Abstract
Although microRNA (miR) 199a-3p functions as a tumor suppressor in multiple malignancies, its expression and role in esophageal cancer have not been studied. Based on our previous observation that miR-199a-3p is markedly downregulated in esophageal cancer cell lines relative to esophageal epithelial cells, we examined the function of miR-199a-3p in these cells. MiR-199a-3p is predicted to bind with high affinity to the mRNA of p21 activated kinase 4 (PAK4). This kinase has been shown to be overexpressed in several malignancies and to modulate proliferation and motility. The current study is designed to determine whether miR-199a-3p regulates the expression of PAK4 in esophageal cancer cells and to understand the functional consequences of this interaction. Herein, we demonstrate reduced expression of miR-199a-3p in human esophageal cancer specimens and cell lines compared to esophageal epithelial cells, with associated increased expression of PAK4. Forced expression of miR-199a-3p decreases expression of PAK4 in esophageal cancer cell lines. Mechanistic studies reveal that miR-199a-3p binds to the 3’UTR of PAK4 mRNA. This interaction results in reduced levels of PAK4 mRNA due to decreased mRNA stability. Downregulation of PAK4 leads to decreased cyclin D1 (CD1) transcription and protein expression, resulting in markedly impaired cellular proliferation. When PAK4 expression is rescued, both CD1 transcription and protein return to baseline levels. Our results show that miR-199a-3p functions as a tumor suppressor in esophageal cancer cells through repression of PAK4. These findings suggest that both miR-199a-3p and PAK4 may be novel therapeutic targets in the treatment of esophageal cancer.
Highlights
Over the past 10 years, the incidence of esophageal cancer has increased by 16% in the United States, with approximately 17,000 cases estimated to be diagnosed in 2018 [1,2]
We demonstrate reduced expression of miR-199a-3p in human esophageal cancer specimens and cell lines compared to esophageal epithelial cells, with associated increased expression of p21 activated kinase 4 (PAK4)
In order to confirm our previous array data [4] showing marked downregulation of miR-199a-3p in the esophageal cancer cell lines TE7 and TE10 compared to esophageal epithelial cells, total RNA was harvested from these cells, as well as the human esophageal adenocarcinoma cell line, FLO1, and quantitative realtime PCR (q-PCR) analysis was performed
Summary
Over the past 10 years, the incidence of esophageal cancer has increased by 16% in the United States, with approximately 17,000 cases estimated to be diagnosed in 2018 [1,2]. Esophageal cancer is the seventh leading cause of death in men in the United States [2]. Unlike other malignancies, such as lung, breast, and colon, no targeted therapies have yet been developed for esophageal cancer. MicroRNAs (miRs) are well recognized as critical post-transcriptional regulators of gene expression in cancer cells [3]. Because of the specificity of the miRtarget interaction, analyzing the roles of individual miRs differentially expressed in esophageal cancer cells can be used to identify molecular targets that directly regulate processes that are critically involved in esophageal cancer oncogenesis. In a previously published array analysis comparing global miR expression in a human esophageal epithelial cell line (hESO) to the human esophageal squamous cancer cell lines TE7 and TE10, we found that miR-199a-3p was one of the most downregulated miRs in the esophageal cancer cells, with a decrease in expression of greater than 3 log fold [4]
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