Background:The regulation of the miR-23a/24-2/27a cluster is largely unknown. Results: EGF induced c-MYC expression to promote the expression of the miR-23a/24-2/27a cluster, resulting in decreased expression of Sprouty2 and increased activation of p44/42 MAPK to stimulate mammary carcinoma cell invasion and subsequent hepatic metastases. Conclusion: EGF promoted mammary carcinoma cell invasion and hepatic metastasis. Significance: The miR-23a/24-2/27a cluster might be used as a biomarker for breast cancer metastasis.

MicroRNAs (miRNAs) are small, non‑coding RNAs which regulate gene expression at the post-transcriptional level. Abnormal expression of miRNAs occurs frequently in tumors. Although the two miRNAs miR‑24‑3p and miR‑27a‑3p come from two duplicated gene clusters of miR‑23a~27a~24‑2 and miR‑23b~27b~24‑1 which are found to be deregulated in a variety of cancers, the role of cooperation of the two clusters and the function of the two miRNAs in tumors have not been completely characterized. Here, we show that overexpression of miR‑24‑3p and miR‑27a‑3p could promote cell proliferation using the MTT assay. By integrated bioinformatic analysis and experimental confirmation, we identified MXI1, which has been found to act as a tumor suppressor gene by affecting c‑Myc, as a direct target of miR‑24‑3p and miR‑27a‑3p. While targeting the MXI1 3' untranslated region by miR‑24‑3p or miR‑27a‑3p, luciferase activity was attenuated. The two miRNAs promote glioma cell proliferation via targeting MXI1 and the experiment was confirmed by the rescue experiments. Furthermore, our results show that two clusters of miR-23a~27a~24-2 and miR‑23b~27b~24‑1 regulate MXI1 synergistically. These findings reveal, for the first time, the novel functions of cooperation of miR‑24‑3p and miR‑27a‑3p from two clusters in promoting cell proliferation through MXI1. Additionally, we observed that miR‑27a‑3p is upregulated in glioma tissues.

Animal microRNA (miRNA) target prediction is still a challenge, although many prediction programs have been exploited. MiRNAs exert their function through partially binding the messenger RNAs (mRNAs; likely at 3′ untranslated regions [3′UTRs]), which makes it possible to detect the miRNA-mRNA interactions in vitro by co-transfection of miRNA and a luciferase reporter gene containing the target mRNA fragment into mammalian cells under a dual-luciferase assay system. Here, we constructed a human miRNA expression library and used a dual-luciferase assay system to perform large-scale screens of interactions between miRNAs and the 3′UTRs of seven genes, which included more than 3,000 interactions with triplicate experiments for each interaction. The screening results showed that the 3′UTR of one gene can be targeted by multiple miRNAs. Among the prediction algorithms, a Bayesian phylogenetic miRNA target identification algorithm and a support vector machine (SVM) presented a relatively better performance (27% for EIMMo and 24.7% for miRDB) against the average precision (17.3%) of the nine prediction programs used here. Additionally, we noticed that a relatively high conservation level was shown at the miRNA 3′ end targeted regions, as well as the 5′ end (seed region) binding sites.

Gliomas are the most common and aggressive primary tumors in the central nervous system. Recently, Max interactor-1 (MXI1), an antagonist of c-Myc that is involved in brain tumor progression, has been reported to be deregulated in a variety of tumors including glioma. However, the mechanism of MXI1 deregulation in gliomas remains unclear. In this study, we show that the relative expression level of MXI1 is markedly down-regulated in glioma cell lines. Using integrated bioinformatic analysis and experimental confirmation, we identified several miRNAs by screening a panel of predicted miRNAs that may regulate the MXI1 3′UTR. The strongest inhibitory miRNA, miR-155, can attenuate the activity of a luciferase reporter gene that is fused with the MXI1 3′UTR and decrease the expression levels of MXI1 mRNA and protein in U87 glioma cells. The potential role of miR-155 in promoting glioma cell proliferation by targeting MXI1 was confirmed in various glioma cell lines by rescue experiments using MTT assays, EdU incorporation assay, and cell counting experiments. In addition, we determined that the level of MXI1 mRNA was inversely correlated with the expression of miR-155 in 18 sets of glioblastoma multiforme specimens. These findings reveal for the first time that the targeting of MXI1 by miR-155 may result in a reduction in MXI1 expression and promote glioma cell proliferation; this result suggests a novel function of miR-155 in targeting MXI1 in glioma-genesis.

