长链非编码RNA WAC-AS1在胰腺癌中的临床意义及分子机制验证

Clinical significance and molecular mechanism validation of long non-coding RNA WAC-AS1 in pancreatic cancer

目的探索长链非编码RNA(lncRNA)WAC-AS1在胰腺癌中的生物学功能和作用机制以及对胰腺癌预后的影响。方法基于在线数据库计算并分析WAC-AS1在胰腺癌组织中相对正常组织的表达以及与其预后、肿瘤微环境、免疫细胞浸润的关系。同时应用基因集差异分析(GSVA)以及基因集富集分析(GSEA)探讨了WAC-AS1在胰腺癌中的生物学作用以及参与的信号通路。构建质粒转染人胰腺癌PANC-1细胞系,分成空载对照组、低表达组、中表达和高表达组,细胞计数试剂盒(CCK-8)实验检测WAC-AS1对胰腺癌细胞的增殖影响,蛋白质印迹法(Western blot)检测WAC-AS1过表达后B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)和细胞周期蛋白(Cyclin)D1的变化,组间比较采用t检验。结果lncRNAWAC-AS1在胰腺癌组织中(P<0.01)相对正常组织呈明显高表达。在胰腺癌中,WAC-AS1主要影响患者的总体生存期以及无进展生存期[P<0.01,风险比(HR)=0.559,95%可信区间(CI):0.368~0.848;P<0.01,HR=0.532,95%CI:0.361~0.783]。并且,WAC-AS1在胰腺癌中的表达与2种免疫浸润细胞相关(P均<0.05)。GSVA与GSEV结果显示,WAC-AS1与炎症信号通路、凋亡以及KRAS信号通路等信号通路呈正相关。CCK-8结果显示WAC-AS1中高表达组的PANC-1细胞增殖能力分别低于对照组,为0.80±0.03,t=28.17,P<0.01;0.71±0.06,t=19.75,P<0.01。Westernblot的结果显示,WAC-AS1中高表达组的CyclinD1表达量低于对照组(0.192±0.039比0.342±0.043,t=3.643,P<0.05;0.126±0.030比0.342±0.043,t=5.781,P<0.01)。结论结果表明lncRNAWAC-AS1与胰腺癌的肿瘤微环境以及免疫浸润相关,同时低表达WAC-AS1与胰腺癌的不良预后相关,细胞模型提示WAC-AS1通过CyclinD1抑制PANC-1细胞增殖。

Objective To explore the biological function and mechanism of long non-coding RNA(lncRNA)WAC-AS1 in pancreatic cancer and its impact on the prognosis of pancreatic cancer based on TCGA and GTEx databases.Methods Based on the online database,the expression of WAC-AS1 in pan-creatic cancer tissues compared to normal tissues and its relationship with prognosis,tumor microenviron-ment,immune cell infiltration were calculated and analyzed.At the same time,gene set variation analysis(GSVA)and gene set enrichment analysis(GSEA)were used to explore the biological role and involved signal pathways of WAC-AS1 in pancreatic cancer.We constructed a plasmid of WAC-AS1,and transfect-ed it into human pancreatic cancer PANC-1 cell line and divided it into control group,low expression group,medium expression group and high expression group.Cell counting kit(CCK-8)was used to detect the effect of WAC-AS1 on proliferation of pancreatic cancer cells.Western blotting was used to detect the changes of B cell lymphoma/leukemia-2(bcl-2),bcl-2 associated X protein(bax)and Cyclin D1 after WAC-AS1 overexpression.The t-test was used for comparison between groups.Results The expression of lncRNA WAC-AS1 in pancreatic cancer was significantly higher than that in normal tissues(P<O.O1).In pancreatic cancer,WAC-ASI mainly affected the overall survival and progression free survival of patients[P<0.01,hazard ratio(HR)=0.559,95%confidence interval(CI):0.368-0.848;P<0.01,HR=0.532,95%Cl:0.361-0.783].Moreover,the expression of WAC-AS1 in pancreatic cancer was related to two kinds of immune infiltrating cells(P<0.05).The results of GSVA and GSEV showed that there was a positive correlation between WAC-ASI and apoptosis,KRAS signaling pathway as well as some inflammatory signaling pathway.CCK-8 results showed that,compared to control group,the proliferation ability of PANC-1 cells in the medium and high expression groups of WAC-AS1 decreased to 0.80±0.03,t=28.17,P<0.01;0.71±0.06,t=19.75,P<0.01.Western blotting results showed that the expression of Cyclin DI in the medium and high expression groups of WAC-AS1 was lower than that in the control group(0.192±0.039 vs.0.342±0.043,t=3.643,P<0.05;0.126±0.030 vs.0.342±0.043,t=5.781,P<0.01).Conclusion The results show that lncRNA WAC-AS1 is related to the tumor microenvironment and immune cell infiltration of pancreatic cancer,and the low expression of WAC-AS1 is related to the poor prognosis of pancreatic cancer.The cell model suggests that WAC-AS1 inhibits the proliferation of PANC-1 cells through Cyclin D1.