Pathogenic Role Of LncRNA-MALAT1 In Microvascular Dysfunction In Diabetes Mellitus

Objective:To investigate the pathogenic role of long noncoding RNAs(lnc RNAs) in the diabetes-related microvascular dysfuncion.Method:(1) The diabetic animal models were built up in Sprague-Dawley rats by a single intravenous STZ injection. Quantitative RT-PCR shows the MALAT1 expression level in the retinas of diabetic rats, non-diabetic rats and db/db mice and controls.RNA fluorescence in situ hybridization(FISH) was performed to the existence of MALAT1 in the RF/6A cells.(2) The wild-type rats, diabetic rats were retreated by an intraocular injection of scramble sh RNA or MALAT1 sh RNA adenovirus. Quantitative RT-PCR was used to detect the MALAT1 level. The visual electrophysicology was conducted to detect the change of retinal function. The TUNEL assay was performed to detect the apoptosis percentage of retina cells.In order to reveal the diabetes induced retinal vessel impairment. The retina trypsin digestion was conducted to detect the change of retinal vessel pericytes and acellular capillaries. And we used the Evans-Blue-albumin method to detect the diabetes induced retinal vascular leakage.To investigate the retinal inflammation affected by MALAT1,we conducted western blots to detect the retinal expression of ICAM,VEGF,TNF-α.(3) The viability of RF/6A was detected by MTT assay. Hoechst,PI,JC-1,Trypan blue staining was used to detect the apoptosis degree of RF/6A cells,which retreated within MALAT1 si RNA,scramble si RNA, or left untreated, followed by high glucose or H2O2 treatment.We investigated the effect of MALAT1 knockdown on VEGF and TNF-αinduced endothelial cell migration and tube formation by cell scratch assay and matrigel-based capillary-genesis assay.(4) We conducted western blotting to examine the activation status of phosphorylated p38, ERK1/2 or JNK1/2to reveal which way to regulate the retinal vessel dysfuction by MALAT1.Results:(1) FISH show the enrichment of MALAT1 in RF/6A. MALAT1 level is up-regulated in diabetic animal model.(2) Quantitative RT-PCR shows MALAT1 sh RNA but not scramble RNA injection downregulates MALAT1 level.MALAT1 knockdown alleviates retinal function and prevents ERG abnormality induced by diabetes.MALAT1 knockdown also reverses the trend of a severe pericytes loss and acellular vascular induced by diabetes,which alleviates retinal vessel leakage in diabetic rats.MALAT1 knockdown decreases the expression of ICAM-1, VEGF,TNF-α in diabetic rats.(3) High glucose or H2O2 decreases the number and viability of RF/6A. MALAT1 knockdown further reduces cell viability and number. MALAT knockdown reduces the number of cell migration and tube formation induced by VEGF and TNF-α.(4) MALAT1 knockdown causes the reduction of phosphorylated p38 level, but has no effect of the levels of phosphorylated ERK1/2 and JNK1/2. Pretreatment of RF/6A with the p38-MAPK pathway inhibitor blocks the effect of MALAT1 induced cell hyperproliferation, whereas ERK inhibitor or JNK inhibitor has no effect of that.Conclusion:MALAT1 up-regulation represents a critical pathogenic mechanism for diabetes-induced microvascular dysfunction. Inhibition of MALAT1 may serve as a potential target for anti-angiogenic therapy for diabetes-related microvascular complications.