Expression of Versican 3′-Untranslated Region Modulates Endogenous MicroRNA Functions

BACKGROUND: Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. METHODS AND FINDINGS: Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector. CONCLUSION: Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities

Versican G3 Domain Modulates Breast Cancer Cell Apoptosis: A Mechanism for Breast Cancer Cell Response to Chemotherapy and EGFR Therapy

Overexpression of EGFR and versican has been reported in association with breast cancers. Considered oncogenic, these molecules may be attractive therapeutic targets. Possessing anti-apoptotic and drug resistant properties, overexpression of these molecules is accompanied by selective sensitization to the process of apoptosis. In this study, we exogenously expressed a versican G3 construct in breast cancer cell lines and analyzed the effects of G3 on cell viability in fetal bovine serum free conditioned media and evaluated the effects of apoptotic agent C2-ceramide, and chemotherapeutic agents including Docetaxel, Doxorubicin, and Epirubicin. Versican G3 domain enhanced tumor cell resistance to apoptosis when cultured in serum free medium, Doxorubicin, or Epirubicin by up-regulating pERK and GSK-3β (S9P). However, it could be prevented by selective EGFR inhibitor AG 1478 and selective MEK inhibitor PD 98059. Both AG 1478 and PD 98059 enhanced expression of pSAPK/JNK, while selective JNK inhibitor SP 600125 enhanced expression of GSK-3β (S9P). Versican G3 promoted cell apoptosis induced by C2-ceramide or Docetaxel by enhancing expression of pSAPK/JNK and decreasing expression of GSK-3β (S9P), an observation blocked by AG 1478 or SP 6000125. Inhibition of endogenous versican expression by siRNA or reduction of versican G3's expression by linking G3 with 3′UTR prevented G3 modulated cell apoptosis. The dual roles of G3 in modulating breast cancer cell resistance to chemotherapeutic agents may in part explain a potential mechanism for breast cancer cell resistance to chemotherapy and EGFR therapy. The apoptotic effects of chemotherapeutics depend upon the activation and balance of down stream signals in the EGFR pathway. GSK-3β (S9P) appears to function as a key checkpoint in this balance of apoptosis and anti-apoptosis. Investigation and potential consideration of targeting GSK-3β (S9P) merits further study

Expression of CD44 3′-untranslated region regulates endogenous microRNA functions in tumorigenesis and angiogenesis

The non-coding 3′-untranslated region (UTR) plays an important role in the regulation of microRNA (miRNA) functions, since it can bind and inactivate multiple miRNAs. Here, we show the 3′-UTR of CD44 is able to antagonize cytoplasmic miRNAs, and result in the increased translation of CD44 and downstream target mRNA, CDC42. A series of cell function assays in the human breast cancer cell line, MT-1, have shown that the CD44 3′-UTR inhibits proliferation, colony formation and tumor growth. Furthermore, it modulated endothelial cell activities, favored angiogenesis, induced tumor cell apoptosis and increased sensitivity to Docetaxel. These results are due to the interaction of the CD44 3′-UTR with multiple miRNAs. Computational algorithms have predicted three miRNAs, miR-216a, miR-330 and miR-608, can bind to both the CD44 and CDC42 3′-UTRs. This was confirmed with luciferase assays, western blotting and immunohistochemical staining and correlated with a series of siRNA assays. Thus, the non-coding CD44 3′-UTR serves as a competitor for miRNA binding and subsequently inactivates miRNA functions, by freeing the target mRNAs from being repressed

A 3′-Untranslated Region (3′UTR) Induces Organ Adhesion by Regulating miR-199a* Functions

Mature microRNAs (miRNAs) are single-stranded RNAs of 18–24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3′UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3′UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3′UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3′UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3′UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3′UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3′UTR may be an approach in the development of gene therapy

MicroRNA miR-328 Regulates Zonation Morphogenesis by Targeting CD44 Expression

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion

Modulation of microRNA Functions by the 3’ Untranslated Regions of CD44 and Tumour Suppressor Candidate 2

