MicroRNA-221 promotes papillary thyroid carcinoma cell migration and invasion via targeting RECK and regulating epithelial-mesenchymal transition.

AIM: The aim of this study was to detect the effects and potential mechanisms of microRNA-221 on a series of biological behaviors of papillary thyroid carcinoma (PTC) cells in vitro and in vivo. METHODS: First, we analyzed the relationship between the expression of miR-221 and several clinicopathological features of PTC patients and then detected the expression of the miR-221 in tumor tissues and cell lines. The effects of miR-221 on proliferation and invasion of PTC cells were verified by cell counting kit-8 (CCK-8) assay, wound healing assay and transwell assay. Western blot assay was applied to explore the correlation between miR-221 and RECK expression in PTC K1 cells. Finally, a xenograft model was established to further confirm the tumor-promoting effects of miR-221 in vivo. RESULTS: Our data indicated that miR-221 was relatively upregulated in metastatic PTC tissues. MiR-221 promoted the proliferation, migration and invasion activities of PTC K1 cells, following variations of epithelial-mesenchymal transition (EMT)-related protein expression. We identified RECK as a direct target of miR-221, revealed its expression to be inversely correlated with miR-221 in PTC samples and showed that its reintroduction reverses miR-221-induced PTC invasiveness. In addition, miR-221 was also verified to promote tumor growth and increase tumor volume and weight in vivo. Taken together, miR-221/RECK axis could be an effective way to regulate biological behaviors of PTC. CONCLUSION: MiR-221 may be involved in PTC cell invasion and metastasis by targeting RECK, indicating that the miR-221/RECK pathway could be studied further as a potential new diagnostic or prognostic biomarker for PTC.

Development of gene and stem cell therapy for ocular neurodegeneration.

Retinal degenerative diseases pose a serious threat to eye health, but there is currently no effective treatment available. Recent years have witnessed rapid development of several cutting-edge technologies, such as gene therapy, stem cell therapy, and tissue engineering. Due to the special features of ocular structure, some of these technologies have been translated into ophthalmological clinic practice with fruitful achievements, setting a good example for other fields. This paper reviews the development of the gene and stem cell therapies in ophthalmology.

Lycium barbarum polysaccharides promotes in vivo proliferation of adult rat retinal progenitor cells.

Lycium barbarum is a widely used Chinese herbal medicine prescription for protection of optic nerve. However, it remains unclear regarding the effects of Lycium barbarum polysaccharides, the main component of Lycium barbarum, on in vivo proliferation of adult ciliary body cells. In this study, adult rats were intragastrically administered low- and high-dose Lycium barbarum polysaccharides (1 and 10 mg/kg) for 35 days and those intragastrically administered phosphate buffered saline served as controls. The number of Ki-67-positive cells in rat ciliary body in the Lycium barbarum polysaccharides groups, in particular low-dose Lycium barbarum polysaccharides group, was significantly greater than that in the phosphate buffered saline group. Ki-67-positive rat ciliary body cells expressed nestin but they did not express glial fibrillary acidic protein. These findings suggest that Lycium barbarum polysaccharides can promote the proliferation of adult rat retinal progenitor cells and the proliferated cells present with neuronal phenotype.

[Screening of key genes and inflammatory signalling pathway involved in the pathogenesis of HLA-B27-associated acute anterior uveitis by gene expression microarray].

