Mature miR-17-5p and passenger miR-17-3p induce hepatocellular carcinoma by targeting PTEN, GalNT7 and vimentin in different signal pathways.

To 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.

Both mature miR-17-5p and passenger strand miR-17-3p target TIMP3 and induce prostate tumor growth and invasion.

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.

The intermediate filament vimentin mediates microRNA miR-378 function in cellular self-renewal by regulating the expression of the Sox2 transcription factor.

MicroRNAs are short noncoding RNAs that are implicated in cell self- renewal and cancer development. We show that miR-378 is up-regulated in human cancers and found that tumor cells transfected with miR-378 acquired properties of tumor stem cells, including cell self-renewal. Overexpression of miR-378 enhanced cell survival and colony formation. Isolated from a single-cell colony, the miR-378-expressing cells formed tumors in nude mice at low cell densities. These cells expressed higher levels of miR-378 and formed more and larger spheres and colonies. We found that the miR-378-expressing cells contained a large number of side population cells and could undergo differentiation. Cells transfected with miR-378 expressed increased levels of Sox2. Expression of miR-378 and Sox2 was found correlated significantly in cancer cell lines and in cancer patient specimens. We also observed opposite levels of vimentin in the cancer cell lines and human breast carcinoma specimens. We further demonstrated that vimentin is a target of miR-378, and ectopic transfection of vimentin inhibited Sox2 expression, resulting in decreased cell survival. Silencing vimentin promoted Sox2 expression and cell survival. Our study demonstrates that miR-378 is a regulator of stem cell marker Sox2 by targeting vimentin, which may serve as a new tool in studying the role of stem cells in tumorigenesis.

The non-coding 3' UTR of CD44 induces metastasis by regulating extracellular matrix functions.

The importance of non-coding RNA transcripts in regulating microRNA (miRNA) functions, especially the 3'-untranslated region (3' UTR), has been revealed in recent years. Genes encoding the extracellular matrix normally produce large mRNA transcripts including the 3' UTR. How these large transcripts affect miRNA functions and how miRNAs modulate extracellular matrix protein expression are largely unknown. Here, we demonstrate that the overexpression of the CD44 3' UTR results in enhanced cell motility, invasion and cell adhesion in human breast carcinoma cell line MDA-MB-231. Furthermore, we found that expression of the CD44 3' UTR enhances metastasis in vivo. We hypothesize that increased expression of the CD44 3' UTR affects miRNA binding and modulates synthesis of the extracellular matrix. Computational analysis indicated that miRNAs that interact with the CD44 3' UTR also have binding sites in other matrix-encoding mRNA 3' UTRs, including collagen type 1α1 (Col1α1) repressed by miR-328 and fibronectin type 1 (FN1) repressed by miR-512-3p, miR-491 and miR-671. Protein analysis demonstrated that expression of CD44, Col1α1 and FN1 were synergistically upregulated in vitro and in vivo upon transfection of the CD44 3' UTR. The non-coding 3' UTR of CD44 interacts with multiple miRNAs that target extracellular matrix properties and thus can be used to antagonize miRNA activities.

Gambogic acid inhibits growth, induces apoptosis, and overcomes drug resistance in human colorectal cancer cells.

The emergence of chemoresistance is a major limitation of colorectal cancer (CRC) therapies and novel biologically based therapies are urgently needed. Natural products represent a novel potential anticancer therapy. Gambogic acid (GA), a small molecule derived from Garcinia hanburyi Hook. f., has been demonstrated to be highly cytotoxic to several types of cancer cells and have low toxicity to the hematopoietic system. However, the potential role of GA in colorectal cancer and its ability to overcome the chemotherapeutic resistance in CRC cells have not been well studied. In the present study, we showed that GA directly inhibited proliferation and induced apoptosis in both 5-fluorouracil (5-FU) sensitive and 5-FU resistant colorectal cancer cells; induced apoptosis via activating JNK signaling pathway. The data, therefore, suggested an alternative strategy to overcome 5-FU resistance in CRC and that GA could be a promising medicinal compound for colorectal cancer therapy.

The pseudogene TUSC2P promotes TUSC2 function by binding multiple microRNAs.

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.

miRNAs regulate expression and function of extracellular matrix molecules.

