Scleral Micro-RNA Signatures in Adult and Fetal Eyes

Introduction

In human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses.

Methods

Scleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained (n=4 to 6, each group), and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions (n=7, each group). Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods.

Results

Human sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10¹¹). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 (1.5 to 4 fold changes, p<0.01). However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples.

Conclusion

For the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia.     

Rapid,Accurate, and Non-Invasive Measurement of Zebrafish Axial Length and Other Eye Dimensions Using SD-OCT Allows Longitudinal Analysis of Myopia and Emmetropization

Refractive errors in vision can be caused by aberrant axial length of the eye, irregular corneal shape, or lens abnormalities. Causes of eye length overgrowth include multiple genetic loci, and visual parameters. We evaluate zebrafish as a potential animal model for studies of the genetic, cellular, and signaling basis of emmetropization and myopia. Axial length and other eye dimensions of zebrafish were measured using spectral domain-optical coherence tomography (SD-OCT). We used ocular lens and body metrics to normalize and compare eye size and relative refractive error (difference between observed retinal radial length and controls) in wild-type and lrp2 zebrafish. Zebrafish were dark-reared to assess effects of visual deprivation on eye size. Two relative measurements, ocular axial length to body length and axial length to lens diameter, were found to accurately normalize comparisons of eye sizes between different sized fish (R² = 0.9548, R² = 0.9921). Ray-traced focal lengths of wild-type zebrafish lenses were equal to their retinal radii, while lrp2 eyes had longer retinal radii than focal lengths. Both genetic mutation (lrp2) and environmental manipulation (dark-rearing) caused elongated eye axes. lrp2 mutants had relative refractive errors of −0.327 compared to wild-types, and dark-reared wild-type fish had relative refractive errors of −0.132 compared to light-reared siblings. Therefore, zebrafish eye anatomy (axial length, lens radius, retinal radius) can be rapidly and accurately measured by SD-OCT, facilitating longitudinal studies of regulated eye growth and emmetropization. Specifically, genes homologous to human myopia candidates may be modified, inactivated or overexpressed in zebrafish, and myopia-sensitizing conditions used to probe gene-environment interactions. Our studies provide foundation for such investigations into genetic contributions that control eye size and impact refractive errors.     

Assessment of the Association of Matrix Metalloproteinases with Myopia,Refractive Error and Ocular Biometric Measures in an Australian Cohort

Extracellular matrix proteins have been implicated in protein remodelling of the sclera in refractive error. The matrix metalloproteinases (MMPs) falling into the collagenase (MMP1, MMP8, MMP13), gelatinase (MMP2, MMP9) and stromelysin (MMP3, MMP10, MMP11) functional groups are particularly important. We wished to assess their association with myopia, refractive error and ocular biometric measures in an Australian cohort. A total of 543 unrelated individuals of Caucasian ethnicity were genotyped including 269 myopes (≤−1.0D) and 274 controls (>−1.0D). Tag single nucleotide polymorphisms (SNPs) (n = 53) were chosen to encompass these eight MMPs. Association tests were performed using linear and logistic regression analysis with age and gender as covariates. Spherical equivalent, myopia, axial length, anterior chamber depth and corneal curvature were the phenotypes of interest. Initial findings indicated that the best p values for each trait were 0.02 for myopia at rs2274755 (MMP9), 0.02 for SE at both rs3740938 (MMP8) and rs131451 (MMP11), 0.01 for axial length at rs11225395 (MMP8), 0.01 for anterior chamber depth at rs498186 (MMP1) and 0.02 at rs10488 (MMP1). However, following correction for multiple testing, none of these SNPs remained statistically significant. Our data suggests that the MMPs in the collagenase, gelatinase and stromelysin categories do not appear to be associated with myopia, refractive error or ocular biometric measures in this cohort.     

Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2^n/n) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2^n/n mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia.     

BMP-2 Is Involved in Scleral Remodeling in Myopia Development

The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2) expression in the sclera of guinea pigs with lens-induced myopia (LIM) and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM) synthesis in human scleral fibroblasts (HSFs) cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups) by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1). Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG) and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia.     

