肝细胞生长因子通过上调环氧合酶2表达增强乳腺癌细胞的侵袭能力

Hepatocyte growth factor induces the invasion ability of breast cancer cells by up-regulating COX2 expression

目的:探讨肝细胞生长因子(hepatocyte growth factor,HGF)对人乳腺癌细胞侵袭能力的影响,以及该作用机制是否与调控环氧合酶2(cyclooxygenase 2,COX2)基因表达有关。方法:用30μg/m L的重组人HGF(recombinant human HGF,rh HGF)处理乳腺癌MDA-MB-231和MCF-7细胞6 h,采用蛋白质印迹法检测HGF受体c-Met及磷酸化c-Met的表达。用不同质量浓度的rh HGF分别处理2种乳腺癌细胞16 h,采用Transwell实验检测细胞侵袭能力的变化。用不同质量浓度的rh HGF分别处理2种乳腺癌细胞不同时间,实时荧光定量PCR和蛋白质印迹法分别检测COX2 m RNA和蛋白表达水平的变化。将携带针对COX2基因的特异性短发夹RNA(short hairpin RNA,sh RNA)的重组质粒psh RNA-COX2分别转染MDA-MB-231和MCF-7细胞,同时用COX2选择性抑制剂塞来昔布(celecoxib)处理作为阳性对照,然后采用Transwell实验检测沉默COX2基因表达后rh HGF对2种乳腺癌细胞侵袭能力的影响,采用蛋白质印迹法检测COX2和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)蛋白表达的改变。结果:MDA-MB-231和MCF-7细胞都能表达HGF受体c-Met,而rh HGF能诱导c-Met磷酸化,提示2种乳腺癌细胞均存在HGF作用靶点。与未处理组相比,rh HGF处理后乳腺癌MDA-MB-231及MCF-7细胞的侵袭能力明显增强(P值均<0.05),COX2 m RNA及蛋白的表达水平明显升高(P值均<0.05),且呈时间和剂量依赖性。而psh RNA-COX2沉默COX 2基因表达或者celecoxib处理后,rh HGF诱导的乳腺癌细胞的侵袭能力明显减弱(P<0.05),COX2和MMP-9蛋白表达水平明显降低(P值均<0.05)。结论 :HGF可通过上调乳腺癌细胞中COX2和MMP-9的表达,增强乳腺癌细胞的侵袭能力。

Objective：To investigate the effect of hepatocyte growth factor（HCF） on the invasion of human breast cancer cells,and to explore whether this mechanism is related to the regulation of cyclooxygenase 2（C0X2） expression.Methods：The human breast cancer MDA-MB-231 and MCF-7 cells were treated with 30ug/mL recombinant human HCF（rhHCF） for 6 h,then the expression levels of HCF receptor c-Met and phospho-c-Met（p-c-Met） were detected by Western blotting.After MDA-MB-231 and MCF-7 cells were treated with various concentrations of rhHCF for 1 6 h,the cell invasion ability was detected by Transwell assay.After the two cell lines were treated with different concentrations of rhHCF for different time,the expressions of COX2 mRNA and protein were analyzed by real-time fluorescence quantitative PCR and Western blotting,respectively.Then the two cell lines were transfected with recombinant plasmid pshRNA-COX2 containing specific short hairpin RNA（shRNA） targeting C0X2 gene,or treated with COX2 selective inhibitor celecoxib as the positive control.The effects of rhHCF on cell invasion ability and the protein expressions of COX2 and matrix metalloproteinase-9（MMP-9） in breast cancer cells with C0X2 gene silencing were detected by Transwell assay and Western blotting,respectively.Results：The expression and rhHCF-induced phosphorylation of c-Met were confirmed in both MDA-MB-231 and MCF-7 cells,which suggested that these two breast cancer cell lines had the target of HCF action.The expression levels of COX2 mRNA and protein as well as the invasion abilities of the two breast cancer cell lines were significantly increased（all P〈0.05）after rhHCF treatment in a concentration and time-dependent manner.After pshRNA-COX2 transfection or celecoxib treatment,the invasion ability and the protein expressions of COX2 and MMP-9 induced by rhHCF were obviously suppressed（all P〈0.05）.Conclusion：HCF can induce the invasion of breast cancer cells through up-regulating COX2 and MMP-9 expressions.