| Background:Acute renal injury（AKI）is a group of clinical syndromes.It was previously said that the occurrence of acute renal failure（ARF）,AKI increases the risk of death in critically ill patients,as well as the risk of CKD and cardiovascular disease.About 13.3 million people develop acute renal injury（AKI）each year,of which about 85percent are from developing countries and are associated with high mortality and morbidity.The main pathological mechanisms of acute renal injury include free radical injury,intracellular calcium overload,inflammatory reaction and apoptosis.Researchers are increasingly aware that apoptosis plays an important role in acute renal injury.Several common renal injuries,including ischemia,toxic injury,radiation and ureteral obstruction,can lead to renal cell apoptosis.The process of apoptosis is regulated by a variety of genes.Therapeutic interventions to inhibit apoptosis can reduce renal dysfunction and accelerate the recovery of acute renal injury.Inhibition of apoptosis may be an effective way to reduce renal ischemia-reperfusion injury.With the further study of circular RNA,circ RNA has been identified as a new therapeutic target and biomarker for kidney disease.Although many circ RNA have been confirmed in kidney tissue,their expression profile and potential role in the development of the disease are still unknown.It is necessary to further explore the role and mechanism of circ RNAs in renal disease.Future studies should confirm the exact mechanism by which circ RNA interacts with specific proteins and other non-coding RNA to mediate their role.Objective:To explore the expression changes of circular RNA circLRIG1 in acute renal injury and its role and potential mechanism in the process of hypoxia-reoxygenation in renal tubular epithelial cells,so as to provide theoretical basis and therapeutic targets for the prevention and treatment of acute renal injury.Methods:Part1:Screening and functional verification of circLRIG1,a key molecule in acute renal injury.1.The up-regulated circular circ RNA in the blood of patients with acute renal injury was screened and do human-mouse homology comparison,circLRIG1,with the highest homology was selected,verify the circular structure by agarose gel electrophoresis test,actinomycin D test and RNase R enzyme digestion test.2.Expression of circLRIG1 in renal tubular epithelial cell hypoxia-reoxygenation model and acute renal injury animal model（1）To establish the model of anoxia and reoxygenation of renal tubular epithelial cells.Methods:HK-2 cells were divided into two groups:hypoxia/reoxygenation group and control group.Before hypoxia/reoxygenation,the complete medium was replaced by glucose-free and serum-free medium.The cells in the hypoxia/reoxygenation group were placed in the hypoxia incubator（5%CO2,1%O2 and 94%N2）and transferred to the normoxic incubator 12 hours later.The cells in the control group grew under normoxic conditions,and there was no need to change the culture medium.（2）To establish an animal model of renal ischemia-reperfusion.Methods:Balb/c mice were randomly divided into sham operation group（control group）and ischemia-reperfusion renal injury model group（I/R group）.At 0,24,48,72 hours after operation,the mice were divided into 4 groups with 6 mice in each group.In I/R group,the bilateral renal pedicles were bluntly separated with elbow tweezers,and the bilateral renal pedicles were clamped with a non-invasive microvascular clamp for 35 minutes.Sham operation group（control group）:anesthesia,fixation and laparotomy were the same as before,only exposing and dissociating the bilateral renal pedicle,without clamping the renal pedicle.BUN,Scr detection,HE staining to confirm renal ischemia-reperfusion animal model was established successfully.（3）q PCR was used to detect the expression of circLRIG1 in renal tubular epithelial cell hypoxia-reoxygenation model and renal ischemia-reperfusion animal model.3.Functional verification of circLRIG1 in acute renal injury（1）Construction of silencing circLRIG1 cell model.Human circLRIG1 targeted small interference RNA（si RNA）,was transfected with50n M concentration for 4-6 hours,and then the complete medium was changed to transfect si-circLRIG1 into HK-2 cells.（2）In the anoxia-reoxygenation HK-2 cell model with circLRIG1 silencing,the cell viability was detected by CCK8.The apoptosis rate was detected by flow cytometry and TUNEL staining.The expression of apoptosis-related proteins BAX,BCL-2,Caspase-3and inflammation-related proteins TNF-α、IL-6 and IL-1βwere detected by Western blot.Part2.Study on the mechanism of circLRIG1 regulating ADAM12 by sponging mi R-1263.1.Screening and prediction of molecular biological information of mi RNA sponged by circLRIG1The intracellular distribution of circLRIG1 was detected by（FISH）.Circbank and targetscan websites predict the possible combination of mi RNA downstream of circLRIG1.2.Identification of mi R-1263 regulated by circLRIG1（1）HK-2 cells were divided into normal HK-2 cell group and（NC）,hypoxia-reoxygenation model group.The expression of mi R-1263 in each group was detected by q PCR.