Background Long noncoding RNAs (lncRNAs) have emerged as critical players in cancer progression, but their functions in colorectal cancer (CRC) metastasis have not been systematically clarified. Methods lncRNA expression profiles in matched normal and CRC tissue were checked using microarray analysis. The biological roles of a novel lncRNA, namely RP11-138 J23.1 (RP11), in development of CRC were checked both in vitro and in vivo. Its association with clinical progression of CRC was further analyzed. Results RP11 was highly expressed in CRC tissues, and its expression increased with CRC stage in patients. RP11 positively regulated the migration, invasion and epithelial mesenchymal transition (EMT) of CRC cells in vitro and enhanced liver metastasis in vivo. Post-translational upregulation of Zeb1, an EMT-related transcription factor, was essential for RP11-induced cell dissemination. Mechanistically, the RP11/hnRNPA2B1/mRNA complex accelerated the mRNA degradation of two E3 ligases, Siah1 and Fbxo45, and subsequently prevented the proteasomal degradation of Zeb1. m 6 A methylation was involved in the upregulation of RP11 by increasing its nuclear accumulation. Clinical analysis showed that m 6 A can regulate the expression of RP11, further, RP11 regulated Siah1-Fbxo45/Zeb1 was involved in the development of CRC. Conclusions m 6 A-induced lncRNA RP11 can trigger the dissemination of CRC cells via post-translational upregulation of Zeb1. Considering the high and specific levels of RP11 in CRC tissues, our present study paves the way for further investigations of RP11 as a predictive biomarker or therapeutic target for CRC. Electronic supplementary material The online version of this article (10.1186/s12943-019-1014-2) contains supplementary material, which is available to authorized users.
MicroRNAs (miRNA) precursor (pre-miRNA) molecules can be processed to release a miRNA/miRNA* duplex. In the canonical model of miRNA biogenesis, one strand of the duplex is thought to be the biologically active miRNA, whereas the other strand is thought to be inactive and degraded as a carrier or passenger strand called miRNA* (miRNA star). However, recent studies have revealed that miRNA* strands frequently play roles in the regulatory networks of miRNA target molecules. Our recent study indicated that miR-17 transgenic mice could abundantly express both the mature miR-17-5p and the passenger strand miR-17-3p. Here, we showed that miR-17 enhanced prostate tumor growth and invasion by increasing tumor cell proliferation, colony formation, cell survival and invasion. miRNA target analysis showed that both miR-17-5p and miR-17-3p repressed TIMP metallopeptidase inhibitor 3 (TIMP3) expression. Silencing with small interfering RNA against TIMP3 promoted cell survival and invasion. Ectopic expression of TIMP3 decreased cell invasion and cell survival. Our results demonstrated that mature miRNA can function coordinately with its passenger strand, enhancing the repressive ability of a miRNA by binding the same target. Within an intricate regulatory network, this may be among the mechanisms by which miRNA can augment their regulatory capacity.
SummaryTo study the physiological role of a single microRNA (miRNA), we generated transgenic mice expressing the miRNA precursor miR-17 and found that the mature miR-17-5p and the passenger strand miR-17-3p were abundantly expressed. We showed that mature miR-17-5p and passenger strand miR-17-3p could synergistically induce the development of hepatocellular carcinoma. The mature miR-17-5p exerted this function by repressing the expression of PTEN. In contrast, the passenger strand miR-17-3p repressed expression of vimentin, an intermediate filament with the ability to modulate metabolism, and GalNT7, an enzyme that regulates metabolism of liver toxin galactosamine. Hepatocellular carcinoma cells, HepG2, transfected with miR-17 formed larger tumors with more blood vessels and less tumor cell death than mock-treated cells. Expression of miR-17 precursor modulated HepG2 proliferation, migration, survival, morphogenesis and colony formation and inhibited endothelial tube formation. Silencing of PTEN, vimentin or GalNT7 with their respective siRNAs enhanced proliferation and migration. Re-expressing these molecules reversed their roles in proliferation, migration and tumorigenesis. Further experiments indicated that these three molecules do not interact with each other, but appear to function in different signaling pathways. Our results demonstrated that a mature miRNA can function synergistically with its passenger strand leading to the same phenotype but by regulating different targets located in different signaling pathways. We anticipate that our assay will serve as a helpful model for studying miRNA regulation.
SummaryMicroRNAs are known to play regulatory roles in gene expression associated with cancer development. We analyzed levels of the microRNA miR-24 in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in benign breast tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer cell invasion and migration. In vivo experiments in mice indicated that the expression of miR-24 enhanced tumor growth, invasion into local tissues, metastasis to lung tissues and decreased overall mouse survival. In the miR-24-expressing cells and tumors, EGFR was highly phosphorylated, whereas expression of the phosphatases tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor-type tyrosine-protein phosphatase F (PTPRF) were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of phosphorylated epidermal growth factor receptor (pEGFR) were higher whereas the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis. Furthermore, we found that MMP2, MMP11, pErk, and ADAM15 were upregulated, whereas TIMP2 was downregulated; all of which supported the roles of miR-24 in tumor invasion and metastasis. Our results suggest that miR-24 plays a key role in breast cancer invasion and metastasis. miR-24 could potentially be a target for cancer intervention.
