Abstract 5906: TIMP-1 expression is inversely correlated with miRNA125a-5p and let-7e in non-small-cell lung carcinoma

Abstract

Abstract Epigenetic alterations are emerging as significant elements in our understanding the biology of lung cancer which remains the leading cause of cancer deaths worldwide. In earlier studies we have shown that tissue inhibitor of metalloproteinase 1 (TIMP-1) overexpression in NSCLC cells results in aggressive tumors in mice. We have also demonstrated TIMP-1’s anti-apoptotic activity in these cells. It is well documented that miRNAs are involved in many physiological and neoplastic processes, including apoptosis. We have previously shown that knocking down TIMP-1 in A549 NSCLC cells alters the miRNA profile in these cells with upregulation of miR-125a-5p. Subsequently, we have identified that miR let-7e is also upregulated in TIMP-1 KD clones. The present study is focused on confirming the involvement of miR-125a-5p with TIMP-1 by using mimics and antagomirs of 125a-5p. Furthermore, we have sought to validate this relationship in vivo by examining these patterns in clinical samples. We added mimics of miR-125a-5p into the parental A549 cells, which resulted in down regulation of TIMP-1 levels. We were then able to rescue TIMP-1 downregulation by adding antagomirs of miR-125a-5p to the same cells. MiR-125a-5p binding sites were identified in TIMP-1 3’ UTR and using luciferase assay, we have confirmed that TIMP-1 is indeed a bona fide target of miR-125a-5p. Translational studies were carried out using archived NSCLC adenocarcinoma tissues from the Surgical Pathology archives at AU Medical Center. We first measured expression level of TIMP-1 by RT-PCR in 20 tumors and paired normal adjacent tissues and divided the samples into 2 groups, those with low and high TIMP-1 expression levels. Tissues were then interrogated for TIMP-1 and miR-125a-5p and let- 7e miRNA expression using chromogenic in situ hybridization. We identified negative correlation pattern between tumor and paired normal adjacent tissue samples. Generally, TIMP-1 was highly expressed in the tumor region, while miRNA expression was relatively decreased when compared with adjacent normal tissues. miRNA 125a-5p and let- 7e are known tumor suppressors in NSCLC. Analyzing secondary data (adenocarcinoma) by KM plotter we found that high TIMP1 correlated with lower patient survival with a hazard ratio of 3.17 (2.3-4.37) and a log rank p value of 8.1e-14. These studies provide additional clarity and further extend our understanding of the relationship between these miRNA families and TIMP-1 expression. Citation Format: Ammar Kutiyanawalla, Sampa Ghoshal-Gupta, Byung R. Lee, Ashis Mondal, Ravindra Kolhe, Amyn M. Rojiani, Mumtaz V. Rojiani. TIMP-1 expression is inversely correlated with miRNA125a-5p and let-7e in non-small-cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5906. doi:10.1158/1538-7445.AM2017-5906