【摘要】 目的脑源性神经营养因子(brain-dervived neurotrophic factor,BDNF)促进肿瘤细胞的生长和转移,反义BDNF(antisense BDNF)是BDNF基因的内源性反义长链非编码RNA,对BDNF表达水平具有调控作用。本研究旨在探讨BDNF AS对肝癌细胞97-H增殖和迁移的作用,以及对BDNF表达水平的影响。方法采用BDNF AS质粒瞬时转染肝癌细胞系97-H,采用qPCR方法检测肝癌细胞中BDNF AS和BDNF mRNA水平,采用蛋白质印迹法检测BDNF蛋白表达水平,采用EdU掺入、划痕实验和Transwell小室研究转染BDNF AS对肝癌细胞增殖和迁移的影响。结果转染BDNF AS质粒的细胞中,BDNF AS水平显著上调,是对照组细胞的(136.97±7.92)倍(F=880.301,P<0.001),而BDNF mRNA和蛋白水平显著降低,分别是对照组细胞的(0.50±0.08)倍和(0.275±0.050)倍,F=59.435,P<0.001;F=566.528,P<0.001。转染BDNF AS细胞的EdU阳性率为(36.1±2.3)%,显著低于对照组细胞(F=40.833,P<0.001),而过表达BDNF AS的细胞经rhBDNF刺激后,细胞增殖百分比增加,P=0.001。BDNF AS转染组细胞的划痕愈合率以及迁移细胞数分别为(6.23±0.30)%和31.0±2.6,均低于对照组细胞(F=25.975,P=0.001;F=15.851,P=0.001),而过表达BDNF AS的细胞经rhBDNF刺激后,迁移细胞数增加,P=0.002。结论肝癌细胞97-H中BDNF AS可能通过下调BDNF mRNA和蛋白水平,实现对肝癌细胞生长、迁移的负性调控作用。更多还原

【Abstract】 OBJECTIVE BDNF facilitates the growth and metastasis of cancer cells.BDNF AS is the endogenous antisense long non-coding RNA of BDNF gene,and plays a regulatory role in BDNF expression.The aim of this study is to investigate the biological effects of BDNF AS on cell proliferation and migration in hepatocellular carcinoma cells of 97-H,and on the expression of BDNF.METHODS The BDNF AS plasmid was transiently transfected into 97-H cells,and the expressions of BDNF AS and BDNF mRNA were detected by qPCR.The BDNF protein level was examined using western blot.The EdU incorporation experiment,wound healing and transwell assay were performed respectively to explore the effects of BDNF AS on cell proliferation and migration.RESULTS The level of BDNF AS in BDNF AS plasmid transfected cells was(136.97±7.92)times as that in control cells(F=880.301,P<0.001),which was significantly upregulated.The levels of BDNF mRNA and protein were(0.50±0.08)and(0.275±0.050)times as that in control cells(F=59.435,P<0.001;F=566.528,P<0.001),which were significantly decreased.The EdU positive rate in BDNF AS transfected cells was(36.1±2.3)% and significantly lower than in control cells(F=40.833,P<0.001).The proliferative percentage of BDNF AS transfected cells was increased by rhBDNF stimulating(P=0.001).The wound healing rate or migratory cell number in BDNF AS transfected cells were(6.23±0.30)% and 31.0±2.6,respectively,which were both lower than that in control cells(F=25.975,P=0.001;F=15.851,P=0.001).The migratory cell number of BDNF AS transfected cells was increased by rhBDNF treatment(P=0.002).CONCLUSION Our data suggests that BDNF AS negatively regulates cell proliferation and migration of 97-H,probably by downregulating the expressions of BDNF mRNA and protein.更多还原