The Role Of MiR-92a In Diabetic Erectile Dysfunction Rats And Underlying Mechanisms

PartⅠEstablishment of a diabetic erectile dysfunction rat modelObjective:To establish a diabetic erectile dysfunction rat model.Methods:A streptozotocin-induced diabetes rat model was established and then erectile function in diabetic rats was evaluated through observing apomorphine (APO)-induced erection. The positive response to APO was defined as glans hyperarmia and appearance of the end of the penis. The effectiveness of PDEi was evaluated in APO-positive and APO-negative diabetic rats through measuring intracavernous pressure (ICP) and mean arterial blood pressure (MAP). Subsequently, the protein expression of eNOS, phospho-eNOS, ROCK2, MYPT1and phosphor-MYPT1was determined and the apoptosis was detected in penile tissues.Results:Compared to normal control, the erectile function of APO-positive and APO-negative diabetic rats was significantly impaired. Furthermore, APO-negative diabetic rats showed lower ICP/MAP than that of APO-positive diabetic rats. After PDE5i treatment (2.08mg/kg), the ICP/MAP of APO-positive diabeitic rats was significantly restored, which reached normal level. Although the ICP/MAP of APO-negative diabeitic rats was significantly elevated after PDE5i treatment (2.08mg/kg), it was still lower than that of normal control. Compared with normal control, P-eNOS/eNOS was dramatically reduced and P-MYPT1/MYPT1, ROCK2/p-actin and apoptotic cells was significantly increased in penile tissues of APO-negative diabetic rat.Conclusions:APO-negative diabetic rats was proved to be unresponsive to PDE5i treatment. Part IIIdentification of miRNAs expression profile in penile tissues of diabetic erectile dysfunction ratsObjective:To find out important miRNAs involved in the pathogenesis of diabetic erectile dysfunction (ED).Methods:Global miRNAs expression in penile tissues from normal rats and APO-negative diabetic rats were measured using Agilent Rat miRNA V18.0and the aberrantly expressed miRNAs were further confirmed by quantitative real-time polymerase chain reaction (PCR). Among these altered miRNAs, the expression of miR-92a was measured in brain, heart, kindey, liver, aorte and penis. The mRNA expression level of integrin alpha5(CD49e), kruppel-like factor2(Klf2), kruppel-like factor4(Klf4), dickkopf3homolog (DKK3) and endothelial nitric oxide synthase (eNOS) were detected in penile tissues using quantitative real-time PCR. Results:Three significantly dysregulated miRNAs were identified in APO-negative diabetic rat’s penile tissues (up-regulated three-fold or down-regulated0.33fold relative to normal control). Among these altered miRNAs, the expression of miR-92a and miR29b was dramatically increased and the expression of miR133b was significantly decreased in penile tissues of APO-negative diabetic rats. MiR-92a was expressed in brain, heart, kindey, liver, aorte and penis. But the expression of miR-92a in penis was relatively low compared to other organs. The mRNA expression level of eNOS and DKK3was significantly decreased in APO-negative diabetic rats’ penes than that in normal control rats. However, the mRNA expression level of CD49e, Klf2and Klf4had no obvious change in APO-negative diabetic rats’ penes.Conclusions:Up-regulation of miR-92a was associated with downregulation of eNOS and DKK3in diabetic ED rat’s penile tissues. Part ⅢStudy on miR-92a and its target genes expression in aortic endothelial cells under high glucose conditionObjective:To explore miR-92a and its target genes expression in aortic endothelial cells under high glucose conditionMethods:Primary aortic endothelial cells were isolated from rat’s aorta and cultured under high glucose condition for48and72hours respectively. The RNA of aortic endothelial cells was extracted to determine the expression of miRNA, eNOS and DKK3. The protein of aortic endothelial cells was also extracted to measure the expression of eNOS and DKK3. Under high glucose condition, aortic endothelial cells were pretreated with antagomir-miR-92a.72hours later, both of RNA and protein of aortic endothelial cells were extracted to determine eNOS and DKK3expression.Results:After48hours and72hours, miR-92a expression in aortic endothelial cells cultured under high glucose enviroment was significantly up-regulated and showed an increasing trend. The mRNA expression of eNOS and DKK3was dramatically down-regulated and showed a decreasing trend. Meanwhile, the protein expression of eNOS and DKK3was aslo significantly reduced72hours after cultured under high glucose condition. Compared to control (antagomir-miR-NC), the expression of eNOS and DKK3was dramatically up-regulated in aortic endothelial cells under high glucose condition72h after pretreatment of antagomir-miR-92a.Conclusions:Under high glucose condition, miR-92a expression was significantly up-regulated, inhibiting eNOS and DKK3expression. Part IVThe effect of miR-92a-inhibition on diabetic erectile dysfunction rats and underlying mechanismsObjective:To explore whether inhibition of miR-92a could improve diabetic erectile dysfunction in rats and underlying mechanisms.Methods:To inhibit miR-92a, a lentiviral vector expressing miR-92a complementary sequence was constructed. Primary aortic endothelial cells were isolated from rat’s aorta and the protein expression of eNOS and DKK3was measured after transfected with Lv-miR-92a-inhibition. A streptozotocin-induced diabetes rat model was established and then erectile function in diabetic rats was evaluated through observing apomorphine (APO)-induced erection.2w after intracavernous injection of Lv-miR-92a-inhibition, the erectile function in APO-negative diabetic rats was determined through measuring intracavernous pressure. Then, the protein expression of eNOS and DKK3was evaluated in penile tissue of rats.Results:96h after transfected with Lv-miR-92a-inhibition in aortic endothelial cells, the mRNA and protein expression of eNOA and DKK3was significantly increased, indicating the successful construction of Lv-miR-92-inhibition.2w after intracavernous injection of Lv-miR-92a-inhibition, the erectile function of APO-negative diabetic rats was significantly improved. APO-negative diabetic rats, treated wih Lv-miR-92a-inhibiton, showed increased eNOS (reached normal level) and increased DKK3(still lower than that of normal control) compared to Lv-miR-NC-inhibition treated rats.Conclusions:miR-92a-inhibition might restore diabetic erectile dysfunction through up-regulate eNOS and DKK3expression in rats.