DDX3 基因和 miR-218 在人類乳突瘤病毒感染之肺腫瘤化之角色

Abstract

Since 2001, our laboratory published a series of papers indicating that HPV 16/18 infection may be involved in lung cancer development in Taiwan. Among these, the negative association between E6 and p53 immunostainings in series tissue-array paraffin sections of lung tumors. Moreover, HPV16 E6-positive TL-1, TL-2, and TL-3 lung adenocarcinoma cells were established from patients’ pleural effusions and demonstrated that p53 was degraded by E6 to downregulate p21 and MDM2 mRNA expression and consequently promoted cell proliferation and colony formation. However, the crucial molecules involved in HPV-associated lung tumorigenesis and may predict patients’ outcome remain to be identified. In recent study, the involvement of DDX3 and miR-218 in viral-associated tumorigenesis has been reported, such as hepatitis virus-infected hepatocellular and cervical carcinoma. However, the role of DDX3 and miR-218 in HPV-associated lung tumorigenesis remains unknown. In the present study, we suspected that the modulation of DDX3 and miR-218 by E6 may promote tumor growth and metastasis; subsequently, the reduction of DDX3 and miR-218 may predict poorer survival and relapse in non-small cell lung cancer (NSCLC). Changes of DDX3 and miR-218 expressions in lung cancer cells were conducted by transfection with E6 cDNA and small interenfence or hairpin RNA plasmids. The p21, MDM2, Slug, E-cadherin, and paxillin expression, candidate downstream gene of DDX3 and miR-218, were examined in cell models by transfection with overexpression and knockdown plasmids. Cell growth and invasion were examined by the doubling time, colony formation assay, soft-agar and Boyden chamber assay. In lung tumors from lung cancer patients, expressions of miR-218, DDX3, p21, MDM2, Slug, and E-cadherin were evaluated by real-time PCR and immunohistochemical analysis, respectively. The impact of DDX3 and miR-218 expression on overall survival (OS) and relapse-free survival (RFS) was analyzed by the Kaplan-Meier test and Cox regression analysis. In present study, we showed that DDX3 was the direct target for p53 regulation in lung cancer by luciferase reporter asssy. DDX3 reduction increased cell proliferation, colony formation, soft-agar colony growth, and invasion capability. Cell growth and invasion capability increased by DDX3 reduction may be through decreased p21 expression and MDM2/Slug/E-cadherin pathway, respectively. Additionally, DDX3 reduction may reduce cisplatin sensitivity via upregulation of cIAP-2. In clinical results, we found that patients with low DDX3 tumors had poorer survival and relapse in lung cancer patients. It suggests that low DDX3 expression in tumor not only promotes tumor growth, but also enhances tumor recurrence and/or metastasis. We also provided the evidence to show that paxillin (PXN) overexpression by miR-218 reduction promotes lung cancer cell growth and invasion capability. In addition, the susceptibility to cisplatin was decreased by PXN overxpression, and this was partially through increased the interaction between PXN and BCL-2. Patients with low miR-218 and PXN-positive had the worst OS and RFS among the four combinations. In conclusion, the reduction of DDX3 and miR-218 by E6 may promote tumor malignancy and patients with poor outcome. Therefore, we suggest that DDX3 and miR-218 molecules may be potential therapeutic targets to improve outcome and life quality in HPV-infected lung cancer patients.