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The role of microRNAs in prognosis and therapy resistance of lung adenocarcinoma

Kaduthanam, Sajo

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Abstract

The diagnosis of non-small cell lung cancer (NSCLC) usually occurs when the disease has locally advanced or spread into distant sites. Patients with early-stage NSCLC have a better overall rate of survival than those patients with advanced stages. Assessment of tumor progression is therefore critical for treatment options. For this purpose, this study used two approaches to analyze the development of NSCLC. First, circulating microRNAs (miRNAs) were examined as prognostic markers from serum samples of early-stage lung adenocarcinoma patients. Secondly, this study investigated the role of miRNAs in the progression of lung adenocarcinoma cells after treatment with the tyrosine-kinase inhibitor (TKI) gefitinib and subsequent resistance. Genome-wide circulating miRNA expression profiling revealed miRNAs out of which miR-142-3p was validated to be associated with poor prognosis in patients with lung adenocarcinoma. In order to improve the prognostic value of circulating miRNAs, a miRNA panel was searched to predict the overall survival and the relapse-free survival. Since no stable set of miRNAs was found, pre-analytical variables were identified as critical for miRNA analysis. In this case, the impact of blood collection and hemolysis were shown to have an influence on the expression level. The miRNAs, which were selected based on their prognostic relevance, showed a tendency to be lesser expressed in arterial blood than miRNAs derived from venipuncture. Moreover, miR-20b-5p and miR-486-5p were affected by hemolysis. In order to further investigate the progression of the tumor, the role of miRNAs and mRNAs in the development of resistance to EGFR-TKI gefitinib was studied. Therefore, a gefitinib resistance model was used by co-culturing lung fibroblasts (MRC-5) with the two different gefitinib-sensitive NSCLC cell lines HCC827 and PC-9. Global miRNA analysis revealed expression changes associated with EGFR-TKI resistance. A total of eleven miRNAs were selected, from which miR-503-5p was validated in both EGFR-TKI NSCLC cell lines. Subsequently, global gene expression profiling revealed 211 differentially expressed mRNAs in the resistant HCC827 and PC-9 cells. Putative target genes of miR-503-5p were determined by comparing global gene expression profiling with miRNA prediction databases. The autophagy gene GABARAPL1 was validated at both the mRNA and protein level. Ectopic overexpression of miR-503-5p resulted in significant reduction of GABARAPL1. Moreover, luciferase reporter assays showed the direct interaction between miR-503-5p and the 3’UTR of GABARAPL1. Different autophagy markers were analyzed in co-cultured HCC827 cells after gefitinib treatment. Thereby, the accumulation of the SQSTM1, as well as a decrease of LC3B-II and GABARAPL1 levels were observed. In accordance with this finding, siRNA-mediated knockdown of GABARAPL1 resulted in an accumulation of SQSTM1 (p62). In conclusion, these data suggest that inhibiting GABARAPL1 through miR-503-5p modulates autophagic activity in gefitinib resistant EGFR-mutant lung adenocarcinoma cells.

Item Type: Dissertation
Supervisor: Angel, Prof. Dr. Peter
Date of thesis defense: 8 April 2016
Date Deposited: 27 Apr 2016 09:18
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > German Cancer Research Center (DKFZ)
Subjects: 570 Life sciences
Uncontrolled Keywords: microRNA, lung cancer, gefitinib resistance, circulating microRNA
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