| Background:Chronic kidney disease(CKD)is a common clinical disease.Because of its high prevalence and lack of effective treatment,it causes glomerulosclerosis and interstitial fibrosis and eventually progresses to end-stage renal disease.Mesangial proliferative nephritis is one of the important causes of CKD.The main pathological features of the disease are mesangial cell proliferation and increased mesangial matrix.At present,the clinical treatment of the disease is general,and the side effects of the drug are large.Failure to stop the progression to end-stage renal disease was fundamentally prevented.Therefore,exploring the molecular mechanism of mesangial cell proliferation and searching for effective methods to inhibit mesangial cell proliferation and matrix hyperplasia has become a new direction in the research of kidney diseases.Transcription factors play a crucial role in the regulation of cell proliferation.Previous studies have found that the transcription factor KLF15 can inhibit the proliferation of mesangial cells in vitro,but its specific molecular mechanism has not yet been fully elucidated.Objective:This article aims to use ChIP-seq and SILAC-LC/MS analysis to determine the target genes and binding sites for KLF15-inhibiting mesangial cell proliferation;To investigate the molecular mechanisms of KLF15-mediated gene targeting inhibition of mesangial cell proliferation in vitro and in vivo.Method:(1)Chromatin co-precipitation technique(ChIP)was used to specifically enrich small fragments of DNA that specifically binds to the transcription factor KLF15 in human primary mesangial cells(HRMC)and then purified.High-throughput sequencing technology(HiSeq)was used to detect the DNA fragments of the samples and match them with the reference genomes.The in-depth analysis of the enriched regions was performed,and finally the gene data that directly combined with KLF15 were obtained.(2)Using the cell culture stable isotope labeling technique(SILAC),the heavy chain and light chain isotope labeled culture medium was used to culture the HRMCs to the sixth passage,and the KLF15 plasmids were transfected with the heavy chain test group(HK)respectively.Group(LC)was transfected with the blank virus,and the collected protein samples(HK:LC = 1:1)were mixed and sent to the Beijing Proteomics Research Center for liquid mass spectrometry(LC/MS)detection.The differential protein data after overexpression of KLF15 was obtained.(3)GO analysis was performed on ChIP-seq results using the www.uniprot.org tool website.SILAC results were analyzed by GO and PATHWAY at the Beijing Computer Center.Finally,the comprehensive gene and protein data were used to screen KLF15 targeted genes.A small Ubiquitin-like Modifier(SUMO1)was used to perform a Motif analysis of SUMO1 using the http://meme.edi.edu.au/website.In addition,analysis of the IPA database and SUMOsp software revealed that P53 is a direct downstream binding molecule of SUMO1.(4)High glucose(30 mmol/L)and platelet-derived growth factor(20 ng/mL)stimulated HRMC(48 h)to induce proliferation of mesangial cells,and Western blot and real-time PCR were used to detect the expression of KLF15 and SUMO1.In the normal proliferation of HRMC,transfected KLF15 plasmid,SUMO1 plasmid,SUMOl siRNA,co-transfected KLF15 and SUMO1 siRNA,the use of Western blot detection of KLF15,SUMO1,P53 expression in each group of samples,Immunoprecipitation(IP)was used to detect the expression of SUMO1-P53 in each group.The proliferation of cells was detected by EDU method.The proliferation of HRMC induced by PDGF was transfected with KLF15 plasmid and SUMO1 plasmid,respectively.The siRNA of SUMO1 was co-transfected with KLF15 and SUMO1 siRNA.The expression of KLF15,SUMO1,and P53 in each sample was detected by Western blot.The expression of SUMO1-P53 was detected by IP,and the cell proliferation was detected by EDU.