Furin通过Hippo-YAP信号传导通路促进胰腺癌细胞上皮间质转化

发布时间：2018-05-09 22:08

本文选题：Furin + 胰腺癌　；参考：《江苏大学》2017年硕士论文

【摘要】：研究目的:本研究旨在探讨前体蛋白加工酶Furin对人胰腺癌细胞生长、转移、侵袭能力等生物学行为的影响,并初步探索其对肿瘤细胞上皮间质转化的影响及可能存在的分子调节机制,为进一步的临床应用研究完善理论基础。研究方法:通过Realtime PCR、Western Blot技术检测Furin在四种人胰腺癌细胞中m RNA和蛋白质的表达差异。构建sh-Furin干扰质粒,通过慢病毒包装感染Furin高表达的胰腺癌细胞,筛选出持续低表达Furin的稳转细胞株,Realtime PCR及Western Blot技术验证干扰效率。构建Flag-Furin过表达质粒,瞬时转染Furin低表达的细胞株,同样在m RNA和蛋白质水平检测过表达效率。通过CCK-8法和细胞克隆形成实验检测Furin对人胰腺癌细胞生长的影响,Transwell migration实验和划痕实验检测Furin对人胰腺癌细胞迁移能力的影响,Transwell invasion实验检测Furin对人胰腺癌细胞侵袭能力的影响,并通过Western Blot技术检测Furin对侵袭相关蛋白的表达量的改变。通过上述实验,初步探索Furin对人胰腺癌细胞生物学行为的影响。Western Blot技术检测上皮表型N-cadherin、Vimentin及间质表型E-cadherin蛋白水平的变化,研究Furin是否促进人胰腺癌细胞的上皮间质转化。同时检测Hippo-YAP通路相关蛋白的改变,初步探明Furin对上皮间质转化作用的分子调控机制。研究结果:成功构建Furin特异性sh-Furin干扰质粒和Flag-Furin过表达质粒。Realtime PCR、Western Blot结果显示在Pa Tu8988和PANC1细胞株中Furin表达量相对较低,而在Bx PC3和SW1990细胞株中表达量相对较高。Sh-Furin慢病毒包装并感染Bx PC3和SW1990细胞株,Flag-Furin瞬时转染Pa Tu8988和PANC1细胞株,Realtime PCR、Western Blot检测结果验证sh-Furin在胰腺癌细胞中良好且稳定的干扰效率,Flag-Furin具有过表达效率。在Bx PC3和SW1990细胞株中下调Furin的表达后,癌细胞的增殖、克隆形成、迁移和侵袭能力显著减弱,细胞中的上皮表型E-cadherin表达水平增加,间质表型N-cadherin和Vimetin表达水平下降。同时Western Blot检测出细胞中总的YAP、p-Mob1表达量明显下降,p-YAP和总的Mob1的表达量显著增加。在Pa Tu8988和PANC1细胞株中过表达Furin后,细胞的的增殖、克隆形成、迁移和侵袭能力增强,而Western Blot检测上皮间质转化和Hippo-YAP通路相应蛋白的表达水平改变与干扰实验结果相反。研究结论:Furin能够促进胰腺癌细胞的增殖、迁移、侵袭能力和上皮间质转化,可能通过Hippo-YAP通路进行调节。
[Abstract]:Objective: to investigate the effects of precursor protein processing enzyme (Furin) on the growth, metastasis and invasion of human pancreatic cancer cells. The effects on epithelial mesenchymal transformation of tumor cells and the possible molecular regulatory mechanisms were explored, which is the theoretical basis for further clinical application. Methods: the expression of m RNA and protein in four kinds of human pancreatic cancer cells was detected by Realtime PCR Western Blot. Sh-Furin interference plasmids were constructed and infected with Furin high expression pancreatic cancer cells by lentivirus packaging. Stable transformed cell lines with low expression of Furin were screened to verify the interference efficiency by using real time PCR and Western Blot techniques. The overexpression plasmid of Flag-Furin was constructed, and the expression efficiency was also detected at the level of m RNA and protein by transient transfection of Furin low expression cell lines. The effects of Furin on the growth of human pancreatic cancer cells were detected by CCK-8 assay and cell clone formation assay. The effects of Furin on the migration ability of human pancreatic cancer cells were detected by transwell migration assay and scratch test. The effect of Furin on the invasion of human pancreatic cancer cells was detected by Transwell invasion assay. Western Blot technique was used to detect the expression of invasion related protein in Furin. The effects of Furin on the biological behavior of human pancreatic cancer cells were studied. Western Blot technique was used to detect the changes of phenotype N-cadherin vimentin and interstitial phenotype E-cadherin protein in human pancreatic cancer cells, and to investigate whether Furin could promote the transformation of human pancreatic cancer cells. At the same time, the changes of Hippo-YAP pathway related proteins were detected, and the molecular regulation mechanism of Furin on epithelial interstitial transformation was preliminarily investigated. Results: the successful construction of Furin specific sh-Furin interference plasmid and Flag-Furin overexpression plasmid. The results showed that Furin expression was relatively low in Pa Tu8988 and PANC1 cell lines. However, the expression level of BX PC3 and SW1990 cells was relatively high. Sh-Furin lentivirus was packaged and infected with Bx PC3 and SW1990 cell lines. Flag-Furin transient transfection of Tu8988 and PANC1 cell lines confirmed the good and stable interference of sh-Furin in pancreatic cancer cells. The efficiency of Flag-Furin was higher than that of Flag-Furin. After down-regulating the expression of Furin in BX PC3 and SW1990 cell lines, the proliferation, clone formation, migration and invasion of cancer cells decreased significantly, the expression of E-cadherin in epithelial cells increased, and the expression of N-cadherin and Vimetin in interstitial cells decreased. At the same time, Western Blot showed that the total expression of YAPP p-Mob1 decreased significantly, and the expression of Mob1 and YAPP-YAP increased significantly. After overexpression of Furin in Pa Tu8988 and PANC1 cell lines, the cell proliferation, clone formation, migration and invasion were enhanced. However, the expression level of Western Blot in the epithelial interstitial transformation and Hippo-YAP pathway was different from the results of interference assay. Conclusion the results suggest that the cell proliferation, migration, invasion and epithelial mesenchymal transformation may be regulated by Hippo-YAP pathway.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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