摘要
目的 探讨血清淀粉样蛋白A1(SAA1)调控胰腺导管腺癌(PDAC)细胞的增殖和转移能力及相应的机制研究.方法 通过从基因表达综合数据库(GEO)数据库下载GSE71729数据集中正常胰腺组织、原位PDAC和肝转移的PDAC组织的RNA表达矩阵数据并使用R软件筛选差异表达基因,设计构建SAA1的小干扰RNA(siRNA)在PDAC细胞中沉默SAA1的表达并通过实时定量反转录聚合酶链反应(RT-qPCR)、细胞计数试剂盒(CCK-8)、Transwell和蛋白质印迹法(Western blot)等实验检测沉默SAA1对PDAC细胞增殖和转移能力的调控作用并探寻作用机制,构建转染沉默SAA1且包含荧光素酶的慢病毒(sh-SAA1)的PDAC细胞并对裸鼠进行皮下及尾静脉注射检测增殖和转移能力变化.两组间数据比较使用独立样本t检验.结果 在GSE71729数据集中,原位PDAC组织中SAA1表达量显著高于正常胰腺组织且在肝转移的PDAC组织中表达量进一步增高(logFC=3.08、4.62,P＜0.05),在 PDAC 细胞系 PANC-1 和 CFPAC-1 中沉默 SAA1,沉默SAA1 组 SAA1 表达量低于对照组(0.584±0.038、0.529±0.032 比 1.045±0.092,t=6.555、7.499,P＜0.01;0.470±0.032、0.501±0.048 比 1.010±0.086,t=8.334、7.339,P＜0.01),沉默 SAA1 组细胞增殖低于对照组(2.254±0.158、1.959±0.144 比 3.214±0.127,t=8.192、11.330,P＜0.01;1.303±0.092、1.591±0.115 比 2.293±0.116,t=11.600、7.456,P＜0.01)、沉默 SAA1 组细胞迁移低于对照组[(137.667±11.441)、(231.000±11.518)个比(775.333±44.289)个,t=19.710、16.820,P＜0.01;(241.667±24.851)、(140.000±21.649)个比(650.000±15.895)个,t=19.580、26.850,P＜0.01],沉默 SAA1 组细胞侵袭低于对照组[(140.667±15.326)、(321.000±20.833)个比(735.000±23.930)个,t=15.640、14.950,P＜0.01;(262.333±17.172)、(275.000±17.205)个比(556.667±20.336)个,t=29.580、18.450,P＜0.01].动物实验结果同样显示沉默SAA1组成瘤体积低于对照组[(828.800±66.110)mm3 比(1 345.000±46.904)mm3,t=12.740,P＜0.01],沉默SAA1 组成瘤重量低于对照组[(0.656±0.154)g 比(1.066±0.125)g,t=4.137,P＜0.01].使用GSEA分析SAA1发挥作用的机制,结果显示SAA1高表达与细胞外基质受体互作通路相关(NS=0.45,P＜0.05)且沉默SAA1组血小板反应蛋白4(THBS4)表达量低于对照组(1.725±0.239比7.285±2.181,t=2.546,P＜0.01),沉默SAA1组基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)表达低于对照组(1.681±0.584 比 2.741±1.052,t=4.148,P＜0.01;5.645±0.647 比7.116±1.375,t=3.764,P＜0.01).结论 在PDAC细胞中,高表达的SAA1通过抑制THBS4促进MMP-2和MMP-9表达从而增强PDAC的增殖和转移能力.
Abstract
Objective To investigate the regulatory effects of serum amyloid Al(SAA1)on the proliferation and metastasis of pancreatic ductal adenocarcinoma(PDAC)cells and correlated mechanism.Methods RNA expression matrix data of normal pancreatic tissues,in situ PDAC and hepatic metastatic PDAC tissues in GSE71729 dataset were downloaded from gene expression omnibus(GEO)database and differentially expressed genes were screened using R software.The small interfering RNA(siRNA)of SAA1 were designed and constructed to silence the expression of SAA1 in PDAC cells.The regulatory effect of SAA1 silencing on the proliferation and metastasis of PDAC cells and related mechanism was ex-plored by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR),cell counting kit-8(CCK-8),Transwell and Western blotting experiments.PDAC cells transfected with SAA1-silenced lentivirus containing luciferase region(sh-SAA1)were constructed,and proliferation and metastasis were detected by subcutaneous and tail vain injection in nude mice.All the data were from 3 groups of independ-ent experiments,and were represented by mean±standard deviation(x±s).Data between the two groups were compared using independent sample t-test,and P＜0.05 was considered statistically significant.Results In the GSE71729 dataset,the expression of SAA1 in in situ PDAC tissue was significantly higher than that in normal pancreatic tissue and its expression was further increased in hepatic metastatic PDAC tissues(logFC=3.08,4.62,P＜0.05).After silencing the expression of SAA1 in PDAC cell lines PANC-1 and CFPAC-1,the expression of SAA1 in the silenced SAA1 group was significantly lower than that in the control group(0.584±0.038,0.529±0.032 vs.1.045±0.092,t=6.555,7.499,P＜0.01;0.470±0.032,0.501±0.048 vs.1.010±0.086,t=8.334,7.339,P＜0.01).The cell prolif-eration in the silenced SAA1 group was lower than that in the control group(2.254±0.158,1.959± 0.144 vs.3.214±0.127,t=8.192,11.330,P＜0.01;1.303±0.092,1.591±0.115 vs.2.293± 0.116,t=11.600,7.456,P＜0.01).The cell migration in the silenced SAA1 group was lower than that in the control group[(137.667±11.441),(231.000±11.518)vs.(775.333±44.289),t=19.710,16.820,P＜0.01;(241.667±24.851),(140.000±21.649)vs.(650.000±15.895),t=19.580,26.850,P＜0.01].The cell invasion in the silenced SAA1 group was lower than that in the control group[(140.667±15.326),(321.000±20.833)vs.(735.000±23.930),t=15.640,14.950,P＜0.01;(262.333±17.172),(275.000±17.205)vs.(556.667±20.336),t=29.580,18.450,P＜0.01].The results of animal experiments also showed that the tumor volume in the silenced SAA1 group was lower than that in the control group[(828.800±66.110)mm3 vs.(1 345.000±46.904)mm3,t=12.740,P＜0.01].The tumor weight in the silenced SAA1 group was lower than that in the control group[(0.656±0.154)g vs.(1.066±0.125)g,t=4.137,P＜0.01].GSEA was used to analyze the mech-anism of SAA1 and the results showed that high expression of SAA1 was correlated with the extracellular matrix receptor interaction pathway(NS=0.45,P＜0.05),the expression of thrombospondin 4(THBS4)in the silenced SAA1 group was higher than that in the control group(1.725±0.239 vs.7.285±2.181,t=2.546,P＜0.01),and the expression of matrix metallopeptidase-2(MMP-2)and matrix metallopeptid-ase-9(MMP-9)in the silenced SAA1 group was lower than that in the control group(1.681±0.584 vs.2.741±1.052,t=4.148,P＜0.01;5.645±0.647 vs.7.116±1.375,t=3.764,P＜0.01).Conclusion In PDAC cells,high expression of SAA1 promotes the expression of MMP-2 and MMP-9 through inhibiting THBS4 and thereby enhancing the proliferation and metastasis of PDAC cells.
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