RESUMO
【Objective】 To investigate the relationship between cerebrospinal fluid exosome-derived miR-630 and the function of brain microvascular endothelial cells （BMECs）. 【Methods】 The subarachnoid hemorrhage endothelial cell model was established to evaluate the effect of hemorrhagic cerebrospinal fluid （BCSF） on BMECs’ proliferation by MTT assay and cell cycle analysis. qRT-PCR and immunofluorescence staining were used to detect the expressions of endothelial cell tight junction protein （ZO-1） and adhesion molecule （ICAM-1 and VCAM-1）. Changes in NOx concentration were detected by radioimmunoassay. The cerebrospinal fluid exosomes in the experimental group （co-incubated with BCSF） and the control group （normal cerebrospinal fluid） were isolated and identified， and differences in the expressions of cerebrospinal fluid exosomal miR-630 between the two groups were compared. BMECs work changes after the intervention with miR-630 analogue were observed. 【Results】 The proliferation of BMECs was significantly inhibited in the experimental group； the mRNA and protein levels of ICAM-1， VCAM-1 and ZO-1 were significantly decreased， and the function of endothelial cells was significantly inhibited （P<0.05）. After the successful separation and identification of cerebrospinal fluid exosomes， the expression of miR-630 was significantly lower in the experimental group than in the control group （P<0.05）. The function of BMECs was significantly improved with miR-630 mimics. 【Conclusion】 The low expression of miR-630 in cerebrospinal fluid exosomes after subarachnoid hemorrhage is closely related to BMECs injury.
RESUMO
Cataract, an eye disease that threatens the health of millions of people, brings about severe economic burden for patients and society. MicroRNA (miR)-378a-5p and miR-630 were recognized as essential regulators in multiple cancers. However, the exact functions of miR-378a-5p and miR-630 in cataract are still unclear. The expression of miR-378a-5p, miR-630, and E2F transcription factor 3 (E2F3) in tissues and cells was measured by quantitative real-time polymerase chain reaction. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was used to evaluate cell viability. Flow cytometry was conducted to analyze cell apoptosis. The interaction between E2F3 and miR-378a-5p or miR-630 was confirmed by dual-luciferase reporter assay. The expression of proteins E2F3, B cell lymphoma (Bcl-2), Bcl-2 associated X (Bax), and cleaved caspase 3 was detected by western blot assay. The expression of miR-378a-5p and miR-630 was up-regulated whereas E2F3 was down-regulated in human cataract lens tissues compared with normal lens tissues. Depletion of miR-378a-5p or miR-630 enhanced proliferation and reduced apoptosis of human lens epithelial cells. Interestingly, up-regulation of E2F3 exhibited the same trend. Next, dual-luciferase reporter assay validated the interaction between E2F3 and miR-378a-5p or miR-630. The rescue experiments further revealed that E2F3 knockdown could recover miR-378a-5p, and miR-630 inhibitor induced promotion of cell proliferation and inhibition of apoptosis in cataract. miR-378a-5p and miR-630 repressed proliferation and induced apoptosis of lens epithelial cells by targeting E2F3 in cataract, representing a prospective alternative therapy for cataract.
Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Catarata/metabolismo , Apoptose , MicroRNAs/metabolismo , Células Epiteliais/metabolismo , Fator de Transcrição E2F3/metabolismo , Regulação para Baixo , Western Blotting , Progressão da Doença , Reação em Cadeia da Polimerase em Tempo Real , Citometria de FluxoRESUMO
Many studies informed that microRNAs (miRNAs) could function as diagnostic and prognostic indicators in several cancers. The aims of this study were to explore the expression of miR-630 in bladder urothelial carcinoma and its clinical significance for the evaluation of cancer prognosis. A total of 116 patients with bladder urothelial carcinoma were obtained in this retrospective study between May, 2012 and Sep. 2015. Quantitative real-time PCR (qRT-PCR) was conducted to evaluate the expression level of miR-630. The chi-square test was used to examine the associations between miR-630 expression and the clinicopathological features. The Kaplan-Meier method was conducted to explore the survival status of urothelial carcinoma patients. The log-rank test was used to analyze differences in survival rate. The results showed an obvious increase in miR-630 expression from normal bladder to bladder urothelial carcinoma (P=0.027). Additionally, patients with higher miR-630 expression had significantly shorter disease-free survival (DFS) (P=0.043) and overall survival (OS) (P=0.038) than those with lower miR-630 expression. Furthermore, multivariate analysis revealed that up-regulation of miR-630 was an independent prognostic factor for both DFS (P=0.042) and OS (P=0.046). It was demonstrated that miR-630 may be a novel and valuable prognostic factor for bladder urothelial carcinoma.