Notch3通过Wnt/β-连环蛋白通路介导基质金属蛋白酶-2/基质金属蛋白酶-9调控肝癌侵袭转移

Notch3 regulate invasion and metastasis of hepatic carcinoma by matrix metalloproteinase-2/matrix metalloproteinase-9 mediated by Wnt/β-catenin pathway

目的观察Notch3通过Wnt/β-连环蛋白（β-catenin）通路介导基质金属蛋白酶（MMP）-2/MMP-9对肝癌侵袭转移的影响。 方法应用实时荧光定量聚合酶连反应（FQ-PCR）方法检测30例肝癌及癌旁组织中Notch3基因的表达。免疫组织化学检测Notch3蛋白在组织样本中的表达。培养肝癌QGY7701细胞，传代细胞培养，并分为3组：空白对照组（Control组）、脂质体转染组（si-NC组）和小干扰RNA（siRNA）-Notch3转染组（si-N3组）。细胞划痕及Transwell小室研究各组细胞侵袭转移能力。Western blot检测Notch3、β-catenin和MMP-2、MMP-9蛋白的表达。 结果Notch3基因在肝癌组织中明显高表达，差异有统计学意义（P＝0.006）；免疫组织化学实验结果示Notch3蛋白在肝癌组织中呈阳性表达；细胞划痕实验示：在不同时间点，si-N3组迁移细胞数较Control组和si-NC组均较少，细胞迁移能力被抑制；Transwell细胞侵袭实验显示：si-N3组侵袭细胞数明显少于Control组和si-NC组[（75±8）个比（125±11）个比（118±10）个，P＝0.001、0.002]；Western blot检测发现下调Notch3后，β-catenin的表达增加，MMP-2、MMP-9的表达减弱。 结论Notch3在肝癌中被激活，Notch3可通过Wnt/β-catenin通路介导MMP-2/MMP-9，促进肝癌细胞侵袭和转移。

ObjectiveTo observe the effect of Notch3 on invasion and metastasis of hepatocellular carcinoma mediated by Wnt/β-catenin pathway. MethodsThe Notch3 gene and protein expression was detected by real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） and Immunohistochemistry. Cultured hepatoma cell lines QGY7701, and then cells were divided into three groups： non transfection group （Control）, Lipo transfection group （si-NC） and small interfering RNA （siRNA）-Notch3 transfection group （si-N3）. Wound healing and transwell assays were used to assess cancer cells the capacity of invasive and metastasis. Western blotting was carried out to analyze the expression of Notch3, β-catenin, matrix metalloproteinase （MMP）-2 and MMP-9 proteins. ResultsNotch3 gene expression levels were significantly increased in the tumor tissue compared with the normal liver tissue from the same patient. The results of immunohistochemistry showed that Notch3 protein expression was positive in the tumor tissue. Wound healing assay showed that the migrating cells of si-N3 group were less than Control group and si-NC group in 12 h and 24 h, which means the capacity of cell migration inhibited. Compared control group, the invasion capacity of QGY7701 cells was significantly decreased [ （75±8） cells vs. （125±11） cells vs. （118±10） cells, P=0.001, 0.002]. After Notch3 protein silenced, the expression level of β-catenin was upregulated and MMP-2/MMP-9 was downregulated. ConclusionNotch3 expression is activated in hepatocellular carcinoma tumor tissues, and Notch3 can mediate MMP-2/MMP-9 through the Wnt/β-catenin pathway to promote the invasion and metastasis of hepatoma cells.