Data Availability StatementAvailability of data and materials: All data generated or analyzed during this study are included in this published article

Data Availability StatementAvailability of data and materials: All data generated or analyzed during this study are included in this published article. Phloretin inhibition measured the cell proliferation rates. Western blot tested the expression status of IGF1/IGF1R-mediated signaling pathway. Dual-luciferase reporter assays demonstrated the molecular mechanism of miR-142-5p. miR-142-5p level was down-regulated in retinal tissues of DR rats and high glucose (HG)-treated HRECs. Insulin-like growth factor 1 (IGF1) was identified as a direct target of miR-142-5p. The reduced miR-142-5p level enhanced HRECs proliferation via activating IGF/IGF1R-mediated signaling pathway including p-PI3K, p-ERK, p-AKT, and VEGF activation, ultimately giving rise to cell proliferation. Either miR-142-5p overexpression or IGF1 knockdown alleviated the pathological effects on retinal tissues in DR rats. Collectively, miR-142-5p participated in DR development by targeting IGF1/p-IGF1R signaling pathway and VEGF generation. This miR-142-5p/IGF1/VEGF axis provided a novel therapeutic target for DR clinical treatment. values 0.05 were considered as statistically significant in this study (* em P /em ? ?0.05, ** em P /em ? ?0.01, *** em P /em ? ?0.001). Results miR-142-5p level was decreased in retinal tissues of DR rats or in HG-induced HRECs First, the pathologic alterations of retinal tissues were compared between DR group and normal group using HE staining. As shown in Figure 1(a), the structure and cell layers of retinal tissue were integrated and clear in the normal group (n?=?2). The cell number was abundant, and the cell morphology was intact and arranged neatly. However, in the DR group, the inner and outer nuclear layers of the retina were blurred, and the nerve fiber layer was edema. Moreover, the number of retinal cells was reduced and the cells arranged sparsely and disorderly. The morphology of HRECs were identified as goose-oval using phase contrast microscope (Figure 1(b)), and the vascular endothelium markers-CD31, von Willebrand factor (vWF) and VE-cadherin were positively expressed in cell lines of HRECs (Figure 1(c)). Next, miR-142-5p expression was detected using qualitative real-time polymerase chain reaction (qRT-PCR) under DR pathological condition in vivo and in vitro. miR-142-5p level was significantly down-regulated in retinal tissues of DR rats compared to that of normal rats (*** em P /em ? ?0.001, n?=?6, Figure 1(d)). In addition, the expression of miR-142-5p was remarkably decreased in HRECs in HG-treated group compared with NG group (*** em P /em ? ?0.001, Figure 1(e)). Taken together, the DR rat model was successfully established and the characteristics of HRECs were well identified, and miR-142-5p level was down-regulated under DR pathological condition. Open in a separate window Figure Phloretin inhibition 1. miR-142-5p level was decreased in retinal tissues Phloretin inhibition of DR-treated rats and in HG-treated HRECs. (a) Representative photos showing the pathological characteristics of retinal tissues in sham and DR group using HE staining. Scale bar?=?50?m. n?=?2. (b) Representative image showing the HRECs. Scale bar?=?50?m. (c) Expression levels of markers including CD31, vWF, and VE-cadherin in HRECs was detected by IF method. Scale bar?=?25?m (CD31, VE-cadherin staining). LT-alpha antibody Scale bar?=?50?m (vWF staining). (d) miR-142-5p levels in retinal tissues Phloretin inhibition in sham and DR group of rats were determined by qRT-PCR (n?=?6). (e) The miR-142-5p levels of HRECs in NG and HG group were tested by qRT-PCR (mean??SD; *** em P /em ? ?0.001). miR-142-5p suppressed HG treatmentCinduced HRECs proliferation Next, the effects of miR-142-5p on the retinal cell growth were examined in cell lines of HRECs. First, we confirmed that the decreased miR-142-5p level was restored by its mimics under HG treatment compared with miR-NC group (*** em P /em ? ?0.001, Figure 2(a)). To determine whether VEGF was associated with the change expression of miR-142-5p, qRT-PCR assays were performed. As shown in Figure 2(b), VEGF level was enhanced upon HG treatment, while decreased by miR-142-5p mimic compared with miR-NC.

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