| Introduction:With the aging process of the global population,aging has become a hot issue in research.Kidney is one of the most obvious organs in the process of aging.It is very important to understand the molecular mechanism of renal cell senescence and find intervention targets to delay renal senescence.Circular ribonucleic acid（circ RNA）is a covalently closed endogenous transcript,which is characterized by covalent linkage at the 3’-5’end.Compared with linear RNA,circ RNA has higher stability,tissue specificity and evolutionary conservation.Previous studies have shown that circ RNAs are involved in the aging process of multiple organs,but the mechanism related to kidney aging is not clear.This study aims to explore the regulatory role of circular RNA in the process of kidney aging,and provide potential therapeutic targets for preventing kidney aging.We found that the expression level of has＿circ＿0127071 was higher than that of young kidney tissue（old group vs.young group,up,logfc3.61,P=0.02;exonic;337）in human aging kidney tissue by transcriptional gene assay.At present,there is no research report on hsa＿circ＿0127071 effect and mechanism on renal aging.Eukaryotic translation initiation factor 4A3（EIF4A3）participates in the regulation of cell cycle and apoptosis process by regulating the attenuation of messenger RNA（m RNA）,and is an important regulator of disease occurrence and development.EIF4A3 is an RNA binding protein that mediates the exon splicing process by binding to RNA to generate an exon junction complex.EIF4A3 can be recruited by long noncoding RNA,affecting the translation of tumor target genes.EIF4A3 also plays an important role in pre m RNA splicing,and can bind to the upstream region of some specific m RNAs,thereby inducing the synthesis of circ RNAs.We performed by RNA protein interaction prediction（RIPseq）（iastate.edu）,the binding prediction of hsa＿circ＿0127071 and EIF4A3 suggested that there might be a binding relationship between the two with 0.5 as the threshold.JAK/STAT signaling pathway is involved in the survival,proliferation and invasion of tumor cells,so it has become an important target for tumor therapy.There are four subtypes of JAK proteins（Jak1,JAK2,JAK3,and Tyk2）,and seven subtypes of STAT proteins（STAT1,stat2,STAT3,STAT4,STAT5A,STAT5b,and STAT6）.JAK2 is an important cytosolic tyrosine kinase,located downstream of cytokine（growth factor）receptors,which promotes the proliferation of normal and tumor cells.JAK2 activates major downstream targets,including STAT5,PI3K,and extracellular signal regulated kinases.JAK2/STAT signaling pathway is involved in many biological processes such as cell differentiation,proliferation,apoptosis,immunity and hematopoiesis.JAK2 and STAT5 are considered as key mediators of tumor cell growth and survival.In PC12 cell model of neurological disease,JAK2/STAT5 pathway activation can improve cell survival and reduce apoptosis.We predicted the mi RNAs adsorbed by circ RNAs based on the target gene prediction software of Miranda and targetscan,using KEGG pathway（http://www.kegg.jp）.The target gene dataset is mapped to the pathway map to explain the biological functions of these molecules at the pathway level.Through the analysis of hsa＿circ＿0127071 corresponding target gene pathway analysis showed that JAK2/STAT5pathway had obvious advantages.So far,the specific mechanism of hsa＿circ＿0127071 and JAK2/STAT5 pathway involved in renal aging has not yet been reported.In conclusion,this study intends to use human kidney tissue and human mesangial cells for validation experiments through transcriptional gene assay,KEGG pathway and other prediction,aiming to explore the induction of hsa＿circ＿0127071 by EIF4A3promotes the aging mechanism of human mesangial cells through JAK2/STAT5 signaling pathway,in order to provide a new therapeutic target for the prevention and treatment of renal aging.Methods:Part Ⅰ:The kidney tissue samples of 76 patients who underwent radical nephrectomy for urological tumors or trauma in the First Affiliated Hospital of China Medical University from March 12,2018 to August 15,2018 were collected.Select qualified human kidney tissue according to the inclusion criteria.During the operation,samples were taken and immediately embedded in paraffin or frozen in liquid nitrogen.The senescence-related marker proteins P53 and P21waf were detected by Masson and immunohistochemistry.Combined with the natural age of the patient,the aging level of the patient’s kidney tissue was comprehensively evaluated,and the patient was divided into aging and young groups（5 cases each）.Renal tissue samples for hsa＿circ＿0127071 were tested by q RT-PCR.Then,we used bioinformatics method to retrieve hsa＿circ＿0127071.We tested hsa＿circ＿0127071 