Jim W. Xuan

MicroRNA MiR-17 retards tissue growth and represses fibronectin expression

MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-17∼92 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We anticipate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.

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

Summary 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.

Versican 3′-untranslated region (3′-UTR) functions as a ceRNA in inducing the development of hepatocellular carcinoma by regulating miRNA activity

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.—Fang, L., Du, W. W., Yang, X., Chen, K., Ghanekar, A., Levy, G., Yang, W., Yee, A. J., Lu, W.‐Y., Xuan, J. W., Gao, Z., Xie, F., He, C., Deng, Z., Yang, B. B. Versican 3′‐untranslated region (3′‐UTR) functions as a ceRNA in inducing the development of hepatocellular carcinoma by regulating miRNA activity. FASEB J. 27, 907–919 (2013). www.fasebj.org

Fibroblast growth factor receptor 2 tyrosine kinase is required for prostatic morphogenesis and the acquisition of strict androgen dependency for adult tissue homeostasis

The fibroblast growth factor (FGF) family consists of 22 members and regulates a broad spectrum of biological activities by activating diverse isotypes of FGF receptor tyrosine kinases (FGFRs). Among the FGFs, FGF7 and FGF10 have been implicated in the regulation of prostate development and prostate tissue homeostasis by signaling through the FGFR2 isoform. Using conditional gene ablation with the Cre-LoxP system in mice, we demonstrate a tissue-specific requirement for FGFR2 in urogenital epithelial cells - the precursors of prostatic epithelial cells - for prostatic branching morphogenesis and prostatic growth. Most Fgfr2 conditional null (Fgfr2cn) embryos developed only two dorsal prostatic (dp) and two lateral prostatic (lp) lobes. This contrasts to wild-type prostate, which has two anterior prostatic (ap), two dp, two lp and two ventral prostatic (vp) lobes. Unlike wild-type prostates, which are composed of well developed epithelial ductal networks, the Fgfr2cn prostates, despite retaining a compartmented tissue structure, exhibited a primitive epithelial architecture. Moreover, although Fgfr2cn prostates continued to produce secretory proteins in an androgen-dependent manner, they responded poorly to androgen with respect to tissue homeostasis. The results demonstrate that FGFR2 is important for prostate organogenesis and for the prostate to develop into a strictly androgen-dependent organ with respect to tissue homeostasis but not to the secretory function, implying that androgens may regulate tissue homeostasis and tissue function differently. Therefore, Fgfr2cn prostates provide a useful animal model for scrutinizing molecular mechanisms by which androgens regulate prostate growth, homeostasis and function, and may yield clues as to how advanced-tumor prostate cells escape strict androgen regulations.

A New Three-Dimensional Ultrasound Microimaging Technology for Preclinical Studies Using a Transgenic Prostate Cancer Mouse Model

Prostate cancer is the most common cancer in adult men in North America. Preclinical studies of prostate cancer employ genetically engineered mouse models, because prostate cancer does not occur naturally in rodents. Widespread application of these models has been limited because autopsy was the only reliable method to evaluate treatment efficacy in longitudinal studies. This article reports the first use of three-dimensional ultrasound microimaging for measuring tumor progression in a genetically engineered mouse model, the 94-amino acid prostate secretory protein gene-directed transgenic prostate cancer model. Qualitative comparisons of three-dimensional ultrasound images with serial histology sections of prostate tumors show the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. Ultrasound imaging identified tumors ranging from 2.4 to 14 mm maximum diameter. The correlation coefficient of tumor diameter measurements done in vivo with three-dimensional ultrasound and at autopsy was 0.998. Prospective tumor detection sensitivity and specificity were both >90% when diagnoses were based on repeated ultrasound examinations done on separate days. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated volume doubling times of 5 and 13 days for two prostate tumors. Compared with other microimaging and molecular imaging modalities, the application of three-dimensional ultrasound imaging to prostate cancer in mice showed advantages, such as high spatial resolution and contrast in soft tissue, fast and uncomplicated protocols, and portable and economical equipment that will likely enable ultrasound to become a new microimaging modality for mouse preclinical trial studies.

Identification, purification and characterization of a novel human blood protein with binding affinity for prostate secretory protein of 94 amino acids

PSP94 (prostate secretory protein of 94 amino acids), an abundant protein within semen, has reported local functions within the reproductive tract and reported systemic functions. Mechanisms of action remain poorly understood, but binding to undefined molecules within the prostate, pituitary, testis and blood may initiate some of these actions. PSP94 serum measurements, especially of bound and free forms, have potential clinical utility in prostate cancer management. Identification of the binding molecules will help in the understanding of PSP94s action, and enable further development of PSP94 serum assays. PSPBP (PSP94-binding protein) was purified from human serum by ammonium sulphate fractionation, ion-exchange and affinity chromatography. The glycosylated protein ran as two bands on SDS/PAGE (70 and 95 kDa). N-terminal sequencing yielded a 30-amino-acid sequence, identical with the translated N-terminal region of a previously published cDNA (GenBank accession number AX136261). Reverse transcriptase PCR and plaque hybridization demonstrated PSPBP mRNA in peripheral blood leucocytes and in a prostate cDNA library. Northern blotting showed 2 kb mRNA species in prostate, testis, ovary and intestine. Immunohistochemistry demonstrated PSPBP in tissues, including pituitary and Leydig cells, supporting a role for PSP94 in hormonal control at the pituitary gonadal axis. ELISA demonstrated that PSPBP levels were significantly lower (P=0.0014) in the serum of a prostate cancer population (n=65) compared with a control population (n=70). PSPBP identification will help the understanding of PSP94s functions and facilitate ELISA development to address the clinical value of PSP94 serum assays.

cDNA, genomic cloning, and gene expression analysis of mouse PSP94 (prostate secretory protein of 94 amino acids).

