RESUMO
Endothelial-to-mesenchymal transition (EndMT) is a pivotal event in diabetic retinopathy (DR). This study explored the role of circRNA zinc finger protein 532 (circZNF532) in regulating EndMT in DR progression. Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to induce the DR cell model. Actinomycin D-treated HRMECs were used to confirm the mRNA stability of phosphoinositide-3 kinase catalytic subunit δ (PIK3CD). The interaction between TATA-box-binding protein-associated factor 15 (TAF15) and circZNF532/PIK3CD was subsequently analyzed using RNA immunoprecipitation (RIP), RNA pull-down. It was found that HG treatment accelerated EndMT process, facilitated cell migration and angiogenesis, and enhanced PIK3CD and p-AKT levels in HRMECs, whereas si-circZNF532 transfection neutralized these effects. Further data showed that circZNF532 recruited TAF15 to stabilize PIK3CD, thus elevating PIK3CD expression. Following rescue experiments suggested that PIK3CD overexpression partially negated the inhibitory effect of circZNF532 silencing on EndMT, migration, and angiogenesis of HG-treated HRMECs. In conclusion, our results suggest that circZNF532 recruits TAF15 to stabilize PIK3CD, thereby facilitating EndMT in DR.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases , Retinopatia Diabética , Células Endoteliais , Transição Epitelial-Mesenquimal , Humanos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , RNA Circular/metabolismo , RNA Circular/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismoRESUMO
Diabetic retinopathy (DR) is one of the leading causes of blindness in diabetic patients. However, the pathogenesis of DR is complex, and no firm conclusions have been drawn so far. It has become a hot spot in ophthalmology research to deeply study the mechanism of DR pathological changes and find effective treatment options. Human retinal microvascular endothelial cells (HRMECs) were induced by high glucose (HG) to construct DR cell model. CCK-8 assay was used to detect the viability of HRMECs. Transwell assay was used to detect the migration ability of HRMECs. Tube formation assay was used to identify the tube formation ability of HRMECs. The expressions of USP14, ATF2 and PIK3CD were detected by Western blot analysis and qRT-PCR assay. Immunoprecipitation (IP) was used to ascertain the relationship of USP14 and ATF2. To explore the regulatory relationship between ATF2 and PIK3CD by dual-luciferase reporter gene assay and Chromatin immunoprecipitation (ChIP) assay. High glucose treatment promoted the proliferation, migration, and tube formation of HRMEC, and the expressions of USP14, ATF2 and PIK3CD were significantly up-regulated. USP14 or ATF2 knockdown inhibited HG-induced HRMECs proliferation, migration, and tube formation. USP14 regulated the expression of ATF2, and ATF2 promoted PIK3CD expression. PIK3CD overexpression attenuated the inhibitory effectiveness of USP14 knockdown on proliferation, migration and tube formation of DR cell model. Here, we revealed that USP14 regulated the ATF2/PIK3CD axis to promote proliferation, migration, and tube formation in HG-induced HRMECs.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , MicroRNAs , Humanos , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Proliferação de Células/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Diabetes Mellitus/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Endoteliais/metabolismo , Glucose , MicroRNAs/genética , Retina/metabolismo , Retina/patologia , Ubiquitina Tiolesterase/metabolismoRESUMO
BACKGROUND: Diabetic retinopathy (DR) is a common complication of diabetes mellitus that poses a threat to adults. MicroRNAs (miRNAs) play a key role in DR progression. However, the role and mechanism of miR-192-5p in DR remain unclear. We aimed to investigate the effect of miR-192-5p on cell proliferation, migration and angiogenesis in DR. METHODS: Expression of miR-192-5p, ELAV-like RNA binding protein 1 (ELAVL1) and phosphoinositide 3-kinase delta (PI3Kδ) in human retinal fibrovascular membrane (FVM) samples and human retinal microvascular endothelial cells (HRMECs) was assessed using RT-qPCR. ELAVL1 and PI3Kδ protein levels were evaluated by Western blot. RIP and dual luciferase reporter assays were performed to confirm the miR-192-5p/ELAVL1/PI3Kδ regulatory networks. Cell proliferation, migration and angiogenesis were assessed by CCK8, transwell and tube formation assays. RESULTS: MiR-192-5p was decreased in FVM samples from DR patients and high glucose (HG)-treated HRMECs. Functionally, overexpressed miR-192-5p inhibited cell proliferation, migration and angiogenesis in HG-treated HRMECs. Mechanically, miR-192-5p directly targeted ELAVL1 and decreased its expression. We further verified that ELAVL1 bound to PI3Kδ and maintained PI3Kδ mRNA stability. Rescue analysis demonstrated that the suppressive effects of HG-treated HRMECs caused by miR-192-5p up-regulation were overturned by overexpressed ELAVL1 or PI3Kδ. CONCLUSION: MiR-192-5p attenuates DR progression by targeting ELAVL1 and reducing PI3Kδ expression, suggesting a biomarker for the treatment of DR.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , MicroRNAs , Adulto , Humanos , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Regulação para Cima , Células Endoteliais , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Proliferação de Células/genética , Diabetes Mellitus/metabolismo , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismoRESUMO
Diabetic retinopathy (DR) is a serious long-term complication of diabetes. However, the current treatment of DR is still challenging. We aimed to investigate the role of lncRNA SNHG1/miR-340-5p/PIK3CA in DR and the mechanisms involved. Blood samples from clinical DR patients and healthy subjects were obtained. HRMECs were induced by high glucose for 24 h to establish the DR model. The vector for interfering or overexpressing lncRNA SNHG1, miR-340-5p, and PIK3CA was constructed. LncRNA SNHG1, miR-340-5p, and PIK3CA expressions were detected by qRT-PCR or Western blot. Cell proliferation and migration were detected by CCK-8 and Transwell assays. Blood vessel formation was detected by angiogenesis assay. Dual-luciferase reporter assay tested the interaction of lncRNA SNHG1 with miR-340-5p and miR-340-5p with PIK3CA. RIP measured the binding of miR-340-5p to PIK3CA. In the blood of DR patients and the DR model, lncRNA SNHG1 was increased and miR-340-5p expression was down-regulated. In the DR model, PIK3CA expression was elevated. Downregulation of lncRNA SNHG1 inhibited HRMECs proliferation, migration, and angiogenesis. LncRNA SNHG1 interacted with miR-340-5p, and up-regulation of miR-340-5p inhibited HRMECs proliferation, migration and angiogenesis. The inhibition of cell proliferation, migration, and angiogenesis of HRMECs caused by down-regulation of lncRNA SNHG1 was reversed by knockdown of miR-340-5p. miR-340-5p targeted PIK3CA, and downregulation of PIK3CA inhibited HRMECs proliferation, migration, and angiogenesis. The inhibition of HRMECs proliferation, migration and angiogenesis caused by down-regulation of lncRNA SNHG1 could be reversed by overexpression of PIK3CA. LncRNA SNHG1 targeted miR-340-5p/PIK3CA axis to regulate microvascular endothelial cell proliferation, migration, and angiogenesis in DR.