OBJECTIVE: To investigate the genes and signalling pathways located upstream of the inflammatory processes in human leukocyte antigen (HLA)-B27-associated acute anterior uveitis by gene expression microarray. METHODS: Experimental study. HLA-B27-positive and-negative monocytes isolated from human peripheral blood were stimulated with Vibrio cholera lipopolysaccharide (LPS). Gene expression microarrays were used to identify the differentially expressed genes. Differentially expressed (DE) genes were testified by real-time PCR and analyzed by a series of bioinformatics-based techniques such as Gene Ontology, Kyoto Encyclopedia of Genes and Genomes. RESULTS: Gene expression microarray analysis revealed marked differences between HLA-B27-positive acute anterior uveitis (AAU) and HLA-B27-negative healthy control peripheral monocytes in the genes that were upregulated in response to LPS stimulation with 1105 genes and 25 genes respectively. Gene Ontology enrichment and pathway analysis indicated that genes participating in protein transport and folding were essential to the inflammatory process. The LPS receptor-Toll-like receptor (TLR)4 induced TLR signalling pathway and pathway related to Vibrio cholerae infection were located upstream of the network and contribute to the overall response. Among the DE genes, PIK3CA, PIK3CB, AKT3, and MAPK1 might play critical roles in inflammation. CONCLUSIONS: Equivalent LPS stimulation induces a different response in HLA-B27-positive peripheral monocytes compared to normal control, suggesting that the TLR pathway is involved in the pathogenesis of HLA-B27-associated AAU.

Enhanced expression of proneurotrophins in elevated introcular pressure-induced rat retinal ischemia.

BACKGROUND: Proneurotrophins such as the precursor of nerve growth factor (proNGF) and the precursor of brain-derived neurotrophic factor (proBDNF) interacted with sortilin and p75(NTR) to form a complex capable of activating an apoptotic signaling. We found that the expression of p75(NTR) and sortilin was increased in ischemic retina induced by elevated intraocular pressure (IOP), but the protein expression changes of proNGF and proBDNF in the same situation were not clear. This study aimed to ascertain the protein expression changes of proNGF and proBDNF in ischemic retina induced by elevated IOP. METHODS: Expression of proBDNF and proNGF was examined by double-labeling immunochemistry in normal rat retina, examined using Western blotting and analyzed using statistical methods in ischemic retina induced by elevated IOP. RESULTS: Immunocytochemistry showed that the proBDNF expressed in the ganglion cell layer (GCL) while the proNGF primarily existed in both the nerve fiber layers (NFL) and large ganglion cell bodies of normal rat retina. Western blotting analysis demonstrated that the molecule weights of 28 kD (proBDNF)/25 kD (proNGF) band were increased significantly (P < 0.05) at days 3, 5 and 7 after retinal elevated-IOP-induced ischemia. CONCLUSION: ProBDNF expressed in the GCL and proNGF primarily presented in NFL and large ganglion cell bodies of normal rat retina, the protein expression forms of 28 kD proBDNF and 25 kD proNGF increased in ischemic retina induced by elevated IOP.

Metformin inhibits growth of hepatocellular carcinoma cells by inducing apoptosis via mitochondrion-mediated pathway.

Recently, population-based studies of type 2 diabetes patients have provided evidence that metformin treatment is associated with a reduced cancer incidence and mortality, but its mode of action remains unclear. Here we report effects of metformin on hepatocellular carcinoma (HCC) Hep-G2 cells and details of molecular mechanisms of metformin activity. Our research indicates that metformin displays anticancer activity against HCC through inhibition of the mTOR translational pathway in an AMPK-independent manner, leading to G1 arrest in the cell-cycle and subsequent cell apoptosis through the mitochondrion-dependent pathway. Furthermore, we showed that metformin strongly attenuated colony formation and dramatically inhibited Hep-G2 tumor growth in vivo. In conclusion, our studies suggested that metformin might have potential as a cytotoxic drug in the prevention and treatment of HCC.

Triptolide regulates T cell-mediated immunity via induction of CD11c(low) dendritic cell differentiation.