Extracellular Matrix Protein Lumican Promotes Clearance and Resolution of Pseudomonas aeruginosa Keratitis in a Mouse Model

Lumican is an extracellular protein that associates with CD14 on the surface of macrophages and neutrophils, and promotes CD14-TLR4 mediated response to bacterial lipopolysaccharides (LPS). Lumican-deficient (Lum ^−/−) mice and macrophages are impaired in TLR4 signals; raising the possibility that lumican may regulate host response to live bacterial infections. In a recent study we showed that in vitro Lum ^−/− macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infection model the Lum ^−/− mice showed poor survival. The cornea is an immune privileged barrier tissue that relies primarily on innate immunity to protect against ocular infections. Lumican is a major component of the cornea, yet its role in counteracting live bacteria in the cornea remains poorly understood. Here we investigated Pseudomonas aeruginosa infections of the cornea in Lum ^−/− mice. By flow cytometry we found that 24 hours after infection macrophage and neutrophil counts were lower in the cornea of Lum ^−/− mice compared to wild types. Infected Lum ^−/− corneas showed lower levels of the leukocyte chemoattractant CXCL1 by 24–48 hours of infection, and increased bacterial counts up to 5 days after infection, compared to Lum^+/− mice. The pro-inflammatory cytokine TNF-α was comparably low 24 hours after infection, but significantly higher in the Lum ^−/− compared to Lum ^+/− infected corneas by 2–5 days after infection. Taken together, the results indicate that lumican facilitates development of an innate immune response at the earlier stages of infection and lumican deficiency leads to poor bacterial clearance and resolution of corneal inflammation at a later stage.     

A 5'-flanking region of embryonic-type myosin heavy chain gene,MYHM743-2, from torafugu Takifugu rubripes regulates developmental muscle-specific expression

The myosin heavy chain gene, MYH_M743-2, is highly expressed in fast muscle fibers of torafugu embryos. However, the regulatory mechanisms involved in its expression have been unclear. In this study, we examined spatio-temporal expression patterns of this gene during development by injecting expression vectors containing the GFP reporter gene fused to the 5'-flanking region of MYH_M743-2 into fertilized eggs of zebrafish and medaka. Although the -2.1 kb 5'-flanking region of torafugu MYH_M743-2 showed no homology with the corresponding regions of zebrafish and medaka orthologous genes on the rVISTA analysis, the torafugu 5'-flanking region activated the GFP expression which was detected in the myotomal compartment for both zebrafish and medaka embryos. The GFP expression was localized to fast and slow muscle fibers in larvae as revealed by immunohistochemical analysis. In addition to the above tissues, GFP was also expressed in jaw, eye and pectoral fin muscles in embryos and larvae. These results clearly demonstrated that the 2.1 kb 5'-flanking region of MYH_M743-2 contains essential cis-regulatory sequences for myogenesis that are conserved among torafugu, zebrafish and medaka.     

Extracellular Matrix Lumican Promotes Bacterial Phagocytosis,and Lum?/? Mice Show Increased Pseudomonas aeruginosa Lung Infection Severity

Phagocytosis is central to bacterial clearance, but the exact mechanism is incompletely understood. Here, we show a novel and critical role for lumican, the connective tissue extracellular matrix small leucine-rich repeat proteoglycan, in CD14-mediated bacterial phagocytosis. In Psuedomonas aeruginosa lung infections, lumican-deficient (Lum^−/−) mice failed to clear the bacterium from lungs, tissues, and showed a dramatic increase in mortality. In vitro, phagocytosis of nonopsonized Gram-negative Escherichia coli and P. aeruginosa was inhibited in Lum^−/− peritoneal macrophages (MΦs). Lumican co-localized with CD14, CD18, and bacteria on Lum^+/+ MΦ surfaces. Using two different P. aeruginosa strains that require host CD14 (808) or CD18/CR3 (P1) for phagocytosis, we showed that lumican has a larger role in CD14-mediated phagocytosis. Recombinant lumican (rLum) restored phagocytosis in Lum^−/− MΦs. Surface plasmon resonance showed specific binding of rLum to CD14 (K_A = 2.15 × 10⁶ m⁻¹), whereas rLumY20A, and not rLumY21A, where a tyrosine in each was replaced with an alanine, showed 60-fold decreased binding. The rLumY20A variant also failed to restore phagocytosis in Lum^−/− MΦs, indicating Tyr-20 to be functionally important. Thus, in addition to a structural role in connective tissues, lumican has a major protective role in Gram-negative bacterial infections, a novel function for small leucine-rich repeat proteoglycans.     