（2）The mice were divided into renal ischemia-reperfusion model group（AKI）and control group（control）,q PCR to detect the expression of mi R-1263 in each group.（3）Circ LRIG1 was overexpressed and silenced in HK-2 cells,and the expression of mi R-1263 was detected by q PCR.overexpression and silencing mi R-1263,the expression of circLRIG1 was detected by q PCR.（4）The co-localization of circLRIG1 and mi R-1263 was verified by fluorescence in situ hybridization（fish）.Luciferase reporter gene confirmed the direct binding of circLRIG1and mi R-1263.3.Screening and identification of target genes of mi R-1263（1）Targetscan and mi Rdb predict genes that directly bind to mi R-1263,respectively.QPCR detection of mi R-1263 over-expressed HK-2 predicted the relative expression of m RNA of the gene,among which the change of ADAM12 expression was the most obvious.（2）The expression of ADAM12 in I/R cells and animal model was detected by q PCR.Immunohistochemistry was used to detect the expression of ADAM12 in I/R animal model.（3）The changes of ADAM12 expression in HK-2 cells were detected by overexpression and inhibition of mi R-1263,q PCR,respectively.Western blot was used to detect the expression of ADAM12 in renal tubular epithelial cells that had over expressed mi R-1263.（4）The detection of double luciferase reporter gene verified whether mi R-1263 could bind to ADAM12 directly.Part 3:Functional verification of circLRIG1 regulating ADAM12 by sponging mi R-1263.1.Effects of inhibition of mi R-1263 or supplementation of ADAM12 on proliferation and apoptosis of I/R cells after silencing circLRIG1（1）Verification of transfection efficiency in order to verify the transfection efficiency of silencing circLRIG1,and inhibiting mi R-1263 or supplementing ADAM12,HK-2 cells were divided into blank control group（Blank）,transfection blank plasmid group（si RNA-NC）,silencing circLRIG1 group（si-circLRIG1）,silencing circLRIG1+mi R-1263 inhibitor group（si-circLRIG1&mi R-1263 inhibitor）and silencing circLRIG1+overexpression ADAM12 group（si-circLRIG1&pc DNA3.1 ADAM12）,q PCR detected circLRIG1,mi R-1263 respectively).The expression of ADAM12.（2）CCK-8 to detect the change of proliferation level in each group,flow cytometry and TUNEL to detect the change of apoptosis rate in each group,and Western blot to detect the change of BAL,BCL-2,caspase-3 expression in each group.2.Renal specific AAV2/-9 adeno-associated virus was injected into tail vein to observe the effect of silencing circLRIG1 on renal ischemia-reperfusion injury（1）Verification of silencing efficiency of AAV-circLRIG1-sh adeno-associated virus injected into mice.（2）Renal ischemia/reperfusion model was established in mice silenced with circLRIG1.The mice were divided into two groups:silencing circLRIG1 virus group（AKI-AAV-circLRIG1-sh）and virus unloaded group（AKI-AAV-NC）.（3）The following indexes were detected in the kidney tissue of AKI mice silenced with circLRIG1:the expression level of mi R-1263,ADAM12 was detected by RT-q PCR method.Blood urea nitrogen（BUN）level,creatinine（Scr）level and renal tubulointerstitial semi-quantitative score were detected by immunohistochemistry.The expression of BAX,BCL-2,cleavedcaspase-3 was detected by immune-histochemistry.Result:Part 1:Circ LRIG1 is a circular structure,up-regulated expression in renal tubular epithelial cells hypoxia-reoxygenation model and ischemia-reperfusion renal injury animal model,silencing circLRIG1 promote hypoxia-reoxygenation renal tubular epithelial cell proliferation,inhibit apoptosis,reduce the level of inflammation1.Circ LRIG1 homology of human and mouse is 83.7%,agarose gel electrophoresis,actinomycin D test,RNase R enzyme digestion test confirmed that circLRIG1 is a circular structure.2.The expression of circLRIG1 in HK2 cells increased significantly in hypoxia-reoxygenation model group（p<0.001）.The expression of circLRIG1 in renal ischemia-reperfusion animal model was significantly increased,and with the extension of time,the expression of circLRIG1 increased gradually（p<0.05）.3.In HK-2 cells transfected with circLRIG1si RNA,CCK8 results showed that the cell viability was significantly increased in silenced circLRIG1 group.The results of flow cytometry and Tunel showed that the level of apoptosis decreased significantly in the silenced circLRIG1 group（p<0.05）.Western blot showed that the expression of BAX and Caspase-3 in the silenced circLRIG1 group was