HYOU1在胰腺癌发生发展中的作用及其调控机制

The role and regulation mechanism of HYOU1 in the pathogenesis and development of pancreatic cancer

目的 探究人低氧上调因子1(HYOU1)调控胰腺癌发生发展的机制。方法 生物信息学和免疫组织化学染色分析HYOU1在胰腺肿瘤组织和正常及癌旁组织中的表达水平以及与患者预后的关系;实时荧光定量PCR(qPCR)、蛋白质印迹(Western blot)检测HYOU1在正常胰腺导管上皮细胞和多种胰腺癌细胞系中的表达水平;利用CRISPR-Cas9技术在表达水平最高的细胞系BXPC-3和Panc-1中敲除HYOU1,通过细胞计数试剂盒8(CCK-8)、集落形成和划痕实验检测细胞存活、增殖和迁移能力,流式细胞术检测细胞抗凋亡能力;通过Western blot实验在野生型和HYOU1敲低细胞中检测PI3K/Akt通路中指标PI3K、p-PI3K、Akt和p-Akt的蛋白水平;利用PI3K/Akt通路激活剂Recilisib处理HYOU1敲低细胞系并检测细胞增殖能力。结果 HYOU1在胰腺肿瘤组织中的表达高于正常组织,HYOU1高表达患者生存期短于低表达患者(P<0.01);免疫组化染色显示,HYOU1在胰腺癌患者肿瘤组织中的表达高于癌旁组织(P<0.01);胰腺癌细胞系中HYOU1 mRNA和蛋白水平高于正常胰腺导管上皮细胞(P<0.001,P<0.01)。敲低HYOU1能抑制BXPC-3和Panc-1细胞存活、增殖和迁移,促进细胞早期凋亡,还能抑制PI3K/Akt信号通路激活,而PI3K/Akt通路激活剂Recilisib能逆转敲低HYOU1对细胞的作用。结论 HYOU1通过激活PI3K/Akt信号通路促进胰腺癌发展。

Objective To investigate the mechanism by which human hypoxia up-regulation 1(HYOU1)regulates pancreatic cancer development.Methods Bioinformatic and immunohistochemical staining analyses of HYOU1 expression level in pancreatic tumor tissues,normal and paracancerous tissues and its correlation with patients′survival.Quantitative real-time PCR(qPCR)and Western blot were used to clarify the expression level of HYOU1 in a number of human pancreatic ductal adenocarcinoma cell lines and a normal human pancreatic ductal cell line.Two cell lines with the highest expression levels of HYOU1,BXPC-3 and Panc-1,were selected to knock out HYOU1 by CRISPR-Cas9,and then cell survival,proliferation and migration of these cells were examined by cell counting kit-8(CCK-8),colony formation assay and wound healing experiment separately,as well as apoptosis was detected by flow cytometry.Subsequently,the protein levels of the PI3K/Akt signaling including PI3K,p-PI3K,Akt,and p-Akt were detected by Western blot in parental and HYOU1-ablated BXPC-3 and Panc-1 cells.Cell proliferation was also examined in HYOU1-ablated cells after treatment of recilisib,an activator of the PI3K/Akt pathway.Results The expression of HYOU1 in pancreatic tumor tissues was significantly higher than that in normal tissues,and the patients with high expression of HYOU1 had a much shorter survival compared to the patients with low HYOU1(P<0.01).Immunohistochemical staining of pancreatic cancer specimens showed that the expression of HYOU1 was higher in tumor tissues than in paracancerous tissues(P<0.01).The mRNA and protein levels of HYOU1 were higher in all pancreatic cancer cell lines compared to the human normal pancreatic ductal cell(P<0.001,P<0.01).HYOU1 ablation inhibited BXPC-3 and Panc-1 cells survival,proliferation and migration,and promoted early cell apoptosis.In addition,loss of HYOU1 decreased PI3K/Akt signaling activity,whereas the PI3K/Akt activator Recilisib reversed the effects of HYOU1 ablation on cell survival and proliferation.Conclusion HYOU1 promotes pancreatic cancer progression by activating the PI3K/Akt signaling pathway.