Immunostimulatory properties of phosphorothioate CpG DNA containing both 3'-5'- and 2'-5'-internucleotide linkages

Synthetic oligodeoxyribonucleotides containing CpG-dinucleotides (CpG DNA) in specific sequence contexts activate the vertebrate immune system. We have examined the effect of 3′-deoxy-2′–5′-ribonucleoside (3′-deoxynucleoside) incorporation into CpG DNA on the immunostimulatory activity. Incorporation of 3′-deoxynucleosides results in the formation of 2′–5′-internucleotide linkages in an otherwise 3′–5′-linked CpG DNA. In studies, both in vitro and in vivo, CpG DNA containing unnatural 3′-deoxynucleoside either within the CpG-dinucleotide or adjacent to the CpG-dinucleotide failed to induce immunostimulatory activity, suggesting that the modification was not recognized by the receptors. Incorporation of the same modification distal to the CpG-dinucleotide in the 5′-flanking sequence potentiated the immunostimulatory activity of the CpG DNA. The same modification when incorporated in the 3′-flanking sequence had an insignificant effect on immunostimulatory activity of CpG DNA. Interestingly, substitution of a 3′-deoxynucleoside in the 5′-flanking sequence distal to the CpG-dinucleotide resulted in increased IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. The incorporation of the same modification in the 3′-flanking sequence resulted in lower IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. These results suggest that site-specific incorporation of 3′-deoxynucleotides in CpG DNA modulates immunostimulatory properties.     

Heparan Sulfate 2-O-Sulfotransferase Is Required for Triglyceride-rich Lipoprotein Clearance

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153–164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st^f/f) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1^f/f) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st^f/fAlbCre⁺ mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1^f/fAlbCre⁺ mice. In contrast, Hs6st1^f/fAlbCre⁺ mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.     

Scleral Cross-Linking Using Riboflavin UVA Irradiation for the Prevention of Myopia Progression in a Guinea Pig Model: Blocked Axial Extension and Altered Scleral Microstructure

PurposeTo develop methods of collagen cross-linking (CXL) in the sclera for the treatment of progressive myopia and to investigate the biomechanical and histological changes that occur in as a result.MethodsTwenty 14-day-old guinea pigs were divided into 3 groups: the cross-linking group (CL, n = 8), non cross-linking group (NCL, n = 8), and control group (n = 4). The scleras of the right eyes of the guinea pigs in the CL group were surgically exposed and riboflavin was dropped onto the irradiation zone for 20 seconds prior to ultraviolet-A (UVA) irradiation. The same procedure was conducted on the NCL group but without UVA irradiation. No procedure was conducted on the control group. The right eyes of the guinea pigs in the CL and NCL groups were then fitted with -10.00DS optics for six weeks. Retinoscopy and the axial lengths (AXL) were measured at baseline, and at the second, fourth and sixth weeks post-treatment in all three groups. All animal subjects were euthanized after the sixth week and then biomechanical and histopathological examinations of the scleras were conducted.ResultsThe mean AXL of the NCL group was longer than both the control and CL groups at six weeks (P = 0.001). The mean refractive error in the NCL group was statistically significantly more negative than both the control and the CL groups at six weeks (P = 0.001). The scleral collagen fiber arrangements of the CL and control groups were denser and more regularly distributed than the NCL group. Ultimate stress of the sclera was lowest in the NCL group, followed by the CL then the control group (P<0.05). Ultimate strain (%) of the sclera was lowest in the CL group followed by the NCL and then the control group (P<0.05).ConclusionOur study demonstrates that scleral CXL using riboflavin UVA irradiation effectively prevents the progression of myopia by increasing scleral biomechanical strength in a guinea pig model.     

The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in Phanerochaete chrysosporium

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3′ untranslated region. In pUGGM3′ and pUMGM3′, the promoters were fused directly with egfp, whereas in pUGiGM3′ and pUMiGM3′, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5′ end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of the egfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5′ intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.     

Glaucomatous-Type Optic Discs in High Myopia

Purpose

To assess the prevalence of glaucoma in patients with high myopia defined as myopic refractive error of >-8 diopters or axial length ≥26.5 mm.

