Hui Ju Lin

Genome-wide association study of diabetic retinopathy in a Taiwanese population.

PURPOSE Diabetic retinopathy (DR) is a microvascular complication of diabetes with a complex multifactorial pathogenesis. The aim of this study was to identify the susceptibility genes that increase the risk of DR in type 2 diabetes (T2D) and to further elucidate the underlying mechanism of DR pathogenesis. DESIGN A case-control study. PARTICIPANTS We included 749 unrelated individuals with T2D (174 with DR and 575 without DR) and 100 nondiabetic controls. METHODS We conducted a genome-wide association study using Illumina HumanHap550-Duo BeadChips. MAIN OUTCOME MEASURES Compared with the genotypic distribution of single nucleotide polymorphisms (SNPs) between subjects with DR and without DR. RESULTS Using statistical models, we selected a total of 12 SNPs with P-values <1 × 10(-6) that were associated with DR. After controlling for diabetes duration and hemoglobin A(1C), 9 of the 12 SNPs located on 5 chromosomal regions were found to be associated with DR. Five loci not previously associated with DR susceptibility were identified in and around the following genes: MYSM1 (Myb-like, SWIRM, and MPN domains 1) located on chromosome 1p (odds ratio [OR], 1.50; 95% confidence interval [CI], 1.03-2.20); PLXDC2 (plexin domain-containing 2) located on the chromosome 10p (OR, 1.67; 95% CI, 1.06-2.65); ARHGAP22 (Rho GTPase-activating protein 22) located on chromosome 10q (OR, 1.65; 95% CI, 1.05-2.60); and HS6ST3 (heparan sulfate 6-O-sulfotransferase 3) located on chromosome 13q (OR, 2.33; 95% CI, 1.13-4.77). The SNPs rs13163610 and rs17376456 located in the unknown gene on chromosome 5q were also associated with DR (OR, 3.63; 95% CI, 1.38-9.58). CONCLUSIONS We identified a genetic association for susceptibility to DR in 5 novel chromosomal regions and PLXDC2 and ARHGAP22, the latter 2 of which are genes implicated in endothelial cell angiogenesis and increased capillary permeability. These findings suggest unsuspected pathways in the pathogenesis of DR.

Association of tumour necrosis factor alpha -308 gene polymorphism with primary open-angle glaucoma in Chinese.

AbstractPurpose Genetic factors are known to play a role in the aetiology of glaucoma, and in particular the role of the immune system is highly suspected. In this study, we evaluated the association between tumour necrosis factor alpha −308 (TNF α−308) and primary open-angle glaucoma (POAG).Methods A total of sixty POAG patients and 103 healthy volunteers as control group were enrolled in this case-controlled study. Furthermore, we used polymerase chain reaction based analysis to resolve the TNF α−308 polymorphism. Statistical analysis for the relative risk of TNF α−308 polymorphism was compared by the χ2 test.Results There were significant differences in the distribution of the polymorphism between the POAG patients and the control subjects (P=0.00016; P<0.05) and it was found that the A−308 allele occurred more frequently in POAG patients (odds ratio: 2.72; 95% confidence interval: 1.66–4.45).Conclusion The results of our study concluded that the distribution of TNF α−308 was significantly higher in the POAG patients than in the control group. Therefore, the A−308 allele appears to be associated with POAG and, therefore, could be used as a genetic marker for disease mapping. POAG is a complex disease, and a single gene could not be responsible. Understanding the role of genetic polymorphisms, like TNF α, could be a prediction of the disease and useful for developing new treatments for POAG.

