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Diabetic retinopathy (DR) is the main complication of diabetes mellitus (DM). Recent studies have implicated microRNAs dysfunction in human retinal microvascular endothelial cell (HRMEC). In this study, we aim to investigate the apoptotic promotion of miR-29b-3p by blocking SIRT1 in HRMEC for DR situation. To identify the regulating relationship between miR-29b-3p and SIRT1, HRMECs were transfected with miR-29b-3p mimics/inhibitors or their negative controls. Cell viability was assessed with the cell counting kit-8 (CCK-8) assay, and apoptotic cells were stained by one-step TUNEL assay kit. Gene and protein expression were assayed by RT-qPCR and Western blotting separately. Dual-luciferase reporter assay using HEK293T cells was performed to show the direct interaction of miR-29b-3p and the 3'-UTR of SIRT1. HRMECs were identified as >95% positive for CD31 and vWF. Upregulated miR-29b-3p decreased the expression of SIRT1 and increased the ratio of Bax/Bcl-2, while downregulated miR-29b-3p increased the expression of SIRT1 protein and downregulated the ratio of Bax/Bcl-2. Dual-luciferase reporter assay showed the direct interaction of miR-29b-3p and SIRT1. The dysregulation of miR-29b-3p/SIRT1 is a potential mechanism of HRMEC apoptosis in DR. miR-29b-3p/SIRT1 may be a potential therapeutic target for DR., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The pathogenesis of type 2 diabetes mellitus (T2DM) is commonly associated with altered gut bacteria. However, whether the microbial dysbiosis that exists in human diabetic patients with or without retinopathy is different remains largely unknown. Here, we collected clinical information and fecal samples from 75 participants, including 25 diabetic patients without retinopathy (DM), 25 diabetic patients with retinopathy (DR), and 25 healthy controls (HC). The gut microbial composition in the three groups was analyzed using 16S ribosomal RNA (rRNA) gene sequencing. Microbial structure and composition differed in the three groups. The α and β diversities in both the DM and DR groups were reduced compared with those in the HC group. Blautia was the most abundant genus, especially in the DM group. In addition, increased levels of Bifidobacterium and Lactobacillus and decreased levels of Escherichia-Shigella, Faecalibacterium , Eubacterium_hallii_group and Clostridium genera were observed in the DM and DR groups compared with the HC group. Furthermore, a biomarker set of 25 bacterial families, which could distinguish patients in the DR group from those in the DM and HC groups was identified, with the area under the curve values ranging from 0.69 to 0.85. Of note, Pasteurellaceae , which was increased in DM and decreased in DR compared with HC, generated a high AUC (0.74) as an individual predictive biomarker. Moreover, 14 family biomarkers were associated with fasting blood glucose levels or diabetes, with most of them being negatively correlated. In summary, our study establishes compositional alterations of gut microbiota in DM and DR, suggesting the potential use of gut microbiota as a non-invasive biomarker for clinical and differential diagnosis, as well as identifying potential therapeutic targets of diabetic retinopathy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Huang, Wang, Ma, Ji, Chen, Cui, Chen and Tang.)

Background: Diabetic retinopathy (DR) is a main complication of diabetes mellitus (DM). Recent studies have implicated microRNAs in human retinal microvascular endothelial cell (HRMEC) dysfunction. In this study, we aim to investigate the apoptotic promotion of miR-29b-3p by blocking SIRT1 in HRMEC for DR situation. Method: Blood samples were obtained from DR patients and controls. Dual-luciferase reporter assay using HEK-293T cells was performed to show the direct interaction of miR-29b-3p and the 3′UTR of SIRT1. HRMECs were exposed to 5.5 mmol/L of glucose (normal control), 5.5 mmol/L of glucose and 24.5 mmol/L of mannitol (osmotic pressure control), 30 mmol/L of glucose [hyperglycemia (HG)], 150 μmol/L of CoCl2 (hypoxia), and 30 mmol/L of glucose plus 150 μmol/L of CoCl2 (HG-CoCl2). To identify the regulating relationship between miR-29b-3p and SIRT1, HRMECs were transfected with miR-29b-3p mimics/inhibitors or their negative controls. SRT1720 was used as a SIRT1 agonist. Cell viability was assessed with the cell counting kit-8 (CCK-8) assay, and apoptotic cells were stained by one-step terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit. Gene and protein expression were assayed by quantitative real-time reverse transcriptase-PCR (RT-qPCR) and western blotting separately. Result: MiR-29b-3p was upregulated to 3.2-fold, and SIRT1 protein was downregulated to 65% in DR patients. Dual-luciferase reporter assay showed the direct interaction of miR-29b-3p and SIRT1. HRMECs were identified as >95% positive for CD31 and von Willebrand factor (vWF). MiR-29b-3p and Bax/Bcl-2 ratio was upregulated, whereas SIRT1 was downregulated in HRMECs in the HG-CoCl2 condition. Decreased cell viability and upregulated apoptosis were also found in HRMECs of the HG-CoCl2 condition. Upregulated miR-29b-3p decreased the expression of SIRT1 and increased the ratio of Bax/Bcl-2, whereas downregulated miR-29b-3p increased the expression of SIRT1 protein and downregulated the ratio of Bax/Bcl-2. SRT1720 rescued miR-29b-3p-induced HRMEC apoptosis via upregulating the expression of SIRT1 protein. Conclusion: The dysregulation of miR-29b-3p/SIRT1 is a potential mechanism of HRMEC apoptosis in DR. MiR-29b-3p/SIRT1 may be a potential therapeutic target for DR. [ABSTRACT FROM AUTHOR]