果显示,下调基因的GO条目共20个,上调条目共13个,其中“negative regulation of canonical Wnt signaling pathway”等条目与肿瘤密切相关。京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析结果显示,下调基因的KEGG条目共11个,其中“TNF signaling pathway”“IL-17 signaling pathway”等条目与肿瘤密切相关。基因集富集分析(Gene Set Enrichment Analysis,GSEA)结果显示,与肿瘤相关条目有“Prostate cancer”“Pathways in cancer”“Leukocyte transendothelial migration”等。结论游泳运动可以抑制HCC模型裸鼠的肿瘤生长,且其干预调控机制可能是多途径的,Wnt信号通路,肿瘤坏死因子(tumornecrosisfactor,TNF)信号通路与磷脂酰肌醇-3-激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B,PI3K/Akt)信号通路等均可能是其潜在的干预机制。

Objective To explore the potential mechanism of swimming inhibiting the tumor growth of hepatocellular carcinoma(HCC)in nude mice based on transcriptome sequencing technology.Methods A total of 124-week-old BALB/C male nude mice were randomly divided into exercise group and control group,with 6 mice in each.After one week of swim-ming adaptation,both the nude mice in the exercise group and the control group received subcutaneous tumor inocula-tion,afterwards,the nude mice in exercise group underwent swimming training(0%body weight load,30 min/d,5 days per week for 4 consecutive weeks).After the training,the nude mice were executed to collect serum and measure tumor weight,at the same time,the cell counting kit 8(CCK-8)test was used to verify the inhibitory ability of swimming exercise serum on proliferation of HCC cell,and transcriptome sequencing was further used to explore the po-tential mechanism of aerobic exercise intervention in HCC.Results The tumor weight of the nude mice in the exercise group significantly decreased compared to the control group[(0.136±0.020)g vs.(0.290±0.099)g,t=26.000,P<0.05];the CCK-8 test results showed that the optical density(OD)of cells in the exercise group was significantly decreased at 36 h[(0.418±0.001)g vs.(0.431±0.002)g,t=6.000,P<0.05]and 72 h[(0.402±0.019)g vs.(0.452±0.022)g,t=2.955,P<0.05]compared to the control group.The results of transcriptome sequencing analy-sis showed that compared with the control group,a total of 1263 differentially expressed genes were screened for sequen-cing in the exercise group,after intersecting with HCC target genes,a total of 89 intersecting target genes were ob-tained.The results of protein-protein interaction(PPI)analysis indicated that genes such as signal transducerand activa-tor of transcription 1(STAT1),transporters associated with antigen processing 1(TAP1),recombinant proteasome subunit beta type 9(PSMB9),human leukocyte antigen B(HLA-B),interferon regulatory factor 1(IRF1)and beta-2 microglobulin(β2 M)and other genes scored the highest in each algorithm.The results of gene ontology(GO)en-richment analysis showed that there were 20 GO items down-regulated and 13 items up-regulated,among which"nega-tive regulation of canonical Wnt signaling pathway"and other items were closely related to tumors.The enrichment anal-ysis results of Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed a total of 11 KEGG i-tems for down-regulated genes,items such as"TNF signaling pathway"and"IL-17 signaling pathway"were closely re-lated to tumors.The results of Gene Set Enrichment Analysis(GSEA)showed that tumor related items included"Pros-tate cancer""Pathways in cancer""Leukocyte transendothelial migration"and so on.Conclusion Swimming exercise can inhibit tumor growth of nude mice in HCC model,and its intervention and regulation mechanism may be multifaceted,the Wnt signaling pathway,tumornecrosisfactor(TNF)signaling pathway and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway may all be potential intervention mechanisms.