with Sanger sequencing,RNase R and actinomycin（D）,to verify whether it has cyclic RNA characteristics.Western blot was used to detect the levels of JAK2 and p STAT5 in kidney tissue of each group.Through the above experiments,it is preliminarily found that the change of hsa＿circ＿012707 and JAK2/STAT5 pathway in aging renal.We put forward assumptions for the second part of the study according it.Part Ⅱ:Human glomerular mesangial cells（HGMC）were cultured in vitro,and the cultured HGMC was stimulated with 250 mg/L advanced glycation end products（AGEs）or equivalent volume of bovine serum albumin（BSA）for 0,24,48,72,96 hours,respectively.Western blot was used to detect the expression of aging related proteins(P53,P21waf,P16)and pathway proteins（JAK2,p-STAT5,STAT5）,the aging level of cells every group and the changes in JAK2/STAT5 pathway activation level between groups were observed and compared.The best conditions for AGEs to induce mesangial cells aging was obtained,and according that the aging model of human mesangial cells can be established and verified,The changes of JAK2/STAT5 pathway during the aging of human mesangial cells were also found.AGEs stimulated HGMC for 72 hours to establish aging group（hsa＿circ＿0127071（-）-NC or hsa＿circ＿0127071（+）-NC）;hsa＿circ＿0127071 was silenced,relevant experimental groups are as follows:after si RNA specific silencing hsa＿circ＿0127071 was stimulated with AGEs for 72 hours,hsa＿circ＿0127071（-）group was established.Hsa＿circ＿0127071 was silenced,the hsa＿circ＿0127071（-）+EPO group was established by co-stimulation of AGEs and 200U/m L EPO for 72 hours,and hsa＿circ＿0127071 was silenced The cells were co-stimulated with AGEs and 1 ug/u L losartan for 72 hours to establish the hsa＿circ＿0127071（-）+losartan group,and the same method was used to establish the hsa＿circ＿0127071（-）+EPO+losartan group;Relevant experimental groups of overexpression of hsa＿circ＿0127071 are as follows:targeting hsa＿circ＿0127071 overexpression plasmid overexpress hsa＿circ＿0127071,hsa＿circ＿0127071（+）group was established after 72hours of AGEs stimulation;and hsa＿circ＿0127071 was overexpressed,hsa＿circ＿0127071（+）+AG490 group was established with AGEs and 10 umol/L AG490 stimulating for 72 hours;and hsa＿circ＿0127071 was overexpressed,hsa＿circ＿0127071（+）+losartan group was established by stimulation with AGEs and 1 ug/u L losartan for 72 hours,and hsa＿circ＿0127071（+）+AG490+losartan group was established by the same method;Western blot was used to detect the expression of aging related proteins(P53,P21waf,P16)and pathway proteins（JAK2,p-STAT5,STAT5）;In situ staining ofβ-galactosidase was used for observation and comparison the aging level of cells in each group.Compare the JAK2,STAT5 level and HGMC aging proteins level between groups.Then it could be further confirmed that has＿circ＿0127071 can induce human renal mesangial cells aging by regulating the JAK2/STAT5 signal pathway.Part Ⅲ:Combined prediction of hsa＿circ＿0127071 and EIF4A3（uniprotid:P38919）was did by RNA-Protein Interaction Prediction（RPISeq）.Pull-down and RIP experiments prove that EIF4A3 and hsa＿circ＿0127071 interact between downstream areas.Hsa＿circ＿0127071 expression changes in the case of EIF4A3 silence or overexpression were detected by PCR.FISH identified the relation of hsa＿circ＿0127071and EIF4A3.All of these have studied the effects of EIF4A3 on hsa＿circ＿0127071 expression.The EIF4A3 was silenced and overexpressed in the cells by the sh EIF4A3 and ov EIF4A3 plasmids,respectively.We tested hsa＿circ＿0127071 expression changes in the case of EIF4A3 silence or overexpression.Sh EIF4A3 and ov EIF4A3 were divided into groups,and the expression of aging related proteins(P53,P21waf,P16)and pathway proteins（JAK2,p-STAT5,STAT5）were detected by Western blot,in situ staining ofβ-galactosidase was used to observe and compare the aging level of cells in each group.Comparing the JAK2-STAT5 level and HGMC aging proteins level between groups,and then verify that EIF4A3 inducing hsa＿circ＿0127071 promotes kidney aging process through JAK2/STAT5 pathway.Results:Part Ⅰ:The degree of glomerulosclerosis in the renal cortex of the elderly group was significantly higher than that of the young group.Compared to the younger group,hsa＿circ＿0127071 was significantly up-regulated in aging kidney tissue.Though retrieval of hsa＿circ＿0127071 by bioinformatics,we found that hsa＿circ＿0127071 formed the 2-3exon of SCD5 gene.Process hsa＿circ＿0127071 with RNase R,it was verified that it had enzyme tolerance.Western blot showed that the levels of JAK2 and STAT5 in the elderly group were significantly higher than those in the young group（P<0.05）.Part Ⅱ:Long-term（≥48h）AGEs stimulation can induce HGMC aging.Western Blot detection showed that the expression of cell