十溴联苯醚灌胃后小鼠宫颈癌皮下移植瘤组织肿瘤相关成纤维细胞标志基因表达变化及其意义

Changes in expression of cancer-associated fibroblast marker genes in mouse cervical cancer subcutaneously implanted tumor tissues after intragastric administration of BDE-209 and its significance

目的观察十溴联苯醚(BDE-209)灌胃后小鼠宫颈癌皮下移植瘤组织肿瘤相关成纤维细胞(CAFs)标志基因表达变化,并探讨其参与宫颈癌发生发展的相关机制。方法将40只雌性C57BL/6小鼠随机分为对照组、BDE-209低剂量组、BDE-209中剂量组、BDE-209高剂量组,每组10只。各组小鼠均采用右前腋下接种宫颈癌U14细胞悬液的方法建立宫颈癌皮下移植瘤模型,BDE-209低剂量组、BDE-209中剂量组、BDE-209高剂量组经口灌胃给予20、100、500 mg/kg BDE-209,对照组给予等体积玉米油,连续21 d。取各组小鼠宫颈癌皮下移植瘤组织,进行转录组测序后筛选差异表达基因,与查阅文献得到的小鼠CAFs标志基因取交集后获得CAFs标志差异表达基因。对CAFs标志差异表达基因进行基因本体论(GO)功能和基因组百科全书(KEGG)通路富集分析,建立蛋白质相互作用网络(PPI)图后采用Betweenness算法确定排名前十的CAFs标志差异表达基因,即为CAFs标志差异核心基因。取BDE-209高剂量组与对照组小鼠部分宫颈癌皮下移植瘤组织,采用实时荧光定量PCR法检测其10个CAFs标志差异核心基因表达。结果共筛选到CAFs标志差异表达基因30个,其中BDE-209干预后上调29个、下调1个。GO功能富集分析结果显示,CAFs标志差异基因主要富集在细胞外基质(ECM)、生殖结构发育、PI3K/Akt信号转导等;KEGG通路富集分析结果显示,CAFs标志差异基因主要富集在补体和凝血级联反应、肿瘤发病途径等信号通路。PPI图和Betweenness算法筛选得到排名前十的CAFs标志差异核心基因分别为核心蛋白聚糖(Dcn)、细胞间黏附分子1(Icam1)、C-X-C基序趋化因子配体12(Cxcl12)、基质金属蛋白酶2(MMP-2)、血小板衍生生长因子受体(Pdgfrb)、Ⅴ型胶原蛋白α2(Col5a2)、补体成分1的子成分1(C1s1)、肝细胞生长因子(Hgf)、补体成分3(C3)、纤溶酶原激活物(Plat)。与对照组比较,BDE-209高剂量组小鼠移植瘤组织Dcn、Icam1、Cxcl12、MMP-2、Pdgfrb、Col5a2、C1s1、Hgf、C3、Plat mRNA相对表达量均升高(P均<0.05),Pdgfrb mRNA相对表达量无明显变化(P>0.05)。结论BDE-209灌胃会导致小鼠宫颈癌皮下移植瘤组织CAFs相关标志基因Dcn、Icam1、Cxcl12、MMP-2、Col5a2、C1s1、Hgf、C3、Plat表达升高,上述基因表达改变可能通过诱导CAFs的ECM变化和激活PI3K/Akt信号通路参与宫颈癌的发生发展。

Objective To observe the changes in expression of cancer-associated fibroblasts(CAFs)marker genes of mouse cervical cancer subcutaneously implanted tumor tissues after intragastric administration of decabromodiphenyl ether(BDE-209)and to explore their possible mechanisms.Methods Forty female C57BL/6 mice were randomly divided into the control group,low-dose BDE-209 group,medium-dose BDE-209 group,and high-dose BDE-209 group,with 10 mice in each group.All mice were used to establish the subcutaneous graft tumor models of cervical cancer by inoculation of cervical cancer U14 cell suspension in the right anterior axilla.Mice in the low-dose,medium-dose,and high-dose BDE-209 groups were given 20,100,and 500 mg/kg BDE-209 by oral gavage,and mice in the control group were given an equal volume of corn oil for 21 consecutive days.Transcriptome sequencing was performed on the subcutaneously transplanted tumor tissues of mice in each group to screen the differentially expressed genes.The differential expressed genes and mouse CAFs marker genes were intersected to obtain CAFs marker differentially expressed genes.Gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed on CAFs marker differentially expressed genes,and the top ten CAFs marker differentially expressed genes were identified as CAFs marker differentially hub genes by using the Betweenness algorithm after the establishment of protein-protein interaction(PPI)network graphs.Some cervical cancer tissues from mice in the high-dose BDE-209 group and control group were taken,and their CAFs differential hub genes expression was detected by real-time fluorescence quantitative PCR.Results Thirty CAFs marker differential genes were screened out,including 29 up-regulated genes and 1 down-regulated gene.GO functional enrichment analysis showed that CAFs marker differential genes were mainly enriched in the extracellular matrix(ECM),reproductive structure development,PI3K/Akt signaling.KEGG pathway enrichment analysis showed that CAFs marker differential genes were mainly enriched in signaling pathways such as complement and coagulation cascade reactions and tumor pathways.The top ten hub genes identified by the PPI graphs and Betweenness algorithm were decorin(Dcn),intercellular adhesion molecule 1(Icam1),C-X-C motif chemokine ligand 12(Cxcl12),matrix metallopeptidase 2(MMP-2),platelet-derived growth factor receptor,beta polypeptide(Pdgfrb),Col5a2,complement component 1,s subcomponent 1(C1s1),hepatocyte growth factor(Hgf),complement component 3(C3),and plasminogen activator(Plat).Compared with the control group,the relative expression levels of Dcn,Icam1,Cxcl12,MMP-2,Pdgfrb,Col5a2,C1s1,Hgf,C3,and Plat mRNAs were elevated in the cancer tissues of mice in the high-dose BDE-209 group(all P<0.05),whereas there was no change in the relative expression of Pdgfrb mRNA(P>0.05).Conclusion Intragastric administration of BDE-209 resulted in elevated expression levels of CAFs-related marker genes Dcn,Icam1,Cxcl12,MMP-2,Col5a2,C1s1,Hgf,C3,and Plat in mouse cervical cancer subcutaneously implanted tumor tissues,and the above-mentioned changes in gene expression may be involved in the occurrence and development of cervical cancer by inducing the ECM changes of CAFs and activating the PI3K/Akt signaling pathway.