significantly decreased,while the expression of BCL-2 protein was increased.4.Western blotting showed that the expression of inflammation-related proteins TNF-α,IL-6 and IL-1βdecreased in silenced circLRIG1 group.Part 2:Study on the Mechanism of circLRIG1 regulating ADAM12 by sponging mi R-12631.Circ LRIG1 is expressed in both nucleus and cytoplasm.Bioinformatics prediction shows mi R-1263 can be combined with circLRIG1 on both circbank and circinteractome websites.2.The expression of mi R-1263 was down-regulated in I/R cells and animals,and circLRIG1 could negatively regulate and direct binding to mi R-1263.The results showed that the expression of mi R-1263 decreased in hypoxia-reoxygenation group（p<0.001）,and the expression of mi R-1263 decreased with the prolongation of ischemia time in the animal model of renal ischemia-reperfusion injury（p<0.001）.After silencing circLRIG1,the expression of mi R-1263 increased significantly.Overexpression of circLRIG1,mi R-1263 decreased significantly（p<0.001）.After silencing mi R-1263,the expression of circLRIG1 increased significantly.Overexpression of mi R-1263,circLRIG1 decreased significantly（p<0.01）.It is suggested that circLRIG1 can negatively regulate mi R-1263,mi R-1263 can affect circLRIG1 in HK-2 cells.FISH results showed that there was obvious co-localization of circLRIG1 and mi R-1263 in the cytoplasm of renal tubular epithelial cells（yellow fluorescence）.The direct binding of circLRIG1 and mi R-1263 was verified by double luciferase reporter gene.3.The expression of ADAM12 was up-regulated in I/R cells and animals,and mi R-1263negatively regulated the expression of ADAM12.Targetscan and mi Rdb predict the intersection of 10 genes that could directly bind to mi R-1263,overexpression of mi R-1263,ADAM12 in HK-2 cells decreased most significantly（p<0.005）.The expression of ADAM12 in hypoxia-reoxygenation HK2cells was significantly increased（p<0.05）,and the expression of ADAM12 in I/R animal model was significantly increased（p<0.001）.The expression of ADAM12 decreased significantly after overexpression of mi R-1263（p<0.001）,and increased significantly after inhibition of mi R-1263（p<0.001）.The protein expression of ADAM12 decreased gradually with the increase of mi R-1263 mimics transfection concentration（p<0.001）.The expression of ADAM12 decreased significantly after silencing circLRIG1,and the direct binding of mi R-1263 and ADAM12 was verified by double luciferase reporter gene.Part 3:Functional verification of circLRIG1 regulating ADAM12 by sponging mi R-1263.1.In the hypoxia-reoxygenation model of HK-2 cells,silencing circLRIG1 promoted cell proliferation and inhibited apoptosis.When inhibiting mi R-1263 or supplementing ADAM12 at the same time,the effects of promoting cell proliferation and inhibiting apoptosis were restored.The results of CCK-8 detection showed that the cell viability was increased after silencing circLRIG1,and the viability was decreased again after inhibiting mi R-1263 or supplementing ADAM12 at the same time.The results of flow cytometry,TUNEL and Western blot showed that the level of apoptosis decreased after silencing circLRIG1,and the level of apoptosis increased again after inhibiting mi R-1263 or supplementing ADAM12 at the same time.The inhibitory effect of silencing circLRIG1 on apoptosis was restored.2.Intravenous injection of AAV2/-9 adeno-associated virus silencing circLRIG1,into the caudal vein could alleviate renal ischemia-reperfusion injury in AKI miceSilenced circLRIG1 AKI mice’s blood urea nitrogen,creatinine,renal tubular injury score and renal injury induced by HE staining were significantly reduced.The expression of mi R-1263 in kidney tissue was significantly up-regulated.The expression of ADAM12 was significantly down-regulated.The staining intensity of BAX,cleaved caspase-3 decreased significantly,while that of BCL-2 increased significantly.Conclusion:1.The expression of circLRIG1 was up-regulated in cell model and animal model of renal ischemia-reperfusion injury.In vitro studies have confirmed that silencing circLRIG1 can promote cell proliferation and inhibit apoptosis in the process of renal ischemia-reperfusion injury.2.In vitro studies confirmed that circLRIG1 negative regulation mi R-1263,mi R-1263negative regulation ADAM12,confirmed the existence of circLRIG1-mi R-1263-ADAM12 regulatory axis in the process of renal ischemia-reperfusion injury.3.In vitro studies have confirmed that circLRIG1 regulates cell proliferation and apoptosis through the circLRIG1-mi R-1263-ADAM12 axis in the process of renal ischemia-reperfusion injury.In vivo studies have confirmed that silencing circLRIG1 can reduce the level of apoptosis and reduce renal injury in the process of renal ischemia-reperfusion injury. |
