Effect Of ShRNA Down-regulation Ezrin On Invasion And Metastasis Ability Of Human Lung Cancer Cell

Objective To explore the expression of Ezrin protein or mRNA in lung cancer cell lines with different metastatic potential, and the correlation between Ezrin protein expression and invasion, metastasis and microfialmant cytoskeleton system, respectively. In addition, we further evaluate the relationship with the expression of Ezrin protein and clinicopatholo gical parameters, metastasis and prognosis of human non-small cell lung cancer tissues(NSCLC). By this study, we try to provide certain theoretical basis for prognosis judgment and new target therapy in lung cancer.Method The expression of Ezrin protein and mRNA in lung cancer cell lines(HLF-1,95C、95D) was detected by RT-PCR and Western blotting. We chose highly metastatic human lung cancer95D cell line, which highly express the Ezrin proteins, as a model to examine the functional roles of Ezrin in tumor suppression. An Ezrin silenced95D cell line was established using lentivirus-mediated short hairpin RNA (shRNA) method. Multiple techniques as cell transfection, CCK-8assay, the scratch test, transwell test, flow cytometry, confocal laser scanning microscopy and other technology were adapted to detecte the biological behaviour of lung cancer cells which has been down-regulated Ezrin protein. Furthermore, Ezrin protein was detected by immuno-histochemical staining SP technique in tumor tissues from150lung cancer cases and in normal lung tissues from30patients.Results1. The expression levels of Ezrin protein and mRNA were up-regulated in highly metastatic human lung cancer.2. Down-regulation of Ezrin significantly suppressed the proliferation of95D cells in vitro, but also affected the cell cycle distribution and eventual apoptosis.3. Down-regulation of Ezrin specific inhibited the cell migration and invasion. It also induced rearrangements of the actin cytoskeleton.4. Down-regulation of Ezrin affected the expression of E-Cadherin and CD44in lung cancer cells.5. The up-regulation of Ezrin expression was significantly correlated with lymph node metastasis(Χ2=9.013, P<0.01), but not correlated with age, sex, tumor size, histological type, clinical TNM system and pathological grading.(P>0.05) Survival analysis showed that the5years survival rate of patients with Ezrin negative expression was29.3%,which was significantly higher than that of patients with Ezrin positive expression(15.22%, Χ2=4.309, P<0.01), and multivariate Cox regression analysis showed that Ezrin expression (RR=3.012,P=0.047) and lymph node metastasis (RR=1.827, P=0.035) were a significantly independent predicitive factors for patients with lung cancer, other factors could not use as an independent predicitive factors.Conclusion: Ezrin played an important role in the tumorigenicity and metastasis of lung cancer cells, which will provide certain theoretical basis for prognosis judgment. Therefore, we proposed that Ezrin could be a potential molecular target of gene therapy by RNAi-targeting in lung cancer cells.