Tpr-Met致NIH3T3细胞恶性转化的分子机制初探

Molecular Mechanism of Tpr-Met-Mediated Malignant Transformation in NIH3T3 Cells

[目的]探讨NIH3T3细胞恶性转化的分子机制。[方法]用表达Tpr-Met的重组质粒转染NIH3T3细胞致其恶性转化,用软琼脂集落实验和3H-TdR掺入DNA的方法检查细胞的生长状态及增殖性,用Westernblot检测其c-Met及P-Erk、P-Akt表达水平,用EMSA实验检测NF-кB的结合活性。[结果]转染Tpr-Met的细胞形态有明显改变,能在软琼脂中形成集落,形成的集落大且明显增多,转染pcDNA3.1/c-Met/Tpr-Met的细胞集落数分别是0,71±10和160±12,说明Tpr-Met能诱导NIH3T3细胞恶性转化。用3H-TdR掺入DNA的方法来检测细胞的增殖性,发现转染Tpr-Met的NIH3T3细胞和转染c-Met的NIH3T3细胞相比,转染Tpr-Met的细胞增殖能力明显增强,差异有极显著性(P<0.01)。转化后的NIH3T3细胞DNA与NF-кB的结合能力明显增强,并且P-Erk和P-Akt的表达水平上调。[结论]NF-кB参与了Tpr-Met致NIH3T3细胞恶性转化的信号调控,提示阻断该信号途径有可能抑制肿瘤的生长和转移。

[Purpose ]To explore the molecular mechanism of NIH3T3 cells malignant transformation induced by Tpr-Met, the mutant of the c-Met receptor. [Methods] The human Tpr-Met gene was cloned into pcDNA3.1 eucaryotic expression vector. The recombinant plasmid and the control pcDNA3.1 vector were introduced into NIH3T3 cells by lipofectin-mediated gene transfection. Then positive colonies were selected by G418 and were tested for their proliferation by colony forming and incorporation of 3H-TdR. The DNA binding activity （nuclear transposal） of NF-κB was detected by electrophoretic mobility shift assay （EMSA）, and c-Met, P-Erk, P-Akt protein expression were detected by Western blotting. [Results ] Comparing with normal cells and c-Met-transfected NIH3T3 cells, the TprMet-transfected NIH3T3 cells had changed evidently in cytomorphology, its ability of forming cell colonies in soft agar（0,71±10 and 160±12, respectively, P〈0.01） increased, which revealed the malignant transformation of NIH3T3 cells induced by Tpr-Met. Taking incorporation of 3H-TdR as the index of proliferation, the ability of cell proliferation of the Tpr-Met-transfected NIH3T3 cells increased significantly. In the transformed cells, the expressions of P-Erk and P-Akt were upregulated at the protein level and the DNA binding activity （nuclear transposal） of NF-κB was increased. [Conclusion] NF-κB pathway plays an important modulating role in the NIH3T3 cell malignant transformation, which suggests that blocking the NF-κB pathway would inhibit the tumor growth and metastasis.