Notch信号通路在胰腺癌中的表达及其作用研究

Expression of Notch signaling pathway in pancreatic cancer and its role

目的研究Notch信号通路成员在胰腺癌组织中的异常表达及其在胰腺癌发生发展中的重要作用。方法应用Affymetrix基因表达谱芯片筛选10例胰腺癌组织及其癌旁组织中Notch信号通路存在差异表达的成员,并应用实时定量PCR和Western blot印迹法加以验证;利用携锯齿2(Jagged 2,JAG2)基因siRNA片段的慢病毒表达载体转染胰腺癌原代细胞构建JAG2基因阻遏表达胰腺癌细胞株,并采用MTT法、流式细胞术、侵袭小室实验法观察与分析该细胞株增殖、细胞周期及侵袭转移能力的变化。结果 Affymetrix基因表达谱芯片共检测到差异表达基因512个,其中表达上调的基因419个,表达下调的基因93个; Notch信号通路中存在差异表达的为JAG2(上调表达8. 20倍)、NOTCH1(上调表达3. 74倍)、HES1(上调表达3. 27倍)、NOTCH2(上调表达3. 16倍); PCR和Western blot法验证结果与基因芯片结果相符;与对照组相比,JAG2基因阻遏表达胰腺癌细胞株的生长明显受到抑制;经流式细胞仪分析两组细胞周期示JAG2基因阻遏表达胰腺癌细胞株的凋亡与S期阻滞明显增强;侵袭小室侵实验结果示JAG2基因阻遏表达胰腺癌细胞株袭转移能力明显减弱(P <0. 05)。结论 Notch信号通路部分成员在胰腺癌组织中存在显著差异表达,且阻遏该成员表达可影响胰腺癌细胞生长、细胞周期及侵袭转移。

Objective The objective of this study was to investigate the abnormal expression of Notch signaling pathway members in pancreatic cancer and its important effect on the development of pancreatic carcinoma.Methods Affymetrix gene expression microarray was used to screen the differentially expressed members of Notch signal pathway in 10 cases of pancreatic carcinoma and its adjacent tissues,and verified by real-time quantitative PCR and Western blotting.The lentivirus expression vector carrying the siRNA fragment of Jagged 2(JAG2)gene was transfected into the pancreatic cancer primary cells to construct the JAG2 gene repression-expressing pancreatic cancer cell line.MTT,flow cytometry and Transwell assays were used to analyze cell proliferation,changes of cell cycle and invasive transfer capabilities.Results A total of 512 differentially expressed genes were detected by Affymetrix gene expression microarray,including 419 up-regulated genes and 93 down-regulated genes.JAG2(up-regulated expression 8.20 times),NOTCH1(up-regulated expression 3.74 times),HES1(up-regulated expression 3.27 times),and NOTCH2(up-regulated expression 3.16 times)were differentially expressed in Notch signaling pathway.The results of PCR and Western blotting were consistent with those of gene chip.The growth curves of JAG2 gene repressed pancreatic cancer cells and pancreatic cancer primary cells were drawn by the standard OD490 value of d1-d5 by MTT assay.JAG2 gene repressor expression vector could significantly inhibit the proliferation of pancreatic cancer cells.The cell cycle analysis showed that the apoptosis and the arrested cell cycle at the S phase were significantly increased in pancreatic cancer cells with JAG2 gene repressor expression.The invasive ability was significantly reduced in JAG2 gene repressor expression pancreatic cancer cells(P<0.05).Conclusion Some members of the Notch signaling pathway are significantly differentially expressed in pancreatic cancer tissues,and repression of this member can affect the growth,cell cycle,invasion and metastasis of pancreatic cancer cells.