The Role And Mechanism Of LncRNA AK154753 In Acute Kidney Injury Induced By Ischemia/Reperfusion

ObjectiveIn our experiment,the role and mechanisms of lncRNA AK154753 in ischemia/reperfusion(I/R)acute kidney injury(AKI)were explored via both in vivo and in vitro AKI models.Methods1.Eight weeks old C57BL/6 male mice were subjected to bilateral renal artery occlusion for 0.5h and reperfusion for 24 h to establish an I/R model to simulate AKI.Serum creatinine(Scr)and blood urea nitrogen(BUN)were detected to indicate renal function.Quantitative real-time polymerase chain reaction(RT-qPCR)was performed to analyze the expression of lncRNA AK154753,miR-345-3p,miR-708-5p,gapdh(reference gene)and u6(reference gene)of kidney tissues,while cleavedcaspase3,BAK,BIM and GAPDH(loading control)were detected by western-blot.Furthermore,renal tissue sections were collected for Hematoxylin and eosin(HE)staining and TUNEL staining.2.BUMPT cells were cultured in complete medium and subjected to glucose-free DMEM medium + antimycin A + oligomycin(5 μM)at a confluence of 70-80% for 2h,then changed back to complete medium and cultured for another 2h to establish an oxygen glucose deprivation/reperfusion(OGD/R)model to simulate AKI.Cells were collected for flow cytometry to detect cell apoptosis.Cellular RNAs were extracted to detect lncRNA AK154753,miR-345-3p,miR-708-5p,gapdh(reference gene)and u6(reference gene)by RT-qPCR.Total proteins were collected for western-blot analysis of cleaved-caspase3,BAK,BIM and β-actin(loading control).3.To further explore the underlying mechanism of lncRNA AK154753,BUMPT cells were infected with lentivirus carrying shAK154753,and the cell line stably expressing sh-AK154753 was established.Cells were randomly divided into the following 4 groups: 1)control group;2)OGD/R group;3)OGD/R+ sh-NC group;4)OGD/R+sh-AK154753 group.the cells were collected for TUNEL staining,RTqPCR and western-blot analysis.4.Four-week-old C57BL/6 male mice were injecting adenoassociated virus(AAV)carrying sh-AK154753 into the tail vein to construct mice stably expressing sh-AK154753.Mice were randomly divided into the following 4 groups: 1)sham group;2)I/R group;3)I/R+sh-NC group;4)I/R+ sh-AK154753 group.Scr and BUN were detected and renal tissues were collected for RT-qPCR,western-blot analysis,HE staining and TUNEL staining.Results1.In I/R mice model,renal tissues were damaged,cell apoptosis and the expression of apoptosis-related proteins were increased,as well as lncRNA AK154753,instead of miR-345-3p and miR-708-5p decreased.In BUMPT cells,OGD/R escalated cell apoptosis,and the expression levels of cleaved-caspase3,BAK and BIM were up-regulated,while lncRNA AK154753 was increased,accompanied by miR-345-3p,miR-708-5p decreased.2.In BUMPT cells,silencing of lncRNA AK154753 attenuates OGD/R induced apoptosis and rescues the down-regulation of miR-345-3p and miR-708-5p.3.In mice,knock-down of lncRNA AK154753 reduces I/R-induced cell apoptosis and renal lesion.ConclusionLncRNA AK154753 may act as a "molecular sponge" to participate in the development of ischemia/reperfusion induced AKI by binding to miR-345-3p and miR-708-5p.