目的:探索2',5'-寡腺苷酸合成酶1(2',5'-oligoadenylate synthase 1,OAS1)在胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)中的表达情况、临床意义以及其对PDAC细胞增殖能力的调控作用及潜在机制.方法:利用高通量基因表达数据库(Gene Expression Omnibus,GEO)和癌症基因图谱(The Cancer Genome Atlas,TCGA)等公共数据库分析OAS1在胰腺癌组织中的表达情况,并通过免疫组织化学染色验证OAS1在PDAC患者组织芯片中的表达情况及其与患者临床预后的相关性;通过实时荧光定量PCR和蛋白质印迹实验检测OAS1 mRNA和蛋白质在不同PDAC细胞系中的表达水平;利用siRNA干扰OAS1在Patu-8988与PDC0034细胞系中的表达,随后通过CCK-8实验和克隆形成实验探究OAS1对PDAC细胞增殖能力的影响;通过基因集富集分析(Gene Set Enrichment Analysis,GSEA)筛选OAS1调控PDAC的可能机制;利用siRNA干扰OAS1在Patu-8988与PDC0034细胞系中的表达的同时对细胞予以哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路激动剂MHY1485处理,通过测定细胞存活能力和细胞中总胆固醇含量来验证OAS1调控PDAC细胞增殖的潜在分子机制.结果:数据库分析结果提示OAS1在胰腺癌组织中的表达显著上调(P＜0.05);PDAC组织芯片的免疫组织化学染色结果显示,相比于配对的癌旁正常组织,OAS1在肿瘤组织中的表达水平明显上调,且其高表达与患者的不良预后呈正相关(P＜0.05);实时荧光定量PCR和蛋白质印迹实验结果显示,OAS1在PDAC细胞中的表达水平普遍高于正常胰腺导管细胞;进一步在Patu-8988与PDC0034细胞系中干扰OAS1表达后,PDAC细胞的增殖能力显著下降(P＜0.001).GSEA结果提示OAS1 可能通过影响mTOR信号通路和胆固醇稳态相关通路来影响PDAC细胞的增殖;干扰OAS1表达后,PDAC细胞中的mTOR信号通路可能被抑制且细胞的胆固醇含量下降,用mTOR信号通路激动剂MHY1485处理细胞可部分逆转OAS1干扰造成的增殖抑制和胆固醇减少.结论:OAS1在PDAC组织和细胞中高表达且与患者的不良预后相关,OAS1可能通过促进mTOR信号通路的激活调节胰腺癌细胞的胆固醇代谢,进而促进胰腺癌细胞的增殖功能.
OAS1 promotes the proliferation of pancreatic cancer cells by enhancing mTOR signaling pathway
Objective:To investigate the expression pattern,clinical significance,and the regulatory role of 2',5'-oligoadenylate synthetase 1(OAS1)in the proliferation of pancreatic ductal adenocarcinoma(PDAC)cells.Methods:Public databases such as Gene Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA)were used to analyze the expression of OAS1 in pancreatic cancer tissues.Immunohistochemical staining was applied to validate the expression level of OAS1 in PDAC tissue microarrays,and the association between OAS1 expression level and the prognosis of patients was analyzed.Real-time fluorescence quantitative PCR was performed to examine the expression level of OAS1 mRNA in different PDAC cell lines.CCK-8 assay and colony formation assay was used to assess the effect of OAS1 on the proliferation of PDAC cells after OAS1 silencing in Patu-8988 and PDC0034 cells by siRNA treatment.Further,Gene Set enrichment analysis(GSEA)was performed to screen for possible molecular mechanism of the regulatory role of OAS1 in PDAC.Cell viability and cholesterol level was analyzed after treatment with mTOR signaling activator MHY1485 in OAS1-silenced Patu-8988 and PDC0034 cells in order to verify the underlying mechanism of the regulatory role of OAS1 in PDAC cell proliferation.Results:Database analysis showed significant upregulation of OAS1 expression in pancreatic cancer tissues(P<0.05).Immunohistochemical staining results from PDAC tissue microarray showed that OAS1 expression was significantly upregulated in PDAC tissues compared with the paired paracancerous tissues,and high OAS1 expression was associated with poor prognosis(P<0.05).Real-time fluorescence quantitative PCR and Western blotting analysis show that OAS1 expression was higher in PDAC cells lines compared with normal ductal cells of the pancreas.The proliferative activity of PDAC cells decreased significantly after OAS1 silencing in Patu-8988 and PDC0034 cells(P<0.001).GSEA results indicated that OAS1 may affect PDAC cell proliferation through mTOR signaling pathway and cholesterol metabolism associated pathway.The mTOR signaling pathway may be inhibited and the total cellular cholesterol decreased after OAS1 silencing.Treatment with mammalian target of rapamycin(mTOR)activator MHY1485 partially reversed the inhibitory effect of OAS1 silencing on the proliferation and cholesterol metabolism of PDAC cells.Conclusion:OAS1 expression is upregulated in PDAC tumor tissues and cells and is associated with poor prognosis.OAS1 may promote the proliferation of pancreatic cancer cells by enhancing cholesterol metabolism through activation of the mTOR signaling pathway.
万方数据
维普