Methods

The hospital-based observational study included 172 patients (336 eyes) with a mean age of 61.9±12.3 years and mean axial length of 30.1±2.3 mm (range: 24.7–39.1mm). Glaucomatous-type optic discs were defined by glaucomatous optic disc appearance. Glaucoma was defined by glaucomatous optic disc appearance and glaucomatous Goldmann visual field defects not corresponding with myopic macular changes.

Results

Larger disc area (mean: 3.18±1.94 mm²) was associated with longer axial length (P<0.001; standardized correlation coefficient: 0.45). Glaucoma was detected in 94 (28%; 95% Confidence intervals: 23%, 33%) eyes. In multivariate analysis, glaucoma prevalence was 3.2 times higher (P<0.001) in megalodiscs (>3.79 mm²) than in normal-sized discs or small discs (<1.51 mm²) after adjusting for older age. Axial length was not significantly (P = 0.38) associated with glaucoma prevalence in that model. Glaucoma prevalence increased by a factor of 1.39 for each increase in optic disc area by one mm². Again, axial length was not significantly (P = 0.38) associated with glaucoma prevalence when added to this multivariate model.

Conclusion

Within highly myopic individuals, glaucoma prevalence increased with larger optic disc size beyond a disc area of 3.8 mm². Highly myopic megalodiscs as compared to normal sized discs or small discs had a 3.2 times higher risk for glaucomatous optic nerve neuropathy. The increased glaucoma prevalence in axial high myopia was primarily associated with axial myopia associated disc enlargement and not with axial elongation itself.     

Genetic variants on chromosome 1q41 influence ocular axial length and high myopia

As one of the leading causes of visual impairment and blindness, myopia poses a significant public health burden in Asia. The primary determinant of myopia is an elongated ocular axial length (AL). Here we report a meta-analysis of three genome-wide association studies on AL conducted in 1,860 Chinese adults, 929 Chinese children, and 2,155 Malay adults. We identified a genetic locus on chromosome 1q41 harboring the zinc-finger 11B pseudogene ZC3H11B showing genome-wide significant association with AL variation (rs4373767, β = −0.16 mm per minor allele, P_meta = 2.69×10⁻¹⁰). The minor C allele of rs4373767 was also observed to significantly associate with decreased susceptibility to high myopia (per-allele odds ratio (OR) = 0.75, 95% CI: 0.68–0.84, P_meta = 4.38×10⁻⁷) in 1,118 highly myopic cases and 5,433 controls. ZC3H11B and two neighboring genes SLC30A10 and LYPLAL1 were expressed in the human neural retina, retinal pigment epithelium, and sclera. In an experimental myopia mouse model, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for the murine genes ZC3H11A, SLC30A10, and LYPLAL1. This supports the likely role of genetic variants at chromosome 1q41 in influencing AL variation and high myopia.     

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

Background

During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3′UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression.

Methodology/Principal Findings

Using a GFP reporter mRNA that was fused with tdrd7 3′UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3′UTR of dazl mRNA, another germline mRNA targeted by miR-430.

Conclusions/Significance

Our present study indicated that DAZL acts as an “anti-miRNA factor” during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.     

Targeting of Integrin ��1 and Kinesin 2�� by MicroRNA 183

MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H₂O₂. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells.     

The TSG-6/HC2-mediated Transfer Is a Dynamic Process Shuffling Heavy Chains between Glycosaminoglycans

The heavy chain (HC) subunits of the bikunin proteins are covalently attached to a single chondroitin sulfate (CS) chain originating from bikunin and can be transferred to different hyaluronan (HA) molecules by TSG-6/HC2. In the present study, we demonstrate that HCs transferred to HA may function as HC donors in subsequent transfer reactions, and we show that the CS of bikunin may serve as an HC acceptor, analogous to HA. Our data suggest that TSG-6/HC2 link HCs randomly on the CS chain of bikunin, in contrast to the ordered attachment observed during the biosynthesis. Moreover, the results show that the transfer activity is indifferent to the new HC position, and the relocated HCs are thus prone to further TSG-6/HC2-induced transfer reactions. The data suggest that HCs may be transferred directly from HA to HA without the involvement of the bikunin CS chain. The results demonstrate reversibility of the interactions between HCs and glycosaminoglycans and suggest that a dynamic shuffling of the HCs occur in vivo.