胰腺癌预后和治疗靶点的生物标志物筛查与验证

Screening and validation for prognostic biomarker and therapeutic target in pancreatic adenocarcinoma

目的寻找一个潜在的能预测胰腺癌(PAAD)患者生存期和作为治疗靶标的生物标志物。方法利用在线数据库GEPIA2和Kaplan-Meier plotter分析序列相似性家族111成员B(FAM111B)基因表达与预后的关系。利用R语言分别下载高通量基因表达(GEO)和癌症基因组图谱(TCGA)数据库中PAAD数据集,根据FAM111B表达量中位值将数据集分为高表达组和低表达组,进一步验证FAM111B表达和预后的关系,并分析FAM111B表达与肿瘤病理分级和分期的关系。基因集富集分析(GSEA)FAM111B富集的信号通路。RT-qPCR、CCK-8实验和流式细胞术实验验证与对照si-NC组相比,si-FAM111B组中FAM111B基因沉默后对PAAD细胞的增殖能力和细胞周期的影响。结果TCGA数据库显示,与正常的癌旁组织相比,FAM111B在PAAD组织中高表达,差异有统计学意义(P<0.05)。进一步分析GEO数据库中的GSE62452和GSE183795数据集,显示FAM111B在肿瘤中表达显著高于癌旁组织,差异均有统计学意义(t=4.617、6.362,P均<0.05);且FAM111B表达与肿瘤病理分级和分期具有正相关性。FAM111B高表达组与PAAD患者预后不良密切相关,与低表达组相比,差异有统计学意义(P均<0.05)。GSEA分析显示,FAM111B高表达组显著富集在细胞周期相关通路上,且FAM111B的表达与细胞周期相关分子的表达成正相关。RT-qPCR结果显示,沉默FAM111B后,与si-NC组相比,si-FAM111B组细胞细胞周期相关分子细胞周期蛋白B1(CCNB)、细胞周期蛋白C(CCNC)、细胞周期蛋白E(CCNE)、细胞分裂周期蛋白25A(CDC25A)、细胞分裂周期蛋白25C(CDC25C)、细胞周期蛋白依赖性激酶1(CDK1)、细胞周期蛋白依赖性激酶6(CDK6)的mRNA表达水平显著下降,差异均有统计学意义(t=13.370、6.769、3.011、6.304、5.441、6.058、11.420,P均<0.05)。CCK-8实验结果显示,与si-NC组相比,si-FAM111B组的细胞增殖能力下降,差异有统计学意义(t=43.710,P<0.05)。流式细胞术检测分析发现,沉默FAM111B后,与si-NC组相比,si-FAM111B组细胞周期的G2/M期比例显著增加,差异有统计学意义(t=8.179,P<0.01)。结论FAM111B是PAAD患者预后差的独立生物标志物和潜在的治疗靶点。

Objective Identify a novel biomarker for prognosticating the survival of patients and therapeutic target in pancreatic adenocarcinoma(PAAD). Methods The prognostic potential of family with sequence similarity 111 member B(FAM111B) was evaluated using GEPIA2 and Kaplan-Meier plotter databases. According to the median of FAM111B expression level, the datasets from Gene Expression Omnibus(GEO) and RNA sequencing data from The Cancer Genome Atlas(TCGA) were divided into high expression group and low expression group, and the datasets were used to further assessed the prognostic value and analyzed the correlation between FAM111B expression and clinical characterization of PAAD patients. Gene set enrichment analysis(GSEA) was used to explore the role of FAM111B in related pathways.Subsequently, RT-qPCR and CCK-8 assays were performed to identify the functions of silencing FAM111B gene group siFAM111B on tumor proliferation in PAAD cells in comparison with control group si-NC. Flow cytometry was applied to detect cell cycle of si-NC group and si-FAM111B group. Results Based on TCGA database analysis, FAM111B was found to be higher in PAAD tissues than that in matched normal tissues;the difference was statistically significant(P<0.05). GEO datasets of GSE62452 and GSE183795 were further demonstrated that FAM111B expression was higher in PAAD tissues than that in normal tissues;the differences were statistically significant(t=4.617, 6.362;all P<0.05). The clinical tumor grade and stage of PAAD was significantly correlated with the FAM111B expression level. And poor prognosis was significantly correlated with overexpression of FAM111B in PAAD patients;the differences were statistically significant(all P<0.05). GSEA results indicated that FAM111B might be involved in PAAD progression through cell cycle signaling pathway. Furthermore, silencing of FAM111B downregulated the mRNA expression of cell cycle related markers, including cyclin B(CCNB),cyclin C(CCNC),cyclin E1(CCNE),cell division cycle 25A(CDC25A),cell division cycle 25C(CDC25C),cyclin dependent kinase 1(CDK1),cyclin dependent kinase 6(CDK6);the differences between si-NC group and si-FAM111B group were statistically significant(t=13.370, 6.769, 3.011, 6.304, 5.441, 6.058, 11.420;all P<0.05).CCK-8 assay indicated that cell proliferation was significantly suppressed in the si-FAM111B group compared to the si-NC group, and the difference was statistically significant(t=43.710, P<0.05). Flow cytometry showed that the proportion of PANC-1 cells in G2/M phase significantly increased in si-FAM111B group after FAM111B knockdown than that in si-NC group(t=8.179,P<0.01). Conclusion FAM111B had the potential to be an independent biomarker of poor prognosis in PAAD patients and the therapeutic target for treating PAAD.