Distributions of p53 codon 72 polymorphism in primary open angle glaucoma

Background: Glaucomatous neuropathy is a type of cell death by apoptosis. The p53 gene is one of the regulatory genes of apoptosis. Recently, p53 codon 72 polymorphism has been extensively studied to determine the risk factors responsible for many diseases. In the p53 gene, a single base change from G to C causes the alternation of amino acid residue 72 from arginine to proline. In this study the association between p53 codon 72 polymorphism and primary open angle glaucoma (POAG) patients was evaluated. Methods: 58 POAG patients and 59 healthy volunteers were enrolled in this study. Polymerase chain reaction based analysis was used to resolve the p53 codon 72 polymorphism. Results: There were significant differences in the distribution of the polymorphism between the control subjects and the POAG patients (p = 0.00782) The proline form of p53 gene codon 72 appears to be a significant risk factor in the development of POAG (odds ratio 2.389, 95% confidence interval: 1.14 to 5.01). Conclusions: Retinal ganglion cells die during POAG by apoptosis. The tumour suppressor protein, p53, is one of the primary regulators steps of apoptosis, and the results of our study are compatible with this concept.

Emodin Has Cytotoxic and Protective Effects in Rat C6 Glioma Cells: Roles of Mdr1a and Nuclear Factor κB in Cell Survival

1,3,8-Trihydroxy-6-methylanthaquinone (emodin) is recognized as an antiproliferative compound. In the present study, however, we show that emodin has both toxic and survival effects in glioma cells and that the survival effects involve Mdr1a. Emodin inhibited the proliferation and induced apoptosis of C6 cells in a 12-h treatment, but C6 cells survived a 72-h drug treatment, indicating resistance to emodin. Emodin-induced apoptosis was reduced by inhibition of the expression and activation of apoptosis-associated proteins including p53, Bax, Bcl-2, Fas, and caspase-3. C6 cells could express antioxidant proteins (superoxide dismutase and catalase) to decrease reactive oxygen species-induced cytotoxicity of emodin and overexpress multidrug resistance genes (Mdr1a, MRP2, MRP3, and MRP6) to decrease the intracellular accumulation of emodin. Electrophoretic mobility shift analysis showed that emodin decreased nuclear factor κB (NF-κB) expression in 24 h of treatment, but in 48 h, emodin increased NF-κB activity. A confocal microscope showed that emodin induced NF-κB translocation from cytoplasm to nuclei. C6 cells would activate the mitogen-activated protein kinase survival pathway and express the DNA repair gene (MGMT) and associated proteins (PARP and XRCC1) to recover the cell activity. C6 cells also expressed GRP78 to decrease emodin-induced endoplasmic reticulum (ER) stress that would cause apoptosis in C6 cells, and GRP78 inhibited the expression of GADD153 to enhance the expression of Bcl-2 that could balance the ER- and mitochondria-induced apoptosis of C6 cells.

A PAX6 gene polymorphism is associated with genetic predisposition to extreme myopia

PurposeThe PAX6 gene is a homeobox gene involved in oculogenesis, ocular growth, and form-deprivation myopia. Our aim was to determine whether PAX6polymorphism at position −12 of intron 9 (IVS9-12C to T, rs667773) is associated with high myopia in Chinese Taiwanese.MethodsThis case–control study compared a study group (n=188) with high myopia whose spherical equivalent was greater than −6.0 D with a control group (n=85) whose spherical equivalent was less than −0.5 D. Genotyping of IVS9-12C to T was conducted by restriction fragment length polymorphism analysis, and results were compared for the two groups.ResultsNo significant difference in genotype and allelic frequency at this position between the study and control groups was detected. However, there was a significantly higher frequency of the CC genotype in extremely myopic (greater than −10 D) patients (P<0.001, odds ratio (OR=5.265), confidence interval (CI=2.0342–13.626)). Furthermore, there was a higher frequency of the C allele in the extreme myopia group than in the control group (P=0.002, OR=3.73, CI=1.57–8.81).ConclusionsThe elevated frequency of the CC genotype within the extreme myopia group indicated that the CC genotype could act as a genetic marker, identifying patients predisposed to develop extreme myopia. Varied expression of this genotype may contribute to the genetic predisposition to high myopia in Chinese Taiwanese.

