The effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells by targeting miR-200b-5p / 中华肿瘤杂志

Objective: To explore the effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells and its possible mechanism. Methods: The tumor tissues of 31 patients with retinoblastoma admitted to Henan Provincial Eye Hospital from February to June 2020 and their corresponding normal tissues adjacent to the cancer were collected. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p in retinoblastoma tissues and normal adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Human retinal epithelial cell ARPE-19, human retinoblastoma cell Y-79 and WERI-Rb-1 were cultured in vitro. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p were detected by qRT-PCR. Y-79 cells were randomly divided into si-con group, si-lncRNA ADPGK-AS1 group, miR con group, miR-200b-5p group, si-lncRNA ADPGK-AS1+ anti-miR con group, and si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group. The proliferation, cloning and apoptosis of cells in each group were detected by tetramethylazol blue method, plate cloning test and flow cytometry, respectively. The targeting relationship between lncRNA ADPGK-AS1 and miR-200b-5p was detected by double luciferase report test, and the expression level of cleaved-caspase-3 protein was detected by western blot. Results: Compared with the adjacent tissues, the expression of lncRNA ADPGK-AS1 in retinoblastoma tissues was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with ARPE-19 cells, the expression of lncRNA ADPGK-AS1 in Y-79 and WERI-Rb-1 cells was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with the si-con group, the cell viability of the si-lncRNA ADPGK-AS1 group was reduced (1.06±0.09 vs 0.53±0.05, P<0.05), the number of cell clone formation was reduced (114.00±8.03 vs 57.00±4.13, P<0.05), while the apoptosis rate [(7.93±0.68)% vs (25.43±1.94)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). Compared with the miR-con group, the cell viability of the miR-200b-5p group was decreased (1.05±0.08 vs 0.57±0.05, P<0.05), the number of cell clone formation was decreased (118.00±10.02 vs 64.00±5.13, P<0.05), while the apoptosis rate [(7.89±0.71)% vs (23.15±1.62)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). lncRNA ADPGK-AS1 could target the expression of miR-200b-5p. Compared with the si-lncRNA ADPGK-AS1+ anti-miR-con group, cell viability of the si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group was increased (0.53±0.04 vs 1.25±0.10, P<0.05), and the number of cell clones was increased (54.00±4.39 vs 125.00±10.03, P<0.05), while the rate of apoptosis [(25.38±1.53)% vs (9.76±0.71)%] and the protein level of cleaved-caspase-3 were decreased (P<0.05). Conclusion: Interfering with the expression of lncRNA ADPGK-AS1 could inhibit the proliferation and clone formation and induce apoptosis of retinoblastoma cells by targeting the expression of miR-200b-5p.

microRNA-144 functions as a diagnostic and prognostic marker for retinoblastoma

OBJECTIVES: Retinoblastoma (RB) is a highly malignant eye tumor with a low survival rate and a high metastatic rate. The current work was designed to investigate the potential roles of microRNA-144 (miR-144) in the diagnosis and prognosis of RB. METHODS: miR-144 expression levels in RB tissues and adjacent normal tissues, as well as serum samples from RB patients and healthy controls were measured. The association between miR-144 expression levels and clinical features were analyzed. Moreover, diagnostic and prognostic values of miR-144 in RB were verified by receiver operating characteristic analysis and Kaplan-Meier survival assays. RESULTS: The expression level of miR-144 was markedly decreased in tumor tissues of RB patients, and the expression level of miR-144 was positively associated with tumor size and metastasis in RB patients. Moreover, miR-144 can distinguish tumor tissues from normal tissues with high specificity and sensitivity, and RB patients with lower miR-144 expression have shorter overall and disease-free survival rates than those with higher miR-144 expression. Alternatively, miR-144 also decreased in the serum of RB patients in comparison with healthy subjects, and miR-144 expression levels in the tissue samples and serum were positively correlated. Furthermore, miR-144 levels in the serum of RB patients sensitively distinguished RB patients from healthy controls. CONCLUSIONS: miR-144 expression was downregulated in serum and tissue samples of RB patients and may function as a diagnostic and prognostic marker for RB.

Uncommon RB1 somatic mutations in a unilateral retinoblastoma patient

Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a developing retina is a crucial event in the initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed the DNA from tumor tissue and peripheral blood of a unilateral retinoblastoma patient to determine the RB1 mutation status and to provide an accurate genetic counseling. A comprehensive approach, based on our previous experience, was used to identify the causative RB1 mutations. Screening for RB1 mutations was performed by PCR direct sequencing, multiplex ligation-dependent probe amplification (MLPA) and Real Time-PCR analyses. Three different mutations were identified in the tumor DNA, which were absent in blood DNA. The somatic origin of these mutations was vital to rule out the heritable condition in this patient.

El retinoblastoma (RB) es el cáncer ocular más común de la niñez. La inactivación somática de ambos alelos del gen supresor de tumores RB1 en la retina en desarrollo es un evento crucial en la iniciación de la tumorigénesis en la mayoría de los casos de retinoblastoma unilateral. Nosotros analizamos el ADN de tumor y de sangre periférica de un paciente con retinoblastoma unilateral para identificar las mutaciones y así proveer un asesoramiento genético a la familia. Para ello utilizamos un protocolo basado en nuestra previa experiencia para identificar todas las mutaciones en el gen RB1 que causaron el RB. El rastreo de mutaciones se realizó por medio de los siguientes análisis: PCR-secuenciación, amplificación multiplex de sondas ligadas (MLPA) y PCR-Tiempo Real. Se encontraron tres mutaciones diferentes en el ADN del tumor, las cuales estaban ausentes en el ADN de la sangre. El origen somático de estas mutaciones es importante para indicar que la enfermedad no es hereditaria.