MicroRNAs (miRNAs) are a family of small non-coding RNA molecules that are made up of 18-25 nucleotides that function in post-transcriptional gene regulation. The expression of miRNAs is highly conserved and essential in regulating many cellular processes including formation, maintenance and the remodelling of the extracellular matrix (ECM). In this review, we examine different ECM molecules and the miRNAs involved in regulating their abundance and how these changes influence cell phenotype. For example, miRNAs and their target messenger RNAs (mRNAs) are involved in cell adhesion, by regulating the synthesis and turnover of key ECM adhesion molecules and their receptors including cadherins, integrins and other non-integrin ECM receptors. Other miRNAs regulate the abundance of cytokines and growth factors which in turn stimulate cells to synthesize and secrete specialized ECMs. For example, miR-125a/b and miR-146a and their downstream target mRNAs influence the production of the epidermal growth factor family which has a significant impact on the nature of the ECM formed. miRNAs affect structural ECM proteins important in the assembly, composition and organization of the ECM. Proteins such as collagen, fibronectin, versican, and nephronectin are targeted by several miRNAs. miRNAs can also control the expression of proteins such as matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs), which are involved in ECM remodelling and are important for tissue development, cell motility and wound healing. It has become clear that many different miRNAs control the balance in ECM composition that determines normal tissue function and alterations in the expression of these miRNAs can lead to pathological consequences.

The involvement of microRNAs in malignant transformation.

In the multiple steps in cancer progression, microRNAs (miRNAs) play significant roles in each stage. Reports of considerable differences in expression levels of miRNAs between normal and malignant tissues are understandable considering miRNAs are key regulators of gene expression. Dysregulation of miRNA expression levels in neoplasia occurs because many miRNAs are located in "fragile sites", which are frequently deleted in cancer. miRNAs are often down regulated in cancerous tissues and target oncogenic proteins are classified as tumour suppressor miRNAs, such as let-7. While, miRNAs that are frequently over-expression in neoplastic tissues compared to normal tissues and regulate tumour suppressor proteins are categorized as "oncomiRs". In this review, we summarize information about microRNAs involved in the emerging field of cancer stem cells, and microRNAs involved in breast cancer, an area of our expertise. The application of miRNAs to cancer therapeutics and diagnostics is emerging as an important field of gene therapy. The diverse nature of miRNAs in cancer is continually being elucidated to lead to the enigmatic treatment options for neoplastic disease.

An anti-let-7 sponge decoys and decays endogenous let-7 functions.

The let-7 family contains 12 members, which share identical seed regions, suggesting that they may target the same mRNAs. It is essential to develop a means that can regulate the functions of all members. Using a DNA synthesis technique, we have generated an anti-let-7 sponge aiming to modulate the function of all members. We found that products of the anti-let-7 construct could bind and inactivate all members of the let-7 family, producing decoy and decay effects. To test the role of the anti-let-7 sponge, we stably expressed the anti-let-7 construct in two types of cells, the breast carcinoma cells MT-1 and the oldest and most commonly used human cervical cancer cell line, HeLa cells. We found that expression of anti-let-7 increased cell survival, invasion and adhesion, which corroborate with known functions of let-7 family members. We further identified a novel target site across all species of the let-7 family in hyaluronan synthase 2 (HAS2). HAS2 overexpression produced similar effects as the anti-let-7 sponge. Silencing HAS2 expression by siRNAs produced opposite effects to anti-let-7 on cell survival and invasion. The ability of anti-let-7 to regulate multiple members of the let-7 family allows us to observe their multiple functions using a single reagent. This approach can be applied to other family members with conserved sequences.

MicroRNA miR-98 inhibits tumor angiogenesis and invasion by targeting activin receptor-like kinase-4 and matrix metalloproteinase-11.

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.

MiR-93 enhances angiogenesis and metastasis by targeting LATS2.

Here we report that miR-93, a miRNA in the miR-106B~25 cluster, a paralog of the miR-17-92 cluster, was significantly upregulated in human breast carcinoma tissues. We stably expressed miR-93 in the MT-1 human breast carcinoma cell line and found that tumors formed by the miR-93 cells contained more blood vessels than those formed by the control cells. Co-culture experiments indicated that the MT-1 cells displayed a high activity of adhesion with endothelial cells and could form larger and more tube-like structures with endothelial cells. Lung metastasis assays were performed in a mouse metastatic model, and it was found that expression of miR-93 promoted tumor cell metastasis to lung tissue. In cell culture, expression of miR-93 enhanced cell survival and invasion. We examined the potential target that mediated miR-93's effects and found that the large tumor suppressor, homology 2 (LATS2) was a target of miR-93. Higher levels of LATS2 were associated with cell death in the tumor mass. Silencing LATS2 expression promoted cell survival, tube formation and invasion, while ectopic expression of LATS2 decreased cell survival and invasion. These findings demonstrated that miR-93 promoted tumor angiogenesis and metastasis by suppressing LATS2 expression. Our results suggest that the inhibition of miR-93 function may be a feasible approach to repress tumor metastasis.