Downregulated miR-495-3p in colorectal cancer targets TGFßR1, TGFßR2, SMAD4 and BUB1 genes and induces cell cycle arrest.

BACKGROUND: Hsa-miR-495 (miR-495) has been extensively investigated in cancer initiation and progression. On the other hand, our bioinformatics analysis suggested that miR-495 exerts its effects through targeting of TGFß signaling components. METHODS & RESULTS: In order to investigate such an effect, miR-495 precursor was overexpressed in HEK293T, SW480, and HCT116 cells, which was followed by downregulation of TGFßR1, TGFßR2, SMAD4, and BUB1 putative target genes, detected by RT-qPCR. Also, luciferase assay supported the direct interaction of miR-495 with 3'UTR sequences of TGFßR1, TGFßR2, SMAD4, and BUB1 genes. Furthermore, a negative correlation of expression between miR-495-3p and some of these target genes was deduced in a set of colorectal and breast cancer cell lines. Then, flow cytometry analysis showed that the overexpression of miR-495 in HCT116 and HEK293T resulted in an arrest at the G1 phase. Consistently, western blotting analysis showed a significant reduction of the Cyclin D1 protein in the cells overexpressing miR-495, pointing to downregulation of the TGFß signaling pathway and cell cycle arrest. Finally, microarray data analysis showed that miR-495-3p is significantly downregulated in colorectal tumors, compared to the normal pairs. CONCLUSIONS: Overall, the results of the current study introduced miR-495-3p as a cell cycle progression suppressor, which may negatively regulate TGFßR1, TGFßR2, SMAD4, and BUB1 genes. This finding suggests miR-495-3p as a tumor suppressor candidate for further evaluation.

Comparative expression analysis of the Atoh7 gene regulatory network in the mouse and chicken auditory hindbrain.

The mammalian and avian auditory brainstem likely arose by independent evolution. To compare the underlying molecular mechanisms, we focused on Atoh7, as its expression pattern in the mammalian hindbrain is restricted to bushy cells in the ventral cochlear nucleus. We thereby took advantage of an Atoh7 centered gene regulatory network (GRN) in the retina including upstream regulators, Hes1 and Pax6, and downstream targets, Ebf3 and Eya2. In situ hybridization demonstrated for the latter four genes broad expression in all three murine cochlear nuclei at postnatal days (P) 4 and P30, contrasting the restricted expression of Atoh7. In chicken, all five transcription factors were expressed in all auditory hindbrain nuclei at embryonic day (E) 13 and P14. Notably, all five genes showed graded expression in the embryonic nucleus magnocellularis (NM). Atoh7 was highly expressed in caudally located neurons, whereas the other four transcription factors were highly expressed in rostrally located neurons. Thus, Atoh7 shows a strikingly different expression between the mammalian and avian auditory hindbrain. This together with the consistent absence of graded expression of GRN components in developing mammalian nuclei provide the first molecular support to the current view of convergent evolution as a major mechanism in the amniote auditory hindbrain. The graded expression of five transcription factors specifically in the developing NM confirms this nucleus as a central organizer of tonotopic features in birds. Finally, the expression of all five retinal GRN components in the auditory system suggests co-options of genes for development of sensory systems of distinct modalities.

Differential expression of microRNAs in the developing avian auditory hindbrain.

The avian auditory hindbrain is a longstanding model for studying neural circuit development. Information on gene regulatory network (GRN) components underlying this process, however, is scarce. Recently, the spatiotemporal expression of 12 microRNAs (miRNAs) was investigated in the mammalian auditory hindbrain. As a comparative study, we here investigated the spatiotemporal expression of the orthologous miRNAs during development of the chicken auditory hindbrain. All miRNAs were expressed both at E13, an immature stage, and P14, a mature stage of the auditory system. In most auditory nuclei, a homogeneous expression pattern was observed at both stages, like the mammalian system. An exception was the nucleus magnocellularis (NM). There, at E13, nine miRNAs showed a differential expression pattern along the cochleotopic axis with high expression at the rostromedial pole. One of them showed a gradient expression whereas eight showed a spatially selective expression at the rostral pole that reflected the different rhombomeric origins of this composite nucleus. The miRNA differential expression persisted in the NM to the mature stage, with the selective expression changed to linear gradients. Bioinformatics analysis predicted mRNA targets that are associated with neuronal developmental processes such as neurite and synapse organization, calcium and ephrin-Eph signaling, and neurotransmission. Overall, this first analysis of miRNAs in the chicken central auditory system reveals shared and strikingly distinct features between chicken and murine orthologues. The embryonic gradient expression of these GRN elements in the NM adds miRNA patterns to the list of cochleotopic and developmental gradients in the central auditory system.

Experimental verification of a predicted novel microRNA located in human PIK3CA gene with a potential oncogenic function in colorectal cancer.

PI3K/AKT signaling is involved in cell survival, proliferation, and migration. In this pathway, PI3Kα enzyme is composed of a regulatory protein encoded by p85 gene and a catalytic protein encoded by PIK3CA gene. Human PIK3CA locus is amplified in several cancers including lung and colorectal cancer (CRC). Therefore, microRNAs (miRNAs) that are encoded within the PIK3CA gene might have a role in cancer development. Here, we report a novel microRNA named PIK3CA-miR1 (EBI accession no. LN626315), which is located within PIK3CA gene. A DNA segment corresponding to PIK3CA-premir1 sequence was transfected in human cell lines that resulted in generation of mature exogenous PIK3CA-miR1. Following the overexpression of PIK3CA-miR1, its predicted target genes (APPL1 and TrkC) were significantly downregulated in the CRC-originated HCT116 and SW480 cell lines, detected by qRT-PCR. Then, dual luciferase assay supported the interaction of PIK3CA-miR1 with APPL1 and TrkC transcripts. Endogenous PIK3CA-miR1 expression was also detected in several cell lines (highly in HCT116 and SW480) and highly in CRC specimens. Consistently, overexpression of PIK3CA-premir1 in HCT116 and SW480 cells resulted in significant reduction of the sub-G1 cell distribution and apoptotic cell rate, as detected by flowcytometry, and resulted in increased cell proliferation, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PIK3CA-miR1 overexpression also resulted in Wnt signaling upregulation detected by Top/Fop assay. Overall, accumulative evidences indicated the presence of a bona fide novel onco-miRNA encoded within the PIK3CA oncogene, which is highly expressed in colorectal cancer and has a survival effect in CRC-originated cells.