Role Of LncRNA-RNCR2 In Diabetes Mellitus-related Retinal Neurodegeneration

Objective:To investigate the role of lncRNA-RNCR2 in diabetes mellitus-related retinal neurodegeneration.Method:1. First, we built diabetic animal models using Sprague-Dawley rats by intraperitoneal (streptozotocin, STZ) injection. The experiment included four groups: non-diabetic rats and STZ-induced diabetic rats (4 W,8 W, and 12 W diabetes induction). Quantitative RT-PCRs were used to detect RNCR2 expression in the retinas of diabetic rats. We also treated retinal ganglion cells (RGC) and Muller cells with high glucose. The experiment also included four groups:medium containing normal glucose (5 mmol/L) and high glucose (30 mmol/L) treatment for 24 h,48 h, and 72 h. Quantitative RT-PCR.s were used to detect RNCR2 expression levels in these groups.2. Animals experiments:Diabetic Sprague-Dawley rats were induced by intraperitoneal STZ injection. The experiment was divided into four groups: non-diabetic rats, STZ-SD rat of 12 W, scrambled siRNA knockdown STZ-SD rats of 12 W and RNCR2 knockdown STZ-SD rats of 12 W. We used immunofluorescence experiments to detect the expression level of GFAP, GS, NeuN, TUBB3, PKC-a, Calretinin and calbindin in these groups.3. In vitro study:We built up high glucose models using RGCs and Muller cells. The experiment included four groups:normal group,30 mmol/L glucose treatment, RNCR2 knockdown with 30 mmol/L glucose treatment, and scrambled siRNA knockdown with 30 mmol/L glucose treatment. We built up oxidation stress models using RGCs and Miiller cells. The experiment included four groups:normal group,200 μmol/L H2O2 treatment, RNCR2 knockdown with 200 μmol/L H2O2 treatment, and scrambled siRNA knockdown with 200 μmol/L H2O2 treatment. We used MTT to detect cell viability, Hoechst 33342, JC-1 and PI staining to detect cell apoptosis, and Ki-67 staining to detect cell proliferation.4. Mechanism study:We used western blots to detect the expression change of BDNF, NT-3 and NGF in retinas.Result:1. RNCR2 expression was significantly down-regulated in diabetic animal models. In hyerglycemia cell models, RNCR2 was significantly down-regulated in RGC and Muller cells.2. There is a significant increase in GFAP and GS in diabetic rats, whereas NeuN and TUBB3 exression was significantly down-regulated. RNCR2 knockdown could significantly reduce the expression level of GFAP, GS, NeuN, and TUBB3. No significant change for PKC-α,Calretinin and Calbindin was detected after RNCR2 knockdown.3. High glucose or oxidative stress can significantly reduced the viability and proliferation of RGCs and Muller cells. RNCR2 knockdown could further down-regulate the viability and proliferation. Moreover, RNCR2 knockdown further increased the death and apoptosis.4. RNCR2 knockdown significantly down-regulated BDNF, NT-3, and NGF expression in diabetic retinasConclusion:High glucose treatment could up-regulate RNCR2 exression. RNCR2 up-regulation could accelerate Miiller cell activation and contribute to the secretion of neurotrophic factors.