蛋白酶激活受体-1促进肺癌细胞侵袭和转移功能的研究

目的研究蛋白酶激活受体1（PAR-1）对人类肺癌细胞侵袭和转移功能的影响。方法采用阳离子脂质体介导法,将正义和反义PAR-1重组质粒“pC／PARls”和“pC／PARlas”分别转染至人肺巨细胞癌低转移株（PLA801C）和高转移株（PLA801D）细胞中;采用逆转录．聚合酶链反应（RT-PCR）和Western印迹检测转染后PAR-1基因和蛋白表达水平变化;通过MTT、软琼脂集落形成、流式细胞仪、细胞-基质黏附和Transwell细胞侵袭实验检测PAR-1对肺癌细胞转移相关功能的影响。结果转染正义和反义PAR-1分别明显上调和下调了PLA801C和PLA801D的PAR-1 mRNA和蛋白表达水平。转染正义PAR-1对细胞的生长和克隆形成具有促进作用;并可明显增强细胞对细胞外基质的黏附和侵袭能力（与空载体对照组比较均P〈0．01）。相反,转染反义PAR-1对细胞模型的生长和克隆形成具有抑制作用;能明显降低细胞的S期和G2／M期（与空载体对照组比较分别为P〈0．05、0．01）、升高G0／G1期所占比例（P〈0．01）;还可使细胞对细胞外基质的黏附力（P〈0．05）和侵袭力明显降低（P〈0．01）。结论正义和反义PAR-1基因能够分别上调和下调PAR-1的表达水平;PAR-1能够影响肺癌细胞的生长和增殖、黏附和侵袭特性。抑制PAR-1的表达可能是一种治疗肺癌的途径。

Functional aspects of protease-activated receptor 1 in promoting metastasis of lung cancer

OBJECTIVE: To study the functional aspects of protease-activated receptor 1 (PAR-1) gene involved in tumor metastasis. METHODS: Two human lung giant cell carcinoma cell lines PLA801C (low metastasis potential) and PLA801D (high metastasis potential) were chosen as in-vitro human cancer model systems. Sense and anti-sense expression constructs of PAR-1 gene (pC/PAR1s and pC/PAR1as) were transfected into PLA-801C and PLA-801D cells by lipofection. PAR-1 expression was determined by RT-PCR and western blot analysis. MTT growth, flow cytometry analysis, fibronectin adhesion, and matrigel invasion assays were used to study the effect of PAR-1 expression on the proliferation, adhesion, and invasion of the transfected cells. RESULTS: Appropriate up-regulation or down-regulation of protein expression of PAR-1 was observed in both transfected cell lines (PLA801C and PLA801D) to express PAR-1s or PAR-1as, respectively. Expression of the sense PAR-1 markedly increased cellular proliferation, adhesion and invasion of PLA-801C cells. In contrast, anti-sense PAR-1 significantly inhibited cell growth, adhesion and invasion capabilities, along with cell arrest at G0/G1 phase of the PLA-801D cells. CONCLUSIONS: Successful up- and down- regulation of expression of PAR-1 can be achieved by in-vitro transfection of sense and antisense PAR-1 constructs. PAR-1 may enhance metastasis of lung cancer through its regulation of cellular proliferation, adhesion and invasion. Down-regulation of expression of PAR-1 may provide a new therapeutic strategy against lung carcinoma.