Novel Direct Targets of miR-19a Identified in Breast Cancer Cells by a Quantitative Proteomic Approach

Abstract

The miR-17–92 cluster encodes 7 miRNAs inside a single polycistronic transcript, and is known as a group of oncogenic miRNAs that contribute to tumorigenesis in several cancers. However, their direct targets remain unclear, and it has been suggested that a single miRNA is capable of reducing the production of hundreds of proteins. The majority of reports on the identification of miRNA targets are based on computational approaches or the detection of altered mRNA levels, despite the fact that most miRNAs are thought to regulate their targets primarily by translational inhibition in higher organisms. In this study, we examined the target profiles of miR-19a, miR-20a and miR-92-1 in MCF-7 breast cancer cells by a quantitative proteomic strategy to identify their direct targets. A total of 123 proteins were significantly increased after the endogenous miR-19a, miR-20a and miR-92-1 were knocked down, and were identified as potential targets by two-dimensional electrophoresis and a mass spectrometric analysis. Among the upregulated proteins, four (PPP2R2A, ARHGAP1, IMPDH1 and NPEPL1) were shown to have miR-19a or miR-20a binding sites on their mRNAs. The luciferase activity of the plasmids with each binding site was observed to decrease, and an increased luciferase activity was observed in the presence of the specific anti-miRNA-LNA. A Western blot analysis showed the expression levels of IMPDH1 and NPEPL1 to increase after treatment with anti-miR-19a, while the expression levels of PPP2R2A and ARHGAP1 did not change. The expression levels of IMPDH1 and NPEPL1 did not significantly change by anti-miR-19a-LNA at the mRNA level. These results suggest that the IMPDH1 and NPEPL1 genes are direct targets of miR-19a in breast cancer, while the exogenous expression of these genes is not associated with the growth suppression of MCF-7 cells. Furthermore, our proteomic approaches were shown to be valuable for identifying direct miRNA targets.

miR-19a Promotes Cell Growth and Tumorigenesis through Targeting SOCS1 in Gastric Cancer

“…Identification of cancerspecific miRNAs as well as their target genes is important for elucidating the roles of miRNAs in tumorigenesis and may provide promising therapeutic targets. In this study, we focused on miR-19a, which has been suggested to promote tumor growth in several human cancers, such as breast cancer (Ouchida et al, 2012), multiple myeloma (Pichiorri et al, 2008), cervical cancer (Xu et al, 2012) and B-cell lymphomas (Liu et al, 2011). Recently, Ueda and colleagues found that miR-19a were upregulated in gastric cancer tissues, and overexpression of miR-19a was also associated with progression and prognosis of gastric cancer (Ueda et al, 2010), yet the detailed mechanisms remain unclear.…”

Role of miR-19a targeting TNF-α in mediating ulcerative colitis

“…Further, E2F transcription factors can regulate miR-17-92 cluster [56,57], indicating the circulating feedback regulation between these genes. miR-19a has been reported to regulate a variety of biological processes, including spinocerebellar ataxi [30], multiple myeloma [31], and breast cancer [32]. However, its role in inflammation especially in IBD is rarely studied.…”

“…In the biology of multiple myeloma, miR-19a is shown to downregulate the expression of SOCS-1 [31]. In breast cancer, four upregulated proteins, PPP2R2A, ARH-GAP1, IMPDH1 and NPEPL1, were shown to have miR-19a binding sites on their mRNAs using quantitative proteomic approach [32]. Other miR-19a targets are predicted using a HeLa cell reporter system, including Bim [33].…”

Low serum miR‐19a expression as a novel poor prognostic indicator in multiple myeloma

“…Moreover, the down-regulation of miR-92a in plasma also correlated with response to treatment. Unfortunately, limited information is available describing the function of miR-19a, the only available information identifies it as up-regulated in many cancers 51–53 . Interestingly, Yu et al showed that over-expression of miR-19a suppressed CD142 expression and inhibited cell migration and invasion in colon cancer 53 .…”