proBDNF通过上调p75NTR表达促进口腔鳞状细胞癌SCC-4细胞迁移侵袭

ProBDNF promotes migration and invasion of SCC-4 cells in oral squamous cell carcinoma by up-regulating p75NTR expression

摘要:

目的：探索前体形式———脑源性神经营养因子前体（precursor brain derived neurotrophic factor，proBDNF）对口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）SCC-4细胞迁移与侵袭能力的影响及其潜在机制。方法：对SCC-4细胞给予20 ng/mL proBDNF刺激后，通过Transwell迁移实验和划痕实验检测SCC-4细胞迁移，通过Transwell侵袭实验检测SCC-4细胞侵袭，通过qRT-PCR 和Western blot实验检测SCC-4细胞p75神经营养素受体（p75 neurotrophin receptor，p75NTR）mRNA与蛋白表达。进一步采用免疫荧光法观察proBDNF与p75NTR在SCC-4细胞的亚细胞定位。敲除p75NTR后，分别通过Transwell迁移实验、划痕实验、Transwell侵袭实验和qRT-PCR检测proBDNF对SCC-4细胞迁移、侵袭及促迁移侵袭相关基因表达的影响。结果：20 ng/mL proBDNF可明显促进SCC-4细胞迁移和侵袭（均P=0.000），且proBDNF与p75NTR在SCC-4细胞存在亚细胞共定位。proBDNF可促进SCC-4细胞p75NTR mRNA和蛋白表达（均P=0.000）。敲除p75NTR能逆转proBDNF引起的SCC-4细胞迁移、侵袭反应，以及降低结缔组织生长因子（connective tissue growth factor，CTGF）、波形蛋白（vimentin，VIM）、酪氨酸蛋白激酶受体UFO（anexelekto，AXL）、高迁移率族蛋白A2（high mobility group A2，HMGA2）、解整合素金属蛋白酶19（a disintegrin and metalloproteinase 19，ADAM19）、纤连蛋白富含亮氨酸跨膜蛋白2（fibronectin leucine-rich transmembrane protein 2，FLRT2）和磷酸二酯酶1C（phosphodiesterase 1C，PDE1C） mRNA表达水平（均P<0.05）。结论：proBDNF依赖上调p75NTR表达来促进SCC-4细胞迁移和侵袭，其机制可能为proBDNF结合p75NTR通过上调下游促迁移侵袭相关基因CTGF、VIM、AXL、HMGA2、ADAM19、FLRT2和PDE1C表达有关。

Abstract:

Objective：To investigate the effect of precursor brain-derived neurotrophic factor（proBDNF） on the migration and invasion of SCC-4 cells in oral squamous cell carcinoma（OSCC），and to explore its underlying mechanism. Methods：After stimulation of SCC-4 cells with 20 ng/mL proBNDF，the cell migration was evaluated by Transwell migration assay and wound healing assay，the cell invasion was evaluated by Transwell invasion assay，and the mRNA and protein expression of p75 neurotrophin receptor （p75NTR） in SCC-4 cells was determined by qRT-PCR and Western blot，respectively. Furthermore，immunofluorescence assay was used to observe the subcellular localization of proBNDF and p75NTR in SCC-4 cells. After knockout of p75NTR，the effects of proB－NDF on cell migration，cell invasion，and expression of genes promoting cell migration and invasion were evaluated by Transwell mi－gration assay and wound healing assay，Transwell invasion assay，and qRT-PCR，respectively. Results：Twenty ng/mL proBDNF signif－icantly promoted the migration and invasion of SCC-4 cells（both P=0.000）. Moreover，pro-BDNF and p75NTR were colo－cated in SCC-4 cells. ProBDNF promoted the mRNA and protein expression of p75NTR in SCC-4 cells（both P=0.000）. Knock－out of p75NTR reversed proBNDF-induced SCC-4 cell migra－tion and invasion and reduced the mRNA expression levels of connective tissue growth factor（CTGF），vimentin（VIM），anex－elekto（AXL），high-mobility group A2（HMGA2），a disintegrin and metalloproteinase 19（ADAM19），fibronectin leucine-rich trans－membrane protein 2（FLRT2），and phosphodiesterase 1C（PDE1C）（all P<0.05）. Conclusion：ProBDNF promotes the migration and invasion of SCC-4 cells by up-regulating p75NTR expression，and its mechanism of action may be related to the up-regulation of the expression of downstream migration and invasion-promoting genes（namely，CTGF，VIM，AXL，HMGA2，ADAM19，FLRT2，and PDE1C） through the binding between proBDNF and p75NTR.