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目的探讨巨噬细胞刺激蛋白(macrophage stimulating protein,Msp)对人非小细胞肺癌细胞株PC14增殖、迁移和侵袭能力影响的研究。方法构建Msp编码基因Mst1真核表达载体,将Mst1导入Msp(-)和RON(-)人非小细胞肺癌细胞株PC14,并用G418筛选出稳定表达的细胞株。通过RT-PCR方法检测已转染Mst1载体PC14细胞中Mst1的表达,用Western blot检测Msp在PC14、PC14-Mst1-pEGFP-N1、PC14-pEGFP-N1中的表达水平,同时检测RON在PC14和RON(+)细胞SKBR-3中的表达情况。通过计算RAW 264.7(小鼠单核巨噬细胞)和SKBR-3细胞在各转染组细胞培养上清液的刺激下穿过Transwell微孔膜的细胞数来评价Msp的生物学活性。利用MTT法检测Msp对PC14细胞增殖力的影响,采用Transwell小室和基质胶侵袭实验观察Msp对PC14细胞迁移和侵袭能力的影响。结果转染Mst1基因后,形成PC14-Mst1-pEGFP-N1,稳定表达Mst1mRNA及蛋白,其本身的增殖能力被明显抑制,迁移能力和侵袭能力均较PC14和PC14-pEGFP-N1降低。它还通过旁分泌的方式对RON(+)阳性的癌细胞RAW264.7和SKBR-3的迁移起促进作用。结论 Msp表达可降低人非小细胞肺癌细胞PC14的增殖、迁移和侵袭能力,却对RON(+)阳性的癌细胞迁移通过旁分泌的方式有促进作用。
Objective To investigate the effect of macrophage stimulating protein (Msp) on proliferation, migration and invasion of human non-small cell lung cancer cell line PC14. Methods Mst1 eukaryotic expression vector was constructed. Mst1 was transfected into MSP (-) and RON (-) human non-small cell lung cancer cell line PC14, and stable cell line was screened by G418. The expression of Mst1 in PC14 cells transfected with Mst1 was detected by RT-PCR and the expression of Msp in PC14, PC14-Mst1-pEGFP-N1 and PC14-pEGFP-N1 was detected by Western blot. RON (+) cells in SKBR-3 expression. The biological activity of Msp was evaluated by counting the number of cells that passed through Transwell microporous membrane with RAW 264.7 (mouse mononuclear macrophage) and SKBR-3 cells stimulated with cell culture supernatants from each transfected group. The effect of Msp on the proliferation of PC14 cells was detected by MTT assay. The effect of Msp on the migration and invasion ability of PC14 cells was investigated by Transwell chamber and matrigel invasion assay. Results The Mst1 gene was transfected into PC14-Mst1-pEGFP-N1 cells and stably expressed Mst1 mRNA and protein. The proliferation of Mst1 was significantly inhibited and the migration and invasion ability of PC14-Mst1-pEGFP-N1 was lower than that of PC14 and PC14-pEGFP-N1. It also promotes the migration of RON (+) -positive cancer cells RAW264.7 and SKBR-3 through a paracrine approach. Conclusion Msp expression can reduce the proliferation, migration and invasion of human non-small cell lung cancer cell line PC14, but promote the migration of RON (+) positive cancer cells through paracrine pathway.