The potential use of prostate secretory protein of 94 amino acids (PSP94) as a diagnostic biomarker or a therapeutic agent for prostate cancer has been reported. In order to establish an animal model to further elucidate on its biological role, we cloned the mouse PSP94 cDNA (approximately 500 bp) by reverse transcriptase-polymerase chain reaction (RT-PCR) and disclosed its genomic structure. The whole mouse PSP94 gene (approximately 23 kb) was amplified by long and accurate-PCR and also cloned by screening of a mouse embryo stem-cell genomic library. Computational and statistical analyses have demonstrated several highly conserved characteristics of PSP94 among different species. Comparison of PSP94 from human, two primates, pig, and rodents revealed that the most significant feature is that PSP94 is rich in cysteines (10% of the total sequence) and their positions are highly conserved. The three intron-four exon structure of the human PSP94 gene and the consensus sequence (....GT-intron-AG...) for mRNA splicing are also strongly conserved. A high divergence in cDNA sequence in the protein-coding region and also in the genomic sequence of PSP94 was also observed among these species. Comparing with alpha-globin, a typical evolutionally conserved gene, with the PSP94 gene, the rate of nonsynonymous changes per site per year (kN) is 2 to 6 times higher, indicating that PSP94 gene has been under far fewer evolutionary constraints than other genes and has a potential role as a species barrier in reproductive biology. In order to test this hypothesis, we investigated the gene expression of PSP94 and its tissue distribution in various rodent tissues by RT-PCR and in situ hybridization (ISH). Gene expression was found only in the prostate, suggesting that PSP94 is probably more tissue specific in the prostate of rodents than in mammals. The ISH analysis also revealed a prostate lobe-specific expression of the PSP94 gene in both mice and rats. It was strongly expressed in the lateral prostate, but the findings were negative in the dorsal and ventral lobe. Therefore, it is hypothesized that one of the primary functions of rodent PSP94, as a major prostate secretory protein, is related to reproductive biology.

Osteopontin inhibits inducible nitric oxide synthase activity in rat vascular tissue

We tested the hypothesis that osteopontin (OPN) can inhibit the induction of inducible nitric oxide synthase (iNOS) in vascular tissue. iNOS activity was induced in rat thoracic aortas by incubation of the tissue with lipopolysaccharide (LPS) and measured by conversion of L-[3H]arginine to L-[3H]citrulline. Addition of >/=1 nM recombinant OPN protein significantly reduced the LPS-induced increase in iNOS activity. Western blotting and the RT-PCR were used to determine the effect of LPS with and without OPN on tissue levels of iNOS protein and RNA, respectively. LPS resulted in an increase in iNOS protein and RNA, whereas OPN dose-dependently reduced tissue levels of iNOS activity, protein, and RNA. Mutated OPN proteins, in which the integrin-binding RGD amino acid sequence was deleted or mutated to RGE, resulted in complete and partial loss, respectively, of the ability of OPN to inhibit LPS-induced iNOS activity, implicating integrin binding in the effect. These results indicate that OPN can prevent induction of iNOS in vascular tissue.

Role of epithelial cell fibroblast growth factor receptor substrate 2α in prostate development, regeneration and tumorigenesis

The fibroblast growth factor (FGF) regulates a broad spectrum of biological activities by activation of transmembrane FGF receptor (FGFR) tyrosine kinases and their coupled intracellular signaling pathways. FGF receptor substrate 2α (FRS2α) is an FGFR interactive adaptor protein that links multiple signaling pathways to the activated FGFR kinase. We previously showed that FGFR2 in the prostate epithelium is important for branching morphogenesis and for the acquisition of the androgen responsiveness. Here we show in mice that FRS2α is uniformly expressed in the epithelial cells of developing prostates, whereas it is expressed only in basal cells of the mature prostate epithelium. However, expression of FRS2α was apparent in luminal epithelial cells of regenerating prostates and prostate tumors. To investigate FRS2α function in the prostate, the Frs2α alleles were ablated specifically in the prostatic epithelial precursor cells during prostate development. Similar to the ablation of Fgfr2, ablation of Frs2α disrupted MAP kinase activation, impaired prostatic ductal branching morphogenesis and compromised cell proliferation. Unlike the Fgfr2 ablation, disrupting Frs2α had no effect on the response of the prostate to androgens. More importantly, ablation of Frs2α inhibited prostatic tumorigenesis induced by oncogenic viral proteins. The results suggest that FRS2α-mediated signals in prostate epithelial cells promote branching morphogenesis and proliferation, and that aberrant activation of FRS2-linked pathways might promote tumorigenesis. Thus, the prostate-specific Frs2αcn mice provide a useful animal model for scrutinizing the molecular mechanisms underlying prostatic development and tumorigenesis.

Prognostic significance of β-microseminoprotein mRNA expression in prostate cancer

Human β‐microseminoprotein (β‐MSP or PSP94) is a small protein secreted by prostatic epithelial cells. We recently reported the presence of low levels of β‐MSP mRNA expression and protein in most prostate cancer tissues.