【作者】 张波；

【导师】 周文策； 白仲添；

【作者基本信息】 兰州大学 ， 临床医学·外科学， 2021， 硕士

【摘要】 目的:探讨长链非编码RNA（lnc RNA）LINC00857在胰腺癌中的作用及机制。阐明LINC00857在人胰腺细胞系中的表达差异及其对胰腺癌PANC-1细胞增殖、迁移和凋亡的影响及作用机制。方法:首先在GEPIA、TCGA及CCLE数据库中分析lnc RNA LINC00857在胰腺癌组织和细胞中的表达情况及其临床价值,通过GSEA和共表达基因富集分析LINC00857可能参与调节的信号通路。体外培养胰腺细胞系,通过q RT-PCR检测LINC00857 m RNA的表达差异。构建慢病毒载体GV112,转染胰腺癌PANC-1细胞株获得LINC00857-sh1和LINC00857-sh2两个实验组,以转染空白质粒为阴性对照组,未干预的细胞作为正常对照组。采用CCK-8实验、Transwell实验、划痕实验、流式细胞术、Western blot检测下调LINC00857对细胞增殖、迁移、凋亡、细胞周期以及上皮-间质转化（EMT）和凋亡相关蛋白的影响,并通过q RT-PCR检测G₁/S期主要相关基因的m RNA变化情况。结果:1.LINC00857在胰腺癌组织和细胞中高表达,其表达水平高居33种肿瘤中的第5位,高表达LINC00857的胰腺癌患者预后较差,且其检测对胰腺癌的诊断有一定的价值。GSEA和共表达基因富集分析到多条肿瘤相关的信号通路,且均包含胰腺癌通路;LINC00857可能通过调控细胞周期、凋亡等通路影响胰腺癌的进展。2.胰腺癌细胞系中LINC00857 m RNA的表达显著高于胰腺正常上皮细胞（P<0.01）。下调LINC00857可显著抑制PANC-1细胞的增殖和迁移能力（P<0.0001）;与阴性对照组相比,实验组细胞的凋亡率明显增加（P<0.05）,G₀/G₁期细胞数量增多（P<0.01）,S期细胞数目减少（P<0.05）,细胞被阻滞在G₁期。3.机制上,下调LINC00857抑制了EMT通路的进展,与阴性对照组相比,实验组细胞的EMT相关蛋白E-cadherin表达上调（P<0.05）,N-cadherin和Vimentin表达下调（N-cadherin P<0.01,Vimentin P<0.05）。下调LINC00857激活了凋亡通路,与阴性对照组相比,实验组细胞的Bax蛋白表达升高（P<0.05）,Bcl-2蛋白表达及Bcl-2/Bax比值均降低（P<0.05）。下调LINC00857可降低G₁/S期相关基因CDK1、CCND1及CCNE1 m RNA的表达（P<0.01）,CDK2和E2F1m RNA仅在LINC00857-sh2组表达下调明显（P<0.05）,而CCNB1 m RNA仅在LINC00857-sh1组表达下调明显（P<0.01）。结论:Lnc RNA LINC00857在胰腺癌组织和细胞中高表达,对胰腺癌的诊断有一定贡献,且其高表达的胰腺癌患者预后较差。LINC00857可通过调控Bcl-2/Bax途径、促进细胞周期G₁/S期转化及激活EMT相关信号分子来促进胰腺癌细胞的增殖和迁移,并抑制其凋亡。因此,LINC00857对胰腺癌的发生发展起促进作用,可能成为胰腺癌诊断及治疗的新靶点。更多还原

【Abstract】 Objective:This study was conducted to explore the role and mechanism of long non-coding RNA（lnc RNA）LINC00857 in pancreatic adenocarcinoma,and elucidate the differential expression of LINC00857 in human pancreatic cell lines and its effect on the proliferation,migration and apoptosis of pancreatic cancer PANC-1 cells and its mechanism.Methods:Firstly,the expression of lnc RNA LINC00857 in pancreatic cancer tissues and cells and its clinical value were analyzed in GEPIA,TCGA and CCLE databases.Furthermore,the signaling pathways that LINC00857 may be involved in regulating pancreatic adenocarcinoma were further analyzed by functional enrichment analysis of GSEA and co-expression genes.The expression of LINC00857 m RNA in pancreatic cell lines was analyzed by q RT-PCR.Lentiviral vector GV112 was constructed and transfected into pancreatic cancer PANC-1 cells to get two experimental groups LINC00857-sh1 and LINC00857-sh2.While the blank plasmid was transfected as a negative control group and the cells without intervention were taken as normal control group.CCK-8 assay,Transwell assay,scratch test,flow cytometry,Western blot were used to detect the effect of LINC00857down-regulation on cell proliferation,migration,apoptosis,cell cycle,EMT-related and apoptosis-related proteins.And then,q RT-PCR was used to detect the m RNA changes of the major genes related to G₁/S phase.Results:1.LINC00857 was highly expressed in pancreatic cancer tissues and cells,ranking the 5^th among 33 tumors.Pancreatic cancer patients with high expression of LINC00857 had a poor prognosis,and its detection had a certain value in the diagnosis of pancreatic adenocarcinoma.GSEA and co-expressed genes enrichment analysis had enriched multiple malignancy related signaling pathways,both including pancreatic cancer pathways.LINC00857 may affect the progression of pancreatic adenocarcinoma through cell cycle,apoptosis and other pathways.2.The m RNA expression of LINC00857 in pancreatic cancer cell lines was significantly higher than that in normal pancreatic epithelial cells（P<0.01）.Downregulation of LINC00857 significantly inhibited the proliferation and migration of PANC-1 cells（P<0.0001）.Compared with the negative control group,the apoptosis rate of the experimental group was significantly increased（P<0.05）,the number of cells in G₀/G₁phase was increased（P<0.01）,the number of cells in S phase was decreased（P<0.05）,and the cells were arrested in G1 phase.3.Mechanically,down regulation of LINC00857 inhibited the progress of EMT pathway.Compared with the negative control group,the expression of EMT-related protein E-cadherin in experimental group was up-regulated（P<0.05）.N-cadherin and Vimentin were down-regulated（N-cadherin P<0.01,Vimentin P<0.05）.Downregulation of LINC00857 activated the apoptosis pathway.Compared with the negative control group,the expression of Bax protein in the experimental group was increased（P<0.05）,and the expression of Bcl-2protein and the ratio of Bcl-2/Bax were decreased（P<0.05）.Downregulation of LINC00857 could decrease the m RNA expressions of CDK1,CCND1 and CCNE1related genes in G₁/S phase（P<0.01）,CDK2 and E2F1 m RNA were only down-regulated in LINC00857-sh2 group（P<0.05）,and CCNB1 m RNA was only down-regulated in LINC00857-sh1 group（P<0.01）.Conclusions:Lnc RNA LINC00857 is highly expressed in pancreatic cancer tissues and cells,contributing to the diagnosis of pancreatic cancer,and patients with high expression of LINC00857 have a poor prognosis.LINC00857 can promote the proliferation and migration of pancreatic cancer cells and inhibit their apoptosis by regulating the Bcl-2/Bax pathway,promoting the G1/S phase transformation of cell cycle and activating EMT-related signaling molecular pathway.Therefore,LINC00857 can promote the occurrence and development of pancreatic adenocarcinoma,and may be a new target for the diagnosis and treatment of pancreatic adenocarcinoma.更多还原