Abstract
Expression of miRNAs in Neuroendocrine Neoplasms (NEN) is poorly characterized. We therefore wanted to examine the miRNA expression in Neuroendocrine Tumors (NETs), and identify their targets and importance in NET carcinogenesis. miRNA expression in six NEN primary tumors, six NEN metastases and four normal intestinal tissues was characterized using miRNA arrays, and validated by in-situ hybridization and qPCR. Among the down-regulated miRNAs miR-129-5p and the let-7f/let-7 family, were selected for further characterization. Transfection of miR-129-5p inhibited growth of a pulmonary and an intestinal carcinoid cell line. Analysis of mRNA expression changes identified EGR1 and G3BP1 as miR-129-5p targets. They were validated by luciferase assay and western blotting, and found robustly expressed in NETs by immunohistochemistry. Knockdown of EGR1 and G3BP1 mimicked the growth inhibition induced by miR-129-5p. let-7 overexpression inhibited growth of carcinoid cell lines, and let-7 inhibition increased protein content of the transcription factor BACH1 and its targets MMP1 and HMGA2, all known to promote bone metastases. Immunohistochemistry analysis revealed that let-7 targets are highly expressed in NETs and metastases. We found down-regulation of miR-129-5p and the let-7 family, and identified new neuroendocrine specific targets for these miRNAs, which contributes to the growth and metastatic potential of these tumors.
Highlights
Gastro-Entero-Pancreatic Neuroendocrine Neoplasms (GEP-NEN) are generally slow growing tumors originating from neuroendocrine cells in the gastro-intestinal tract and pancreas [1]
Given the reduced expression of miR-129-5p in Neuroendocrine Tumors (NETs) we examined the expression of Early Growth Response 1 (EGR1) and G3BP1 in NETs and we found the two genes highly expressed in NETs by immunohistochemistry (Figure 4a)
To characterize the biology of EGR1 and G3BP1 in NETs, we examined their importance for growth of carcinoid cell lines
Summary
Gastro-Entero-Pancreatic Neuroendocrine Neoplasms (GEP-NEN) are generally slow growing tumors originating from neuroendocrine cells in the gastro-intestinal tract and pancreas [1]. A deeper knowledge about the genetic changes leading to the development of carcinoid tumors is still lacking [1] thereby distinguishing GEP-NENs from the majority of cancer diseases of which there has been a substantial increase in knowledge [3]. RAS signaling is attenuated by GTPase activating Proteins (GAPs) [7]. (G3BP1-3) binds the RasGAP and is important for transducing RAS signals in a MAPK-independent manner [8]. Activation of MAPK increases the expression of the Early Growth Response 1 (EGR1) transcription factor which in turn is. In carcinoids MAPK activation leads to increased tumor cell migration [12]. Inhibition of MAPK suppresses invasion and metastasis in part by increasing let-7 expression, which leads to suppression of the let-7 targets. We identified two down-regulated miRNAs in NETs and metastases, characterized their actions in vitro and identified some of their targets in order to understand how dysregulation of these miRNAs contributes to NET carcinogenesis
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