Triptolide(TPT) isolated from one of the Chinese herbs, Tripterygium wilfordii Hook. F. (TWHF), are known to have a variety of immunomodulatory activities. This study was performed to investigate the effect of TPT on the differentiation of splenic DCs and its influence on T cell-mediated immunity regarding to DC subsets CD11c(low)I-a/e(low)CD45RB(+)(CD11c(low) DCs) and CD11c(high)I-a/e(high)CD45RB(-) (CD11c(high) DCs) in male C57BL/6 mice spleens in vitro. The percentage of CD11c(low) DCs was significantly increased after treatment with TPT compared to their counterparts (CD11c(high) DCs). It was found that unlike the gradually decreasing interleukin (IL)-12 secretion of CD11c(high) DCs induced by TPT, CD11c(low) DCs showed a obvious dose-dependent response between the increasing of IL-10 production and TPT stimulation. After treatment with anti-IL-12R or anti-IL-10 monoclonal antibody in CD4(+) T cells+CD11c(high) DCs or CD11c(low) DCs mixed lymphocyte reaction, the induction of these DCs on T cells was inhibited dramatically. These data demonstrated that TPT might induce the differentiation of splenic DCs to CD11c(low) DCs followed by shifting of Th1 to Th2 with enhancement of T lymphocyte immune function in vitro.

Association between EGF, TGF-ß1 and TNF-α gene polymorphisms and hepatocellular carcinoma.

INTRODUCTION: Up to present, EGF 61*A/G, TGF-ß1 -509*T/C and TNF-α -308*A/G gene polymorphisms have been analysed in other cancer entities than hepatocellular carcinoma (HCC). We here investigated the frequency of these gene polymorphisms among HCC patients. MATERIALS AND METHODS: A total of 73 HCC patients and 117 cancer-free healthy people were recruited at the Surgical Department of Zhongshan Hospital. Genomic DNA was isolated from peripheral blood and gene polymorphisms were analyzed by PCR-RFLP. RESULTS: The distribution of EGF 61*G/G homozygotes among HCC patients was more frequent than that in the control group (24.7% vs 11.1%, OR=2.618, 95%CI=1.195-5.738). In parallel, the frequency of the "G" allele in the HCC patient group was also higher than that in the control group (45.9% vs 33.3%, OR= 1.696, 95%CI=1.110-2.592). No difference could be found for the TGF-ß1-509 and TNF-α -308 genotypes. CONCLUSION: EGF 61*G/G genotype and G allele are significantly increased among patients with HCC. TGF-ß1-509*T/C and TNF-α -308*A/G gene polymorphisms are not related to this cancer entity.

Gene expression profile changes caused by the dysfunction of Mer during retinal pigment epithelium phagocytosis.

BACKGROUND: Studies indicated that Mer might be the main contributor to the specific internalization of photoreceptor outer segments (POS) in retinal pigment epithelium (RPE). It is very important to understand the mechanism of POS phagocytosis under the pathway of Mer and its ligands. The objective of this study was to identify changes in gene expression profiles caused by Mer gene knockout (Mer-/-) during phagocytosis of POS in RPE. METHODS: RPE from both Mer-/- and wild-type (WT) mice were isolated and cultured to the 3rd passage. POS were subjected to culture medium with 20 nmol/L Gas6 and protein S to activate specific mer-mediated phagocytosis. RPE phagocytosis was evaluated by phagocytosis assays and differential gene expression identified by microarray at 3 and 12 hours; the 0-hour time point served as the control. Three independent samples for each Mer-/- or WT RPE were subjected to the same protocol of microarray. Five genes were confirmed by real-time quantitative PCR (QPCR). RESULTS: The Mer-/- RPE had less internalized POS than WT RPE after both 3 and 12 hours in phagocytosis assay. Compared to WT RPE and the 0-hour control, 38 and 45 different known genes were increased and 68 and 59 known genes were decreased in Mer-/- RPE after 3 and 12 hours, respectively. Abnormal POS phagocytosis in Mer-/- RPE was associated with significant gene expression changes in, for example, signal transduction (WNT, MAPK), phagocytosis (Vav3, Hsd11b1), cytoskeleton components (Myo7a), and metabolism, in a time-specific manner. QPCR results showed Vav3, Hsd11b1, Myo7a, Rtn2 and Itga8 in those independent samples were consistent with microarray. CONCLUSION: Gene expression profiles modulated in a time-specific manner in Mer-/- RPE indicate a possible internalization mechanism for abnormal POS phagocytosis, which gives insight into the mechanism of retinitis pigmentosa caused by the mutation of MerTK in humans.