BackgroundMicroRNAs (miRNAs) are ubiquitous non-coding RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies has revealed that viruses can also encode miRNAs, which are proposed to be involved in viral replication and persistence, cell-mediated antiviral immune response, angiogenesis, and cell cycle regulation. Singapore grouper iridovirus (SGIV) is a pathogenic iridovirus that has severely affected grouper aquaculture in China and Southeast Asia. Comprehensive knowledge about the related miRNAs during SGIV infection is helpful for understanding the infection and the pathogenic mechanisms.Methodology/Principal FindingsTo determine whether SGIV encoded miRNAs during infection, a small RNA library derived from SGIV-infected grouper (GP) cells was constructed and sequenced by Illumina/Solexa deep-sequencing technology. We recovered 6,802,977 usable reads, of which 34,400 represented small RNA sequences encoded by SGIV. Sixteen novel SGIV-encoded miRNAs were identified by a computational pipeline, including a miRNA that shared a similar sequence to herpesvirus miRNA HSV2-miR-H4-5p, which suggests miRNAs are conserved in far related viruses. Generally, these 16 miRNAs are dispersed throughout the SGIV genome, whereas three are located within the ORF057L region. Some SGIV-encoded miRNAs showed marked sequence and length heterogeneity at their 3′ and/or 5′ end that could modulate their functions. Expression levels and potential biological activities of these viral miRNAs were examined by stem-loop quantitative RT-PCR and luciferase reporter assay, respectively, and 11 of these viral miRNAs were present and functional in SGIV-infected GP cells.ConclusionsOur study provided a genome-wide view of miRNA production for iridoviruses and identified 16 novel viral miRNAs. To the best of our knowledge, this is the first experimental demonstration of miRNAs encoded by aquatic animal viruses. The results provide a useful resource for further in-depth studies on SGIV infection and iridovirus pathogenesis.

Telomeric repeat binding factor 2 (TRF2) is essential for telomere maintenance and has been implicated in DNA damage response and aging. Telomere dysfunction induced by TRF2 inhibition can accelerate cellular senescence in human fibroblasts. While previous work has demonstrated that a variety of factors can regulate TRF2 expression transcriptionally and post-translationally, whether microRNAs (miRNAs) also participate in post-transcriptionally modulating TRF2 levels remains largely unknown. To better understand the regulatory pathways that control TRF2, we carried out a large-scale luciferase reporter screen using a miRNA expression library and identified four miRNAs that could target human TRF2 and significantly reduce the level of endogenous TRF2 proteins. In particular, our data revealed that miR-23a could directly target the 3′ untranslated region (3′UTR) of TRF2. Overexpression of miR-23a not only reduced telomere-bound TRF2 and increased telomere dysfunction-induced foci (TIFs), but also accelerated senescence of human fibroblast cells, which could be rescued by ectopically expressed TRF2. Our findings demonstrate that TRF2 is a specific target of miR-23a, and uncover a previously unknown role for miR-23a in telomere regulation and cellular senescence.

During infection, triggering receptor expressed on myeloid cells-2 (TREM-2) restrains dendritic cells (DCs) and macrophages (MΦs) phagocytosis, as well as reduces pro-inflammatory cytokines release through DNAX-activation protein 12 (DAP12) signaling. However, the role of TREM-2 signaling in cancer has never been elucidated. In the current study, we found that TREM-2 was up-regulated on peripheral blood monocytes in tumor-bearing host. More TREM-2+DCs were detected in the lung of 3LL tumor-bearing mice. On the other hand, the level of TREM-2 on pulmonary MΦs positively correlated with the pathological staging of lung cancer. However, surgical or chemotherapeutic reduction of tumor burden led to the obvious decline of TREM-2. In vitro, TREM-2 expression of bone marrow (BM)-derived DCs and MΦs was induced by conditional medium (CM) containing the supernatant of 3LL cells. TREM-2+DCs from CM and/or tumor-bearing mice held altered phenotypes (CD80LowCD86LowMHCIILow) and impaired functions, such as, reduced interleukin (IL)-12 secretion, increased IL-10 production, and weakened ovalbumin (OVA)-endocytic capacity; also developed potent inhibitory effect on T cell proliferation that could be partially reversed by TREM-2 blockage. Moreover, spleen tyrosine kinase (Syk) inhibitor restrained IL-10 production of TREM-2+DC. Remarkably, IL-10 neutralizing antibody and Syk inhibitor both lowered the suppressive potential of TREM-2+DCs in T cell proliferation. Also, adoptive transfer of this TREM-2+DCs accelerated the tumor growth rather than jeopardized survival in lung cancer-bearing mice. In conclusion, these results indicate that TREM-2 might act as a negative immuno-regulatory molecule through Syk pathway in an IL-10 dependent manner and partially predicts prognosis in lung cancer patients.

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