senescence related protein P53/P21waf/P16 was up-regulated and HGMC aging relatedβ-galactosidase staining was deepened,and the JAK2/STAT5 pathway protein was up-regulated,which was time-dependent within 72 hours.Silent hsa＿circ＿0127071,western blot showed that compared with hsa＿circ＿0127071（-）-NC group,JAK2/STAT5 signal pathway protein was down-regulated and HGMC aging marker protein P53/P21waf/P16 was down-regulated.The positive rate ofβ-galactosidase staining decreased;the up-regulation or down-regulation of JAK2/STAT5 signal pathway protein in hsa＿circ＿0127071（-）+EPO group or hsa＿circ＿0127071（-）+losartan group compared with Sh RNA group affects the up-regulation or down-regulation of HGMC aging marker protein P53/P21waf/P16 expression,and the positive rate ofβ-galactosidase staining.Compared with hsa＿circ＿0127071（-）+EPO group or hsa＿circ＿0127071（-）+losartan group,the expression level of JAK2/STAT5signal pathway protein and HGMC aging marker protein P53/P21waf/P16 and the positive rate ofβ-galactosidase staining in hsa＿circ＿0127071（-）+EPO+losartan group intervened between the two groups.Overexpression of hsa＿circ＿0127071,western blot showed that compared with hsa＿circ＿0127071（+）-NC group,JAK2/STAT5 signal pathway protein and HGMC aging marker protein P53/P21waf/P16 were up-regulated,The positive rate ofβ-galactosidase staining increased;Compared with hsa＿circ＿0127071（+）group,the expression of JAK2/STAT5 signal pathway protein and HGMC aging marker protein P53/P21waf/P16were down-regulated in hsa＿circ＿0127071（+）+AG490 group or hsa＿circ＿0127071（+）+losartan group,the positive rate ofβ-galactosidase staining decreased.Compared with hsa＿circ＿0127071（+）+AG490 or hsa＿circ＿0127071（+）+losartan group,in ov RNA+AG490+losartan group the expression of JAK2/STAT5 signal pathway protein and aging marker protein P53/P21waf/P16 were down-regulated,the positive rate ofβ-galactosidase staining decreased.Part Ⅲ:silencing and overexpressing EIF4A3 in cells by si-EIF4A3 and ov-EIF4A3plasmids,respectively.RIP experiment showed that in sh EIF4A3 cell,hsa＿circ＿0127071was down-regulated.In the cells overexpressing ov EIF4A3,the expression of hsa＿circ＿0127071 was up-regulated.FISH identification hsa＿circ＿0127071 and EIF4A3.RIP,pull down and FISH experiments showed that EIF4A3 can combine with hsa＿circ＿0127071and promote its expression.Compared with EIF4A3（-）-NC group,the expression of EIF4A3 was down-regulated,and the expression of JAK2/STAT5 signal pathway protein and aging marker protein P53/P21waf/P16 was down-regulated,the positive rate ofβ-galactosidase staining decreased.Compared with EIF4A3（-）+EPO group or EIF4A3（-）+losartan group and EIF4A3（-）group,the up-regulation or down-regulation of JAK2/STAT5 signal pathway protein affected the up-regulation or down-regulation of HGMC aging marker protein P53/P21waf/P16 expression,and the positive rate ofβ-galactosidase staining;Compared with EIF4A3（-）+EPO group or EIF4A3（-）+losartan group,in EIF4A3（-）+EPO+losartan group the expression level of JAK2/STAT5 signal pathway protein and HGMC aging marker protein P53/P21waf/P16 and the positive rate ofβ-galactosidase staining intervened between the two groups.Western blot showed that the expression of JAK2/STAT5 signal pathway protein and HGMC aging marker protein P53/P21waf/P16 were up-regulated in EIF4A3（+）group compared with EIF4A3（+）-NC group,and the positive rate ofβ-galactosidase staining increased.Compared with EIF4A3（+）+AG490 group or EIF4A3（+）+losartan group and EIF4A3（+）group,JAK2/STAT5 signal pathway protein and HGMC aging marker protein P53/P21waf/P16 expression were down regulated,and the positive rate ofβ-galactosidase staining decreased.Compared with EIF4A3（+）+AG490 or EIF4A3（+）+losartan group,the expression of JAK2/STAT5 signal pathway protein and aging marker protein P53/P21waf/P16 in EIF4A3（+）+AG490+losartan group were down-regulated,and the positive rate ofβ-galactosidase staining decreased.Conclusion:Part Ⅰ:Hsa＿circ＿0127071 has cyclic RNA characteristics,hsa＿circ＿0127071 and JAK2/STAT5 pathway are involved in the aging process of human glomerulus.Part II:JAK2/STAT5 signal pathway positively regulates the aging process of human mesangial cells.The activator and blocker of JAK2/STAT5 signal pathway will affect the activity of the pathway,and then affect the aging of HGMC.The activating or blocking effect is not affected by silent hsa＿circ＿0127071.Hsa＿circ＿0127071 regulates the activity of JAK2/STAT5 signal pathway in a positive way,thereby positively regulating the occurrence of HGMC aging.Part III:RNA binding protein EIF4A3binding to hsa＿circ＿0127071 promotes its expression.EIF4A3 regulates the activity of JAK2/STAT5 signal pathway in a positive way,thereby positively regulating the occurrence of HGMC aging. |
