Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion. The expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay. In this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells. Together, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through exosomes. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.

Functional genomics studies have led to the discovery of a large amount of non-coding RNAs from the human genome; among them are long non-coding RNAs (lncRNAs). Emerging evidence indicates that lncRNAs could have a critical role in the regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. As master gene regulators, lncRNAs are capable of forming lncRNA–protein (ribonucleoprotein) complexes to regulate a large number of genes. For example, lincRNA-RoR suppresses p53 in response to DNA damage through interaction with heterogeneous nuclear ribonucleoprotein I (hnRNP I). The present study demonstrates that hnRNP I can also form a functional ribonucleoprotein complex with lncRNA urothelial carcinoma-associated 1 (UCA1) and increase the UCA1 stability. Of interest, the phosphorylated form of hnRNP I, predominantly in the cytoplasm, is responsible for the interaction with UCA1. Moreover, although hnRNP I enhances the translation of p27 (Kip1) through interaction with the 5′-untranslated region (5′-UTR) of p27 mRNAs, the interaction of UCA1 with hnRNP I suppresses the p27 protein level by competitive inhibition. In support of this finding, UCA1 has an oncogenic role in breast cancer both in vitro and in vivo. Finally, we show a negative correlation between p27 and UCA in the breast tumor cancer tissue microarray. Together, our results suggest an important role of UCA1 in breast cancer.

Protein-coding genes account for only a small part of the human genome, whereas the vast majority of transcripts make up the non-coding RNAs including long non-coding RNAs (lncRNAs). Accumulating evidence indicates that lncRNAs could play a critical role in regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. LncRNA loc285194 was previously shown to be within a tumor suppressor unit in osteosarcoma and to suppress tumor cell growth. However, it is unknown regarding the regulation of loc285194. Moreover, the underlying mechanism by which loc285194 functions as a potential tumor suppressor is elusive. In this study, we show that loc285194 is a p53 transcription target; ectopic expression of loc285194 inhibits tumor cell growth both in vitro and in vivo . Through deletion analysis, we identify an active region responsible for tumor cell growth inhibition within exon 4, which harbors two miR-211 binding sites. Importantly, this loc285194-mediated growth inhibition is in part due to specific suppression of miR-211. We further demonstrate a reciprocal repression between loc285194 and miR-211; in contrast to loc285194, miR-211 promotes cell growth. Finally, we detect downregulation of loc285194 in colon cancer specimens by quantitative PCR arrays and in situ hybridization of tissue microarrays. Together, these results suggest that loc285194 is a p53-regulated tumor suppressor, which acts in part through repression of miR-211.

The major function of Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is to regulate cell metabolism. However, emerging evidence indicates that IGF2BP2 plays a role in cancer, but the underlying mechanism is largely unknown. Here we showed that upregulation of IGF2BP2 is associated with poor outcomes of pancreatic cancer patients and suppression of IGF2BP2 inhibits cell proliferation. We further showed that IGF2BP2 regulates lncRNA DANCR. Ectopic expression IGF2BP2 enhances, whereas knockdown (KD) or knockout (KO) of IGF2BP2 suppresses DANCR expression. Moreover, in vivo RNA precipitation and reciprocal RNA immunoprecipitation revealed that IGF2BP2 interacts with DANCR. DANCR promotes cell proliferation and stemness-like properties. Experiments with xenograft models revealed that while ectopic expression of DANCR promotes, DANCR KO suppresses tumor growth. Mechanistically, DANCR is modified at N6-methyladenosine (m6A) and mutagenesis assay identified that adenosine at 664 of DANCR is critical to the interaction between IGF2BP2 and DANCR where IGF2BP2 serves a reader for m6A modified DANCR and stabilizes DANCR RNA. Together, these results suggest that DANCR is a novel target for IGF2BP2 through m6A modification, and IGF2BP2 and DANCR work together to promote cancer stemness-like properties and pancreatic cancer pathogenesis.

It is well known that upon stress, the level of the tumor suppressor p53 is remarkably elevated. However, despite extensive studies, the underlying mechanism involving important inter-players for stress-induced p53 regulation is still not fully understood. We present evidence that the human lincRNA-RoR (RoR) is a strong negative regulator of p53. Unlike MDM2 that causes p53 degradation through the ubiquitin-proteasome pathway, RoR suppresses p53 translation through direct interaction with the heterogeneous nuclear ribonucleoprotein I (hnRNP I). Importantly, a 28-base RoR sequence carrying hnRNP I binding motifs is essential and sufficient for p53 repression. We further show that RoR inhibits p53-mediated cell cycle arrest and apoptosis. Finally, we demonstrate a RoR-p53 autoregulatory feedback loop where p53 transcriptionally induces RoR expression. Together, these results suggest that the RoR-hnRNP I-p53 axis may constitute an additional surveillance network for the cell to better respond to various stresses.