Enfermedad de von Hippel Lindau en una familia chilena: diagnóstico clínico y genético / Von Hippel Lindau disease: report of an affected family

Von Hippel Lindau disease is a hereditary syndrome characterized by the appearance of benign and malignant tumors in different organs. Its incidence is 1 case per 36000 born alive. We report a family with the disease. The index case was a male with a bilateral pheochromocytoma and cerebelar and retinal hemagioblastomas that had a sudden death due to a cerebrovascular accident at the age of 52 years. One sibling had central nervous system and retinal hemangioblastomas and other was operated for an unilateral pheochromocytoma. Both siblings had the R167Q VHL mutation of the syndrome. Other family members did not have the mutation.

Ki-67 cell proliferation in familial and in esporadic unilateral retinoblastoma: case report

PURPOSE: Ki-67 is a nuclear protein that is expressed at all phases of the cell cycle except the resting phase. This study is a clinicopathologic observational case report that aims to report on the cell proliferation rates, as measured by the Ki-67 antigen, in two enucleated retinoblastoma eyes. METHODS: One unilateral familial (mother with unilateral disease - patient 1) and one unilateral sporadic retinoblastoma (patient 2) patients were submitted to enucleation without previous treatment. The tumor cell proliferation rate was assessed by the Ki-67 antigen labeling index (stained cells / 100 cells) in five different fields of the tumor. RESULTS: Patient 1 was 23 months old and the tumor was exophytic with associated neovascularization of the iris; patient 2 was 6 years old and the tumor was endophytic with coarse vitreous seeds. Both enucleated eyes presented optic nerve with free surgical margins. Positive Ki-67 cell index in patient 1 varied from 75 to 90 (MD ± SD: 79.5 ± 6.61) and in patient 2 from 38 to 60 (MD ± SD: 46.6 ± 8.2). CONCLUSIONS: The familial retinoblastoma, besides the earlier age presentation, showed 45.8 percent more Ki-67 positive cells than the same stage sporadic one. This proliferation rate may explain the earlier presentation age of the tumor in the inherited disease.

OBJETIVO: O Ki-67 é antígeno nuclear que se expressa em todas as fases do ciclo celular, exceto no período de repouso. Este é um estudo de casos com correlação clínico-patológica que visa avaliar a taxa de proliferação celular, medida pelo antígeno Ki-67, em 2 olhos enucleados com retinoblastoma. MÉTODOS: Um paciente com retinoblastoma unilateral familiar (mãe com doença unilateral - paciente 1) e outro com retinoblastoma unilateral esporádico (paciente 2) foram submetidos à enucleação ocular sem outro tratamento prévio. A taxa de proliferação celular foi avaliada segundo índice obtido pela contagem de células marcadas com Ki-67, em 5 campos sob microscópia óptica (células marcadas/100 células). RESULTADOS: O paciente 1, com 23 meses de idade, apresentou tumor exofítico com neovascularização de íris associada; o paciente 2, de 6 anos, apresentou tumor de crescimento endofítico, com sementes vítreas importantes. Ambos os olhos enucleados apresentaram margens cirúrgicas do nervo óptico livres de neoplasia. O índice de células positivas no paciente 1 variou de 75 a 90 (Média ± DP: 79,5 ± 6,61), e no paciente 2, de 38 a 60 (Média ± DP: 46,6 ± 8,2). O retinoblastoma familiar, além de sua manifestação em idade mais precoce, apresentou 45,8 por cento mais células positivas que o retinoblastoma esporádico com o mesmo estadiamento. CONCLUSÃO: O retinoblastoma familiar, além de surgimento mais precoce, apresentou 45,8 por cento mais células em proliferação que o retinoblastoma esporádico em mesmo estádio. Essa taxa de proliferação pode explicar a menor idade de aparecimento do tumor nos casos de doença herdada.

Karyotyping in retinoblastoma--a statistical approach.

PURPOSE: Karyotype analysis in hereditary retinoblastoma is considered to be of marginal value in risk prediction due to uncertainties in the assessment of 13q14 deletions. However, it is a low cost genetic test for retinoblastoma in developing countries. In the present study, the results of karyotype analysis were refined by a statistical method to overcome limitations. METHODS: Karyotype analysis was performed by trypsin-Giemsa banding and naked eye karyotyping for 33 bilateral, 25 unilateral and one regressed retinoblastoma patients. The percentage of metaphases with 13q14 deletions in each case was plotted on a scatter diagram. Normalization of the data was achieved by log transformation and the results were statistically analyzed by one-sample 't' test using SPSS version 9.0. RESULTS: Seven samples had 13q14 deletion percentages above the cutoff value. One-sample 't' test showed significance (p< 0.001). By this method, two unilateral and five bilateral patients had 13q14 deletions, constituting 11.8 % of cases. CONCLUSION: For accuracy, statistical analysis should be considered as an adjunct in karyotyping.