Baicalein-Induced Apoptosis via Endoplasmic Reticulum Stress Through Elevations of Reactive Oxygen Species and Mitochondria Dependent Pathway in Mouse-Rat Hybrid Retina Ganglion Cells (N18)

Studies were designed to investigate the effects of baicalein on mouse–rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, reactive oxygen species (ROS), cytoplasmic Ca2+, mitochondrial membrane potential (MMP), apoptosis induction, and caspases-3 activity were examined by flow cytometric assay. Apoptosis-associated proteins such as p53, Bax, Bcl-2, cytochrome c, and caspase-3 were examined by Western blot. We demonstrated the increase in the levels of p53, Bax, and cytochrome c and decrease in the level of Bcl-2, which are associated with the induction of apoptotic cell death after 24 h treatment with baicalein in N18 cells. Baicalein induced an increase in the cytoplasmic levels of ROS and Ca2+ in 1 h and reached their peak at 3 h, and thereafter a loss of MMP by flow cytometry. We also demonstrated a release of the cytochrome c from mitochondria into cytosol and an activation of caspase-3, which led to the occurrence of apoptosis in N18 cells treated with baicalein by Western blot. Pretreatment was conducted with BAPTA (intracellular calcium chelator) in baicalein-treated cells, the decline of MMP was recovered, and the increase in the level of cytoplasmic Ca2+ was suppressed, and the proportion of apoptosis was also markedly diminished. In conclusion, our data suggests that oxidative stress and cellular Ca2+ modulates the baicalein-induced cell death via a Ca2+-dependent mitochondrial death pathway in N18 cells.

Involvement of Matrix Metalloproteinases on the Inhibition of Cells Invasion and Migration by Emodin in Human Neuroblastoma SH-SY5Y Cells

Emodin (1,3,8-trihydroxy-6-methylanthaquinone), an active component present in the root and rhizome of Rheum palmatum L. (Polygonaceae) has anti-bacterial, anti-tumor, diuretic and vasorelaxant effects. However, its mechanism of action on the cell migration and invasion of human neuroblastoma cancer SH-SY5Y cells is not fully understood. In this study, firstly, the effects of emodin on the percentage of viable cells were examined by using MTT assay and it was found that emodin induced dose-and time-dependent inhibition in human neuroblastoma SH-SY5Y cells. Second, the effects of emodin on the migration and invasion of SH-SY5Y cells were examined by using wound assay and matrigel counting and the results showed that emodin suppressed the migration and invasion of SH-SY5Y cells. Third, we examined the effect of emodin on the levels of associated proteins by using Western blotting and the results indicated that emodin inhibited the levels of GRB2, RhoA, HIF-1α, VEGF, FAK, iNOS, COX2, p-p38, p-c-jun, MMP2, MMP9 and MMP7 but promoted the levels of PKC, PI3K, MEKK3 and NF-κB p65 that led to the inhibition of migration and invasion of SH-SY5Y cells in vitro.

Association of the lumican gene functional 3'-UTR polymorphism with high myopia.

PURPOSE The lumican gene (LUM) encodes a major extracellular component of the fibrous mammalian sclera. Alteration in the expression levels of extracellular matrix components may influence scleral shape, which in turn could affect visual acuity. Single-nucleotide polymorphisms (SNPs) in the LUM gene were determined in an investigation of whether LUM gene polymorphisms correlate with high myopia. METHODS Sequences spanning all three exons, intron-exon boundaries, and promoter regions were determined in 50 normal individuals. Five SNPs were identified, one of which was found to be a newly identified polymorphism. Genomic DNA was prepared from peripheral blood obtained from 201 patients with high myopia and 86 control subjects. Genotypes of the SNPs -1554 T/C (rs3759223), -628 A/-(rs17018757), -59 CC/-(rs3832846), c.601 T/C (rs17853500), and the novel SNP c.1567 C>T were determined by polymerase chain reaction. RESULTS Of the five SNPs, one showed a significant difference between patients and control subjects (c.1567 C>T, P = 0.0016). Haplotype analysis revealed a significantly higher presence of polymorphisms in patients with myopia (P < 0.0001). Moreover, the c.1567 T polymorphism was determined to have lower reporter gene activity than that of c.1567 C. CONCLUSIONS These observations suggest that LUM gene polymorphisms contribute to the development of high myopia.