The role of CCR1 expression in the retinal degeneration in rd mice.

PURPOSE: Chemokine receptors are reported to be involved in neuronal cell death and CNS neurodegenerative diseases. The aim of the current study was to investigate the expression of CCR1, a major chemokine receptor for CC chemokines in retinal dystrophy in rd (retinal degeneration) mice and further explore its role in photoreceptor degeneration. MATERIALS AND METHODS: The expression levels of CCR1 mRNA in the whole control and rd retinas at postnatal days (P) 8, 10, 12, 14, 16, and 18 were determined by RT-PCR assay. Location of CCR1 in the retina of rd mice at each age group was studied by immunohistochemical analysis. Expression of CCR1 in the photoreceptor cells and apoptotic cells was determined by double labeling. RESULTS: Expression of CCR1 mRNA was noted in both control and rd retinas at each age group. CCR1-positive cells started to emerge in the outer nuclear layer (ONL) in rd retinas at P8 and reached a peak at P12 and P14. Double labeling of CCR1 with rhodopsin, CD11b, or TUNEL staining showed expression of CCR1 in the photoreceptor cells, rather than in the microglial cells. Partial CCR1 expression was observed in some of the apoptotic photoreceptor cells. CONCLUSIONS: Expression of CCR1 in the photoreceptor cells was increased with the progress of retinal degeneration in rd mice. Activation of CCR1 may play a role in the photoreceptor apoptosis.

Expressions of Axl and Tyro-3 receptors are under regulation of nerve growth factor and are involved in differentiation of PC12 cells.

OBJECTIVE: Tyro-3 and Axl receptors are expressed in brain in a region-specific manner and their bioactivities in the central nervous system remain still elusive. The aim of the present study was to investigate their functions in neuronal differentiation. METHODS: PC12 cells overexpressing Tyro-3 or Axl were established by transfection with full-length CMV-Tyro-3-eCFP or CMV-Axl-eGFP plasmid, respectively. CMV-eGFP plasmid served as a control vector. After that, the fluorescence intensity and distributions of green fluorescent protein (GFP) and cyan fluorescent protein (CFP) in the cells with or without nerve growth factor (NGF) treatment were real-time monitored. RESULTS: Expressions of Tyro-3 and Axl receptors were under the regulation of NGF and associated with neuronal differentiation. This was not observed in CMV-eGFP-transfected PC12 cells. Besides, confocal microscopy revealed that NGF affected intracellular localization of full-length Axl-eGFP and Tyro-3-eCFP in PC12 cells. Moreover, the development of outgrowth of differentiated PC12 cells under stimulation of NGF was promoted by overexpression of Tyro-3 or Axl. CONCLUSION: Expressions of Tyro-3 and Axl receptors are under the regulation of NGF and are involved in NGF-induced neuronal differentiation of PC12 cells.

Prostaglandin induces the expression of matrix metalloproteinase-1 in ciliary melanocytes.

