活化血管内皮生长因子受体-1诱导肝癌细胞MHCC97-H上皮-间叶表型转化的实验研究

Vascular endothelial growth factor receptor-1 activation induces epithelial to mesenchmal transition in hepatocellular carcinoma cell line MHCC97-H

目的探讨血管内皮生长因子受体-1（VEGFR-1）促肝细胞癌（HCC）侵袭转移的作用机制。方法用VEGFR—1的特异性配体血管内皮生长因子-B（VEGF—B）诱导活化肝癌细胞MHCC97-H，观察MHCC97-H细胞形态学改变，RT—PCR和Weaemblot检测MHCC97-H细胞上皮标志物钙黏蛋白（E—cad）、连环蛋白-α（α-cat）in间叶标志物波形蛋白、神经钙黏蛋白（N—cad）的mRNA和蛋白质表达改变，细胞荧光免疫组织化学法检测E—cad、α-cat和波形蛋白、N—cad表达部位改变，细胞侵袭和迁移试验检测MHCC97-H细胞侵袭和运动能力的改变。组间比较采用单因素方差分析。结果VEGFR—1活化后MHCC97-H细胞变成梭形、纺锤状，细胞间隙增宽，有的伸出伪足；活化前上皮标志物E—cad、α-cat的mRNA吸光度值（4值）分别为12．55±2．98、14．23±1．36，活化后E—cad、α-cat的月值分别为6．78±3．66、6．18±0．92，活化后上皮标志物的mRNA表达显著下调，F=17．21，P〈0．05。活化前上皮标志物E-cad、α-cat蛋白的A值分别为20878±11．54、7520．45±8．66，活化后E—cad、α-cat的A值分别为8031．23±10．44、5425．15±7．37，活化后上皮标志物的蛋白表达显著下调，F=30．49，P〈0．05。波形蛋白、N-cad蛋白的A值分别为6100．22±12．73、1244．64±10．27，活化后的分别为12836．99±9．67、4586．70土8．58，活化后间叶标志物的蛋白表达显著上调，F=19．16，P〈0．05。上皮标志物E-cad和α-cat在胞膜表达减少，胞质中的表达增加，波形蛋白和N—cad在胞质中表达显著增加；MHCC97-H细胞运动和侵袭能力显著增强，与活化前相比，F=20．13，P〈0．05，差异有统计学意义。结论VEGFR—1促进肝细胞癌侵袭和转移是通过诱导肝癌细胞发生上皮-间叶表型转化实现的。

Objective To evaluate the mechanism of increased invasion and migration of hepatocellular carcinoma （HCC） cells induced by vascular endothelial growth factor receptor-1 （VEGFR-1） activation. Methods Vascular endothelial growth factor-B （VEGF-B） was used to induce and stimulate hepatocellular carcinoma cell line MHCC97-H. Morphologic changes of MHCC97-H were investigated. The expression of E-cadherin and α-catenin （two epithelial markers） and Vimentin and N-cadherin （two mesenchymal markers） was detected by reverse transcriptase polymerase chain reaction （RT- PCR）, western blotting, and immunof-luorescence staining. Cell invasion and migration test was performed. Results Treatment of MHCC97-H cells with VEGF-B led to morphologic changes characteristic of epithelial to mesenchymal transition （EMT）, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Expression of the epithelial adhesion molecules, including E-cadherin and α-catenin, was decreased after VEGF-B treatment.Conversely, an increase in the expression of the mesenchymal cell markers, including N-cadherin and vimentin, was observed after VEGF-B treatment （P 〈 0.05）. VEGF-B-treated cells exhibited a change in E-cadherin from an organized, membrane-bound structure to a disorganized state in which it was noted to be dispersed throughout the cytoplasm. Pretreatment with VEGFR-1 blocking antibody 18F1 inhibited the change in localization of E-cadherin induced by VEGF-B treatment. The ability of invasion and migration of MHCC97- H was enhanced by VEGF-B reatment （P 〈 0.05）. Conclusion Increased invasion and migration of HCC cells induced by VEGFR-1 activation was mediated by epithelial to mesenchymal transition.