RESUMEN
BACKGROUND: Age-related macular degeneration (AMD) may lead to irreversibly vision loss among aging populations. In this work, in an in vitro AMD cell model, we examined the expression and function of long non-coding RNA, Prader-Willi Region Non-Protein Coding RNA 2 (PWRN2) in injured human retinal pigment epithelial cells. METHOD: ARPE-19 cell line was maintained in vitro and treated with multi-module stressful conditions, including hydrogen peroxide (H2O2) tert-butylhydroperoxide (t-BuOOH) and ultraviolet B (UVB). Multi-module-stressor-induced cell death was monitored by a viability assay, and PWRN2 expression by qRT-PCR. PWRN2 was either downregulated or upregulated in ARPE-19 cells. The effects of PWRN2 downregulation or upregulation on t-BuOOH-induced cell death, cellular apoptosis and mitochondrial injuries were then quantitatively evaluated. RESULTS: Multi-module stressful conditions induced cell death and PWRN2 upregulation in ARPE-19 cells in vitro. We created ARPE-19 subpopulations with either downregulated or upregulated PWRN2 expressions. Quantitative assays demonstrated that, PWRN2 downregulation effectively alleviated t-BuOOH-induced cell death, apoptosis and various-type of mitochondrial injuries. On the other hand, PWRN2 upregulation worsened t-BuOOH-induced cellular damages in ARPE-19 cells. CONCLUSION: We demonstrated that downregulating PWRN2 protected multi-module-stressor-induced cell death, apoptosis and mitochondrial injuries in human retinal pigment epithelial cells, suggesting PWRN2 may be an active factor in human AMD.
Asunto(s)
Degeneración Macular/genética , Modelos Biológicos , ARN Largo no Codificante/metabolismo , Muerte Celular , Línea Celular , Supervivencia Celular/genética , Regulación hacia Abajo/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Degeneración Macular/patología , Mitocondrias/metabolismo , ARN Largo no Codificante/genética , Epitelio Pigmentado de la Retina/patología , Regulación hacia Arriba/genética , terc-ButilhidroperóxidoRESUMEN
High-glucose-induced retinal tissue impairment is the major pathological phenotype of diabetic retinopathy. In an in vitro diabetic apoptosis cell model, we evaluated the function of long noncoding RNA, insulin growth factor 2 antisense (IGF2-AS) in high-glucose-injured human retinal pigment epithelial cells. A human retinal pigment epithelial cell line, ARPE-19 was incubated with high-glucose in vitro to induce apoptosis. SiRNA-mediated IGF2-AS downregulation was conducted in ARPE-19 cells to evaluate its effect on high-glucose induced apoptosis, assessed by a TUNEL assay. qRT-PCR and western blot assays were applied to examine the functional effect of IGF2-AS on IGF2/AKT/Casp-9 expressions in glucose-injured ARPE-19 cells. ART was further knocked down, specifically in IGF2-AS-downregualted ARPE-19 cells, to investigate its functional involvement in IGF2-AS-inhibition-mediated apoptotic protection in glucose-injured ARPE-19 cells. High-glucose induced apoptosis in ARPE-19 cells, and upregulated IGF-2AS in a dose-dependent manner. SiRNA-mediated IGF2-AS downregulation ameliorated apoptosis, upregulated IGF2/AKT and decreased Casp-9, in high-glucose-treated ARPE-19 cells. AKT knockdown was shown to dramatically reverse the preventive effect of IGF2-AS-downregulation on high-glucose-induced apoptosis in ARPE-19 cells. Moreover, it was demonstrated that AKT knockdown directly upregulated Casp-9 in IGF2-AS-downregulated and high-glucose-treated ARPE-19 cells. We demonstrated that inhibiting IGF2-AS, possibly also through activation of AKT signaling pathway, has a protective function in high-glucose-induced apoptosis in human retinal pigment epithelial cells in diabetic retinopathy.
Asunto(s)
Apoptosis/genética , Retinopatía Diabética/genética , Proteínas/genética , Epitelio Pigmentado de la Retina/metabolismo , Caspasa 9/genética , Retinopatía Diabética/patología , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Neuronas/metabolismo , Neuronas/patología , Proteínas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Epitelio Pigmentado de la Retina/patología , Transducción de Señal/genéticaRESUMEN
The study aimed to examine the impact of parental influence and media richness on gender stereotypes and career decisions among students at the secondary level in Pakistan. The sample size was 200 students, selected through a simple random sampling technique from government and private schools. Four questionnaires were used to gather data. The data was analyzed quantitatively using the Statistical Package for the Social Sciences (SPSS). Regression analyses were used to investigate the impact of parental influence (ß = 0.50) on gender stereotypes and media richness influence (ß = 0.26) on gender stereotype beliefs. Furthermore, parental, media, and gender stereotype behavior all have a significant impact on students' career choices (R 2 = 0.694). On the scale of the parental influence and media richness, no significant gender differences were found. It is concluded that parental influence has a greater effect on students' gender stereotyping behavior and career choices.