Recently, the importance of non-coding regions of the genome, which were once presumed to be “junk” DNA, has been revealed. The non-coding 3’ untranslated region (3’UTR) plays a significant role in the regulation of microRNA (miRNA) functions. The 3’UTR is hypothesized to function in the feed-back regulation of miRNA functions, since it can bind and inactivate multiple miRNAs. In this study, the effects of the exogenous over-expression of two 3’UTRs, CD44 and Tumour Suppressor candidate 2 (TUSC2), on miRNA functions in breast cancer carcinogenesis were investigated. A series of cell function assays in human and mouse breast cancer cell lines, MT-1, MDA-MB-231 and 4T1 showed phenotypic changes caused by the 3’UTRs. A variety of cell function characteristics were affected, including cell proliferation, colony formation, cell survival, angiogenesis, tumour growth, cell migration, invasion and adhesion. These results were hypothesized to occur due to the interaction of the 3’UTRs with multiple miRNAs. The 3’UTRs were able to antagonize cytoplasmic miRNAs, as demonstrated by luciferase activities. In the case of the CD44 3’UTR, downstream target mRNAs, CDC42, Col1a1 and FN1 had an increased expression along with CD44. When the 3’UTR of TUSC2 was over-expressed, there was an increased translation of TUSC2 and the downstream targets, tissue inhibitor of metalloproteinases (TIMP) -2 and -3.The 3’UTRs of these target mRNAs can bind and target multiple miRNAs in common with the TUSC2 3’UTR, which were confirmed with luciferase activity assays and correlated with a series of siRNA and miRNA assays. During the study of the TUSC2 3’UTR, a potential pseudogene, TUSC2P, was discovered. Interestingly, TUSC2P mRNA expression was found to be decreased in cancer cells compared to normal cells and had similar cell functional characteristics as that of the TUSC2 3’UTR. These findings suggest that the over-expressed non-coding transcript can serve as a competitor for miRNA binding, which freed the potential targets of the miRNAs and led to an up-regulation of multiple protein levels. The non-coding transcript can thus be used as a functional miRNA inhibitor that is capable of modulating multiple miRNAs, which can be applied towards treating cancer in the form of gene therapy.Ph

Modulation of microRNA Functions by the 3’ Untranslated Regions of CD44 and Tumour Suppressor Candidate 2

Recently, the importance of non-coding regions of the genome, which were once presumed to be “junk” DNA, has been revealed. The non-coding 3’ untranslated region (3’UTR) plays a significant role in the regulation of microRNA (miRNA) functions. The 3’UTR is hypothesized to function in the feed-back regulation of miRNA functions, since it can bind and inactivate multiple miRNAs. In this study, the effects of the exogenous over-expression of two 3’UTRs, CD44 and Tumour Suppressor candidate 2 (TUSC2), on miRNA functions in breast cancer carcinogenesis were investigated. A series of cell function assays in human and mouse breast cancer cell lines, MT-1, MDA-MB-231 and 4T1 showed phenotypic changes caused by the 3’UTRs. A variety of cell function characteristics were affected, including cell proliferation, colony formation, cell survival, angiogenesis, tumour growth, cell migration, invasion and adhesion. These results were hypothesized to occur due to the interaction of the 3’UTRs with multiple miRNAs. The 3’UTRs were able to antagonize cytoplasmic miRNAs, as demonstrated by luciferase activities. In the case of the CD44 3’UTR, downstream target mRNAs, CDC42, Col1a1 and FN1 had an increased expression along with CD44. When the 3’UTR of TUSC2 was over-expressed, there was an increased translation of TUSC2 and the downstream targets, tissue inhibitor of metalloproteinases (TIMP) -2 and -3.The 3’UTRs of these target mRNAs can bind and target multiple miRNAs in common with the TUSC2 3’UTR, which were confirmed with luciferase activity assays and correlated with a series of siRNA and miRNA assays. During the study of the TUSC2 3’UTR, a potential pseudogene, TUSC2P, was discovered. Interestingly, TUSC2P mRNA expression was found to be decreased in cancer cells compared to normal cells and had similar cell functional characteristics as that of the TUSC2 3’UTR. These findings suggest that the over-expressed non-coding transcript can serve as a competitor for miRNA binding, which freed the potential targets of the miRNAs and led to an up-regulation of multiple protein levels. The non-coding transcript can thus be used as a functional miRNA inhibitor that is capable of modulating multiple miRNAs, which can be applied towards treating cancer in the form of gene therapy.Ph

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