MicroRNA gene silencing in NSCLC patients

MicroRNAs (miRNAs) are short, noncoding RNAs which act as post-transcriptional regulators of gene expression, thereby affecting various biological processes. So far, in humans ~ 1000 miRNA genes have been identified and it has been shown that altered expression of certain miRNA genes may be involved in tumorigenesis. In lung cancer, downregulated expression of numerous miRNA genes has been reported recently. However, knowledge about the mechanisms of miRNA gene silencing is still limited. It has been suggested that besides others, epigenetic changes, particularly DNA methylation (referred to as methylation), may lead to miRNA gene silencing in lung cancer. Thus, in a preliminary study we investigated expression of ~ 800 miRNA genes before and after treatment of non- small cell lung cancer (NSCLC) A549 cells with the DNA methyltransferase inhibitor 5-aza-2`-deoxycytidine (Aza-dC) or the combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. We observed that the expression of 41 miRNA genes was upregulated after drug treatment suggesting that the expression of certain miRNA genes is regulated by epigenetic mechanisms. In addition, using methylation-specific PCR analysis we found miR-193a frequently methylated in primary NSCLCs. Overall our preliminary results demonstrate that certain miRNA genes are methylated in primary NSCLCs. The method for detecting genome-wide methylation used in our preliminary study requires proliferating cells and thus is only suitable to analyse cell lines but not to analyse tissues samples. However, a new method for detection of genome-wide methylation which can also be used to analyse tissue samples was described recently. This method combines methylated DNA immunoprecipitation with microarray (MeDIP-chip) analysis. To obtain further insight into the significance of methylation mediated miRNA gene silencing in NSCLCs, we plan to investigate genome-wide methylation of miRNA genes in primary tumors and matching non-malignant lung tissue samples of 50 NSCLC patients by MeDIP-chip analysis. Certain miRNAs will be selected for further analyses in NSCLC cell lines as well as in additional tumor samples. Major aims of this study are 1. to analyse if miRNA genes are tumor-specifically methylated in primary NSCLCs and if methylation leads to miRNA gene silencing, 2. to investigate if tumor-specific methylation of certain miRNA genes is of prognostic and/or clinical significance for NSCLC patients, and 3. to determine if certain by tumor-specific methylation silenced miRNA genes have tumor cell growth suppressing properties. Overall, we expect that the results of this project will enhance the understanding of the impact of methylation mediated miRNA gene silencing on the pathogenesis of NSCLCs and may potentially influence future treatment strategies of NSCLC patients.

The FWF project P24130 lead by Ao. Univ. Prof. Dr. Sabine Zöchbauer-Müller started in 2012 and continued till 2017. The major goal of this project was to identify genes which are epigenetically altered in primary tumors (TU) but not in non-malignant lung tissue (NL) samples of non-small cell lung cancer (NSCLC) patients. Our work was mainly focused on the analyses of short, noncoding RNAs, called microRNAs (miRNAs) which act as post-transcriptional regulators of gene expression and hereby affect various biological processes. In cancer cells expression of certain miRNA encoding genes may be deregulated. Although downregulated expression of numerous miRNA genes was reported in NSCLCs, knowledge about the mechanisms of miRNA gene silencing is still limited. Preliminary results suggested that epigenetic changes, particularly DNA methylation (referred to as methylation), may lead to miRNA gene silencing in lung cancer. To obtain further insight into the significance of methylation mediated miRNA gene silencing in NSCLC patients, we performed methylation microarray analyses of miRNA encoding genes in 50 TU and 50 NL samples in this project. By this approach, we identified 34 miRNA encoding genes with increased methylation in TU specimens. While some of these miRNA genes were already known to be methylated in NSCLCs, methylation of the vast majority of them was unknown so far. In a next step, we developed gene-specific methylation-sensitive high resolution melting assays (MS-HRM) to investigate methylation of selected miRNA encoding genes in TU and NL samples of in total 108 NSCLC patients. Consistent with our MeDIP-chip data, we observed a statistically significant increase of methylation in TU samples of all miRNA encoding genes investigated by MS-HRM analyses. A correlation of our methylation data with clinico-pathological characteristics of NSCLC patients revealed that the percentage of miR-129-2 methylation was statistically significantly higher in patients with advanced disease. Functional analyses using cell line models demonstrated a statistically significant growth reduction of with miR-1179 transfected NSCLC cells compared to control cells. Thus, miR-1179 is a potential epigenetically regulated tumor cell growth suppressor in NSCLCs. Similar results in NSCLC patients were observed for the protein encoding genes ZNF677 and L1TD1. Overall, our findings may be helpful to better understand the molecular mechanisms involved in the pathogenesis of NSCLCs. Our results were either already published in international scientific journals or are currently under review.