Flavones inhibit breast cancer proliferation through the Akt/FOXO3a signaling pathway

BackgroundFlavones found in plants display various biological activities, including anti-allergic, anti-viral, anti-inflammatory, anti-oxidation, and anti-tumor effects. In this study, we investigated the anti-tumor effects of flavone, apigenin and luteolin on human breast cancer cells.MethodsThe anti-cancer activity of flavone, apigenin and luteolin was investigated using the MTS assay. Apoptosis was analyzed by Hoechst 33342 staining, flow cytometry and western blot. Cell migration was determined using the culture inserts and xCELLigence real-time cell analyzer instrument equipped with a CIM-plate 16. Real-time quantitative PCR and western blot were used to determine the signaling pathway elicited by flavone, apigenin and luteolin.ResultsFlavone, apigenin and luteolin showed potent inhibitory effects on the proliferation of Hs578T, MDA-MB-231 and MCF-7 breast cancer cells in a concentration and time-dependent manner. The ability of flavone, apigenin and luteolin to inhibit the growth of breast cancer cells through apoptosis was confirmed by Hoechst33342 staining and the induction of sub-G1 phase of the cell cycle. Flavone, apigenin and luteolin induced forkhead box O3 (FOXO3a) expression by inhibiting Phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB)/Akt. This subsequently elevated the expression of FOXO3a target genes, including the Cyclin-dependent kinase inhibitors p21Cip1 (p21) and p27kip1 (p27), which increased the levels of activated poly(ADP) polymerase (PARP) and cytochrome c.ConclusionTaken together, these data demonstrated that flavone, apigenin and luteolin induced cell cycle arrest and apoptosis in breast cancer cells through inhibiting PI3K/Akt activation and increasing FOXO3a activation, which suggest that flavone, apigenin and luteolin will be the potential leads for the preventing and treating of breast cancer.

The association between lumican gene polymorphisms and high myopia.

PurposesLumican (LUM) is one of the major extracellular matrix components of the sclera. Increasing evidence suggests that changes in the structure and composition of the sclera are major factors in regulating scleral integrity and axial elongation of the eye, as in myopia.Patients and methodsPatients (n=182; age range, 17–24 years) were with a myopic spherical equivalent (SE) >6.5 diopters (D) and the control group comprised individuals (n=78; age range, 17–25 years) were with a myopic SE <0.5 D. The DNA fragments were separated by horizontal electrophoresis on 3% agarose gels. The forward primer was labelled with a 5′ FAM and the reaction products were detected using a 3100 Genetic Analyzer.ResultsThe polymorphisms detected in this study were LUMc.601, LUM−59, LUM−628, and LUM−1554. Moreover, the haplotype distributions of Ht1 (C/A/CC/T), Ht2 (C/A/--/T), Ht3 (T/A/CC/C), Ht4 (T/--/CC/T), Ht5 (T/--/CC/C), and Ht6 (T/--/--/C) of these polymorphisms were compared between the two groups. The haplotype frequencies of Ht1, Ht2, Ht5, and Ht6 differed significantly between the two groups (P=2.08 × 10−5, odds ratio (OR): 2.19, 95% confidence interval (CI): 1.52–3.15; P=2.2 × 10−5, OR: 0.39, 95% CI: 0.25–0.61; P=2.7 × 10−5, OR: 0.36, 95% CI: 0.22–0.59; P=3.7 × 10−5, OR: 4.71, 95% CI: 2.12–10.5, respectively).ConclusionsThese observations suggest that the four polymorphisms of the LUMpromoter contribute to the pathogenesis of high myopia. Understanding the functions of LUM in myopia helps us design new methods in treating and preventing myopia.