Differential Expression Of Circular RNA In Rat Model Of Chronic Glomerulonephritis And Intervention Effect Of Qi Teng Xiao Zhuo Granule

ObjectiveIn the early stage,gene chip were used to screen gene expression patterns in chronic glomerulonephritis(CGN)rat models induced by adriamycin(ADR)and CGN rats after intervenion with Qitengxiaozhuo granules.Combined with the research basis of the research group mentioned above,high through-put sequencing(HTS)technology was used to detect the changes of circular RNA(circ RNA)expression profile in rat kidney tissues of control group,CGN model group and Qitengxiaozhuo granules group,to explore the possible pathogenesis of CGN and the possible mechanism of action of Qitengxiaozhuo granules.MethodFifty male Sprague-Dawley rats,10 were randomly selected as the control group,and the other rats were injected with ADR on the first and fourteenth day respectively.On the first day,the dose of ADR was 4.0 mg/kg and the ADR dose of the 14 th day was 3.5 mg/kg.On the 21 st day,all the rats were placed in metabolic cages and the urine was collected for 24 hours to measure the total amount of 24-hour urine protein.The criteria for judging the successful models was the total 24-hour urinary protein >50 mg/kg.Subsequently,30 successful model rats were randomly selected and randomly divided into model group,Qitengxiaozhuo granules group(10.8g/kg)and Huangkui capsule group(1g/kg),with 10 rats in each group.The rats in the Qitengxiaozhuo granules group and Huangkui capsule were given the corresponding doses of the drugs,and the other groups were given the same amount of normal saline,once a day for 30 days.After the last dose,all rats were placed in metabolic cages again for collecting the 24-hour urine and the total amount of 24-hour urine protein were measured.At the end of the experiment,the renal organ index was calculated by weighing the bilateral kidneys weight of rats and then dividing it by the corresponding body weight of rats.;hematoxylin-eosin staining(HE)staining and electron microscopic were used to observe the pathological changes of renal tissue in each group;HTS was applied to detect the circ RNA expression profile in renal cortex of each group;the screening criteria for the circ RNA with significant difference is FC> 2 and P<0.05.Based on mi Randa's proprietary mi RNA target prediction software(http://www.microrna.org)predicts all differentially expressed circ RNAs-targeted mi RNAs.The potential mi RNA-targeted m RNAs were predicted using mi RTar Base,and the circ RNA-mi RNA-m RNA ce RNA network was further constructed by Cytoscape(http://cytoscape.github.io/)software.Gene Ontology(GO)and pathway analysis were performed on m RNAs in the above circ RNA-mi RNA-m RNA ce RNA network using the clusterprofiler software.q RT-PCR(quantitative real-time polymerase chain reaction)verification were performed in all the control group,model group and Qitengxiaozhuo group.Results1.Results of high-throughput sequencing: a total of 5363 circular RNA were detected in 9 groups of samples.After filtering by the screening criteria: FC> 2 and P<0.05,31 circ RNA were differentially expressed between the control group and the CGN model group,among which 14 circ RNA were up-regulated and 17 circ RNA were down-regulated in the CGN model group.The 31 circ RNA were widely distributed on all chromosomes.21 circ RNA were differentially expressed between the model group and the Qitengxiaozhuo granules group.Comparing the 21 circ RNA with the above 31 circ RNA,there were 4 circ RNAs that were reversed by the Qitengxiaozhuo granules due to the imbalance caused by the replication of ADR induced CGN.2.Bioinformatics analysis of differentially expressed circ RNAs: there were at least 4 potentially targeted mi RNAs per differentially expressed circ RNA between the model and the control groups,and more than 83% of circ RNAs had at least 23 potentially targeted mi RNAs.Further bioinformatics analysis of differentially expressed circ RNAs showed that the potentially targeted mi RNAs and m RNAs were closely related to the occurrence and development of CGN;similarly,the potentially targeted mi RNAs and m RNAs of the 4 circ RNAs reversed by Qitengxiaozhuo granules are also closely related to the development of CGN.3.q RT-PCR verification results of differentially expressed circ RNA: 2 up-regulated circ RNA:rno-circ RNA-689,rno-circ-RNA-3217 and 2 down-regulated circ RNA: rno-circ RNA-1327 and rno-circ-RNA-5001 were verified by q RT-PCR on all the rats in the control group and the model group,the results were consistent with HTS results.In addition,q RT-PCR was used to verify the expression of rno-circ RNA-213,rno-circ RNA-689,rno-circ RNA-3217 and rno-circ RNA-5001 in all rats of the control group,the model group and the Qitengxiaozhuo granules group.The results were consistent with the HTS results too.Conclusions1.Circ RNAs are differentially expressed during the pathogenesis of CGN.2.Qiteng Xiaozhuo granules have a certain therapeutic effect on CGN,and its mechanism may be related to the regulation of differentially expressed circ RNAs.