Abstract 3086: Modulation of microRNA profile in lung adenocarcinoma cell line following TIMP-1 downregulation.
Authors: Sampa Ghoshal-Gupta, Ammar Kutiyanawalla, Shahanawaz Jiwani, Juhi Ojha, Ravindra Kolhe, Amyn M. Rojiani, Mumtaz V. Rojiani
Journal: Cancer Research (2013)

Abstract Non-small cell carcinoma accounts for 85% of lung cancer cases and has a 5 year survival rate of only 17%. In recent years, tissue inhibitor of metalloproteinase-1 (TIMP-1) has emerged as an important prognostic marker. In several cancers including non-small cell lung cancer, high serum levels of TIMP-1 have been associated with poor prognosis. Previously, we have shown that overexpressing TIMP-1 in H2009, a lung adenocarcinoma cell line resulted in aggressive tumors when implanted in the mouse brain. In the present study, we have knocked down TIMP-1 expression in the non-small cell lung cancer cell line A549 using shRNA. In vitro assays using the TIMP-1 knockdown clones defined decreased migration in wound assays (p&amp;lt;0.01), as well as reduced invasion (p&amp;lt;0.05) using Boyden chamber assays. We investigated these functional alterations by examining microRNAs in these cell lines. MicroRNAs represent a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level and control crucial signaling and physiological processes functioning either as oncogenes or tumor-suppressors. The miRNA expression profile of A549 cell line and its TIMP1 knockdown clone were evaluated using the Affymetrix miRNA microarray platform on Gene-Chip miRNA 3.0 array in A549 cell line. Following hybridization and data acquisition, we used Partek Genomics Suite ® software for RNA normalization and to determine statistically significant differences in miRNA expression between experimental groups by ANOVA and pairwise comparisons (two-sidedα=0.05). Using the Affymetrix miRNA array we have identified altered miRNAs expression in the clones, with 72 differentially regulated miRNAs following TIMP-1 knockdown. Using quantitative real time PCR we have selectively validated several of the miRNAs that showed the maximal difference. We have confirmed a 2 to 4-fold down regulation of MiR-10b, miR-29a and miR-30 and a 3-fold upregulation of miR-125a-5p. Based on KEGG pathway analysis, this group of validated miRNAs are involved in the MAPK signaling pathway. Western blot analysis showed activation of the apoptosis inducing p38 MAPK pathway in the knockdown clones. Additional mechanistic dissection of these changes and involved pathways will contribute further to our understanding of the role of TIMP-1 in lung cancer. (This research was supported by a Georgia Distinguished Cancer Scholar award to AMR). Citation Format: Sampa Ghoshal-Gupta, Ammar Kutiyanawalla, Shahanawaz Jiwani, Juhi Ojha, Ravindra Kolhe, Amyn M. Rojiani, Mumtaz V. Rojiani. Modulation of microRNA profile in lung adenocarcinoma cell line following TIMP-1 downregulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3086. doi:10.1158/1538-7445.AM2013-3086