肝细胞生长因子对卵巢癌细胞侵袭的促进作用及信号转导途径

Effect of Hepatocyte Growth Factor on Invasion of Ovarian Cancer Cell Line SKOV3 and Its Signal Transduction Pathway

背景与目的:肝细胞生长因子(hepatocytegrowthfactor,HGF)与其受体c-met特异性结合,激活多条信号转导通路,在调节肿瘤侵袭、转移和血管形成中起重要作用。HGF和c-met在卵巢癌组织中异常表达。本研究拟探讨HGF在卵巢癌的侵袭中的作用、可能机制及其信号转导途径。方法:Boyden小室体外侵袭实验检测HGF对卵巢癌SKOV3细胞穿透人工重组基底膜能力的影响;应用荧光实时定量逆转录聚合酶链反应(real-timereversetranscriptionpolymerasechainreaction,realtimeRT-PCR)、Westernblot和流式细胞术检测HGF和核转录因子(nuclearfactorκB,NFκB)抑制剂吡咯醛二硫氨基甲酸(pyrrolidinedithiocarbamate,PDTC)作用前后SKOV3细胞c-met、明胶酶B(gelatinaseB,MMP-9)mRNA和蛋白表达的变化;采用免疫细胞化学方法和Westernblot检测HGF对SKOV3细胞核NF&B蛋白的影响。结果(:1)HGF作用后,SKOV3细胞侵入基底膜的细胞数为343±13,较对照组(167±11)明显升高(P<0.01),而PDTC可以减弱HGF的促进作用(241±10,P<0.01)。(2)HGF作用后,SKOV3细胞MMP-9mRNA水平升高(5.66±0.10)倍(P<0.01),而经PDTC处理后只升高(2.75±0.80)倍(P<0.01),c-metmRNA无变化。(3)HGF作用后,SKOV3细胞c-met、MMP-9蛋白阳性率为(96.6±2.0)%和(74.6±4.4)%,较对照组(73.3±2.4)%和(16.0±2.9)%显著增高(P<0.01),c-met、MMP-9蛋白表达强度分别为2.84±0.18和2.94±0.13,较对照组1.65±0.19和0.54±0.18显著增高(P<0.01)。(4)PDTC可显著抑制HGF对SKOV3细胞蛋白表达的促进作用,MMP-9蛋白阳性率和蛋白表达强度分别为(25.8±3.7)%和0.87±0.14,与对照组和HGF组的差异有显著性(P<0.05);对c-met蛋白表达无影响(P>0.05)。(5)HGF作用后细胞核NF&B蛋白表达随时间延长而增加,于1h达高峰,表达强度为1.16±0.21,较无HGF作用的0.42±0.11明显升高(P<0.01),PDTC可显著抑制HGF对NF&B的活化作用,使NF&B的表达强度降为0.38±0.12(P<0.01)。结论:HGF促进SKOV3细胞的侵袭作用,其机制可能为HGF通过c-met调控NF&B的活化,间接激活MMP-9的基因,促进MMP-9蛋白的合成。

BACKGROUND ＆ OBJECTIVE： Hepatocyte growth factor （HGF） specifically binds to its receptor c-met, activates a complex set of intracellular pathways, and plays important roles in regulating tumor invasion, metastasis, and angiogenesis. HGF and c-met are overexpressed in ovarian cancer. This study was designed to investigate the role of HGF in ovarian cancer invasion and its signal transduction pathway. METHODS： HGFinduced invasion of ovarian cancer cell line SKOV3 was analyzed with Boyden chamber invasion assay. The expression changes of c-met and matrix metalloproteinase-9 （MMP-9） in SKOV3 cells before and after treatment with HGF and nuclear factor κB （NF-κB） inhibitor pyrrolidine dithiocarbamate （PDTC） were measured by real-time reverse transcription-polymerase chain reaction （RT-PCR）, Western blot, and flow cytometry. The expression of NF-κB in SKOV3 cells was evaluated by immunocytochemistry and Western blot. RESULTS： The invasive cells were significantly more in HGF group than in control group and PDTC plus HGF group （343±13 vs. 167±11 and 241±10, P〈0.01）. HGF promoted the expression of MMP-9 mRNA by 5.66±0.10 folds, but had no effect on c-met mRNA expression; PDTC suppressed the HGF-driven expression of MMP-9 mRNA by 2.75±0.80 folds. The positive rates of c-met and MMP-9 were significantly higher in HGF-treated cells than in control cells [ （96.6±2.0）% vs. （73.3±2.4）%, P〈0.01; （74.6±4.4）% vs. （16.0± 2.9）%, P〈0.01]. The protein levels of c-met and MMP-9 were significantly higher in HGF-treated cells than in control cells （2.84±0.18 vs. 1.65±0.19, P〈 0.01; 2.94±0.13 vs. 0.54±0.18, P〈0.01）. PDTC significantly suppressed the HGF-driven expression of MMP-9 protein： the positive rate and protein level of MMP-9 were （25.8±3.7）% and 0.87±0.14 （P〈0.05）. HGF promoted the expression of NF-KB protein in a time-dependant manner. The expression peak appeared 1 h after treatment with HGF （from 0.42±0.11 to 1.16±0.21, P〈0.01）. PDTC significantly inhibited the HGF-driven expression of NF-KB protein （0.38±0.12, P〈0.01）. CONCLUSION： HGF might stimulate the invasion of SKOV3 cells by up-regulating the expression of MMP-9 via NF-κB pathway.