There is an urgent clinical need for safe and effective treatment agents and therapy targets for estrogen receptor negative (ER−) breast cancer. G protein-coupled receptor 30 (GPR30), which mediates non-genomic signaling of estrogen to regulate cell growth, is highly expressed in ER− breast cancer cells. We here showed that activation of GPR30 by the receptor-specific agonist G-1 inhibited the growth of ER− breast cancer cells in vitro. Treatment of ER− breast cancer cells with G-1 resulted in G2/M-phase arrest, downregulation of G2-checkpoint regulator cyclin B, and induction of mitochondrial-related apoptosis. The G-1 treatment increased expression of p53 and its phosphorylation levels at Serine 15, promoted its nuclear translocation, and inhibited its ubiquitylation, which mediated the growth arrest effects on cell proliferation. Further, the G-1 induced sustained activation and nuclear translocation of ERK1/2, which was mediated by GPR30/epidermal growth factor receptor (EGFR) signals, also mediated its inhibition effects of G-1. With extensive use of siRNA-knockdown experiments and inhibitors, we found that upregulation of p21 by the cross-talk of GPR30/EGFR and p53 was also involved in G-1-induced cell growth arrest. In vivo experiments showed that G-1 treatment significantly suppressed the growth of SkBr3 xenograft tumors and increased the survival rate, associated with proliferation suppression and upregulation of p53, p21 while downregulation of cyclin B. The discovery of multiple signal pathways mediated the suppression effects of G-1 makes it a promising candidate drug and lays the foundation for future development of GPR30-based therapies for ER− breast cancer treatment.
Angiogenesis and invasion are essential processes for solid tumor growth and dissemination. The tumor development process can be dependent on the activation of a series of signaling pathways, including growth factor-activated pathways. MicroRNAs have been shown to be critical for tumorigenesis, but their roles in cancer angiogenesis, invasion and other signaling pathways important for tumor development are still unclear in the context of tumor biology. We investigated the role of microRNA miR-98 in regulating tumor growth, invasion, and angiogenesis using a highly aggressive breast cancer model in vitro and in vitro. We found that the expression of miR-98 inhibited breast cancer cell proliferation, survival, tumor growth, invasion, and angiogenesis. Conversely, inhibition of endogenous miR-98 promoted cell proliferation, survival, tumor growth, invasion, and angiogenesis. It appeared that miR-98 inhibited angiogenesis by modulating endothelial cell activities including cell spreading, cell invasion and tubule formation. Interestingly, miR-98 reduced the expression of ALK4 and MMP11, both of which were potential targets of miR-98. Transfection of an anti-miR-98 construct increased the expression of both targets. We confirmed that mir-98 targeted the 3'-untranslated regions of ALK4 and MMP11. Finally, ALK4- and MMP11-specific siRNAs inhibited breast cancer cell proliferation, survival, and angiogenesis. Rescue experiments with ALK4 and MMP11 constructs reversed the anti-proliferative, anti-invasive and anti-angiogenic effects of miR-98. Our findings define a regulatory role of miR-98 in tumor angiogenesis and invasion through repressed ALK4 and MMP11 expression.
This study was designed to explore the role of versican in the development of hepatocellular carcinoma (HCC). Ectopic expression of the versican 3'-untranslated region (3'-UTR) was studied as a competitive endogenous RNA for regulating miRNA functions. We used this approach to modulate the expression of versican and its related proteins in 3'-UTR transgenic mice and in the liver cancer cell line HepG2, stably transfected with the 3'-UTR or a control vector. We demonstrated that transgenic mice expressing the versican 3'-UTR developed HCC and increased expression of versican isoforms V0 and V1. HepG2 cells transfected with versican 3'-UTR displayed increased proliferation, survival, migration, invasion, colony formation, and enhanced endothelial cell growth, but decreased apoptosis. We found that versican 3'-UTR could bind to miRNAs miR-133a, miR-199a*, miR-144, and miR-431 and also interacted with CD34 and fibronectin. As a consequence, expression of versican, CD34, and fibronectin was up-regulated by ectopic transfection of the versican 3'-UTR, which was confirmed in HepG2 cells and in transgenic mice as compared with wild-type controls. Transfection with siRNAs targeting the versican 3'-UTR abolished the effects of the 3'-UTR. Taken together, these results demonstrate that versican V0 and V1 isoforms play important roles in HCC development and that versican mRNAs compete with endogenous RNAs in regulating miRNA functions.
Various non-coding regions of the genome, once presumed to be ‘junk’ DNA, have recently been found to be transcriptionally active. In particular, pseudogenes are now known to have important biological roles. Here we report that transcripts of the two tumour suppressor candidate-2 pseudogenes (TUSC2P), found on chromosomes X and Y, are homologous to the 3′-UTR of their corresponding protein coding transcript, TUSC2. TUSC2P and the TUSC2 3′-UTR share many common miRNA-binding sites, including miR-17, miR-93, miR-299-3p, miR-520a, miR-608 and miR-661. We find that ectopic expression of TUSC2P and the TUSC2 3′-UTR inhibits cell proliferation, survival, migration, invasion and colony formation, and increases tumour cell death. By interacting with endogenous miRNAs, TUSC2P and TUSC2 3′-UTR arrest the functions of these miRNAs, resulting in increased translation of TUSC2. The TUSC2P and TUSC2 3′-UTR could thus be used as combinatorial miRNA inhibitors and might have clinical applications.
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