BACKGROUND: Latanoprost, a prostaglandin F2alpha analog, has been shown to be an effective intraocular pressure lowering agent which acts by inducing ciliary muscle cells to synthesise matrix metalloproteinases. However, the response of ciliary melanocytes to latanoprost has never been reported. This research has investigated the ability of latanoprost to induce matrix metalloproteinase-1 expression in human ciliary melanocytes, and thereby advance the understanding of the mechanism of PGF(2alpha) in decreasing intraocular pressure. METHODS: In vitro human ciliary melanocytes were treated for 48 hours with five different concentrations of latanoprost (100, 150, 200, 500, and 1000 nmol/L). Ciliary melanocytes treated with 0.01% ethanol (vehicle) were used as a control. The expression of matrix metalloproteinase-1 in ciliary melanocytes was determined by Western blotting and immunofluorescent staining. RESULTS: Western blotting showed that the expression of matrix metalloproteinase-1 in ciliary melanocytes was induced by latanoprost, and the level of expression was dependent on the concentration of latanoprost in the culture medium. Immunofluorescent staining showed that matrix metalloproteinase-1 was confined to the ciliary melanocyte cytoplasm. CONCLUSIONS: Latanoprost induced the expression of matrix metalloproteinase-1 in human ciliary melanocytes in a dose-dependent manner. Ciliary melanocytes, as well as ciliary muscle cells, may also play an important role in uveoscleral outflow modulation.

Loss of melanopsin-containing retinal ganglion cells in a rat glaucoma model.

BACKGROUND: Glaucoma can cause progressive damage to retinal ganglion cells. These cells can be classified as cells projecting to the superior colliculus and melanopsin-containing retinal ganglion cells, which project to the suprachiasmatic nucleus. This study was to investigate the effects of chronic intraocular pressure elevation on melanopsin-containing retinal ganglion cells in rats. METHODS: Chronic intraocular pressure elevation was induced in one eye of adult Wistar rats by cauterization of three episcleral veins. Intraocular pressure was measured at different intervals with a rebound tonometer. Superior collicular retinal ganglion cells were retrogradely labeled from the superior colliculus with Fluorogold. Melanopsin-containing retinal ganglion cells were visualized by free-floating immunohistochemistry on whole-mount retinas. The number of labeled superior collicular and melanopsin-containing retinal ganglion cells were counted in the sample areas on flat-mounted retinas. RESULTS: Compared with contralateral control eyes, the numbers of both superior collicular and melanopsin-containing retinal ganglion cells were significantly reduced after 12 weeks of experimental intraocular pressure elevation ((2317.41 +/- 29.96)/mm(2) vs (1815.82 +/- 24.25)/mm(2); (26.20 +/- 2.10)/mm(2) vs (20.62 +/- 1.52)/mm(2), respectively). The extent of cell loss of the two types of retinal ganglion cells was similar. However, no morphologic changes were found in melanopsin-containing retinal ganglion cells. CONCLUSION: Both melanopsin-containing and superior collicular retinal ganglion cells were damaged by chronic ocular hypertension, indicating that glaucomatous neural degeneration involves the non-image-forming visual pathway.

Effects of seawater immersion on the functions of mitochondria of myocardium and hepatocyte in hemorrhagic shock rats.

OBJECTIVE: To investigate the effects of seawater immersion on the function of myocardium and hepatocyte mitochondria in experimental hemorrhagic shock rats. METHODS: Twenty-four male Wistar rats were divided into three groups (n=8 in each group): control group, HSL group (hemorrhagic shock group on land) and HSS group (hemorrhagic shock group in seawater). The hemodynamic parameters, activities of H(+)-ATPase (adenosinetriphosphatase), succinate dehydrogenase (SDH) and Ca(2+)-Mg(2+)-ATPase, the calcium contents in myocardium and hepatocyte mitochondria were measured and the changes of proton translocation across the inner mitochondrial membrane were analyzed. RESULTS: The hemodynamic indexes and the activities of H+-ATPase, SDH, Ca(2+)-Mg(2+)-ATPase in HSS group were significantly lower than those in control group and HSL group (P<0.05). In HSS group the calcium levels in tissue and mitochondria of myocardium and hepatocyte were elevated significantly compared with control group and HSL group (P<0.05). There was no significant difference in proton translocation among three groups. CONCLUSIONS: This investigation demonstrates that seawater immersion can aggravate the conditions of hemorrhagic shock rats.