(5)The Western blot of kidney tissue in Thy-1 nephritis model indicated that the expression of KLF15,SUMO1 and P53 in each group gradually decreased from the 0th,3d,and 5th day,the lowest on the 5th day,and gradually increased on the 7th and 10th day.Immunohistochemistry indicated that the expression of PCNA in glomerular mesangial cells gradually increased from day 0,day 3,and day 5,and was highest on the 5th day,and gradually decreased on the 7th and 10th days.The results showed that the expression of KLF15,SUMO1 and P53 gradually decreased with the progress of mesangial proliferative nephritis,and the proliferation of mesangial cells was gradually aggravated.The self-restricted model of Thy-1 nephritis showed that the indexes gradually recovered after the 10th day.Result:(1)ChIP-seq obtained 2478 possible target genes directly regulated by KLF15.SILAC-LC/MS detected 1357 differential proteins after overexpression of KLF15.Combined with ChIP and SILAC results,we analyzed 52 common differential expressions.The gene/protein and its 3 Modifs,of which 5 proteins involved in cell proliferation-related processes,combined with GO and Pathway analysis results,I finally screened for SUMO1 as a KLF5-regulated mesangial cell proliferation targeting gene.Motif analysis found that KLF15 binding sequence was present in the SUMO1 promoter region.The use of ChIP-PCR and dual luciferase reporters verified that the KLF15 transcription factor can directly bind to the SUMO1 promoter region and play a role.We used IPA database analysis to find that P53 may be a direct downstream molecule of SUMOl and participate in cell proliferation process.Using SUMOsp software,we found that K53 of P53 has the typical sequence of SUMO1 binding Ψ-K-x-D/E(Ψ is a hydrophobic group,K is the SUMOs modified lysine site,x is any amino acid,and D/E is any acidic amino acid in aspartic acid or glutamic acid).(2)After high glucose and PDGF induced mesangial cell proliferation by RT-PCR and Western blot,the expression of KLF15 and SUMOl gene and protein were decreased.It shows that there is indeed an interaction between KLF5 and SUMO1.(3)Under the normal proliferation condition of HRMC,the expression of KLF15,SUMO1 and P53 in the transfected group was increased after transfection of KLF15 plasmid,and the SUMOylation(SUMO1-P53)level of P53 was increased.The cell proliferation was decreased.After transfection of SUMO1 plasmid,there was no significant difference in the expression of KLF15 in the transfection group compared with the control group.The SUMOI and P53 expressions were increased,and the SUMOylation level of P53 was also increased,and the cell proliferation was reduced.After transfected with siRNA of SUMO1,the expression of KLF15 in the transfection group had no significant difference compared with the control group.The expression of SUMO1 and P53 was decreased,the SUMOylation of P53 was decreased,and the cell proliferation was increased;and KLF15 and SUMOl were co-transfected.After siRNA,compared with the control group,there was no significant difference in the expression of KLF15 in the co-transfection group.The expression of SUMO1 was slightly decreased,the expression of P53 was slightly decreased,the SUMOylation of P53 was decreased,and the cell proliferation was increased.(4)Under the condition of PDGF induced HRMC proliferation,KLF15,SUMO1,P53,and SUMO1-P53 expression were increased and the cell proliferation was decreased after KLF15 plasmid transfection.After transfection of SUMO1 plasmid,KLF15 expression did not change significantly.The expressions of SUMO1,P53,and SUMO1-P53 were increased and the cell proliferation was decreased.After transfected with siRNA of SUMO1,the expression of KLF15 was not changed,and the expression of SUMO1,P53,and SUMO1-P53 were decreased,and the cell proliferation was increased;and KLF15 was co-transfected.After the siRNA of SUM01,the expression of KLF15 did not change significantly,SUMO1,P53,and SUMO1-P53 slightly decreased,and cell proliferation increased.(5)The Western blot of kidney tissue in Thy-1 rat model indicated that the expression of KLF15,SUMO1 and P53 in each group gradually decreased from the 0th,3d,and 5th day,the lowest on the 5th day,and gradually increased on the 7th and 10th day.Immunohistochemistry indicated that the expression of PCNA in glomerular mesangial cells gradually increased from day 0,day 3,and day 5,and was highest on the 5th day,and gradually decreased on the 7th and 10th days.The results showed that the expression of KLF15,SUMO1 and P53 gradually decreased with the progress of mesangial proliferative nephritis,and the proliferation of mesangial cells increased gradually.The most obvious was on the 5th day,and gradually recovered on the 10th day.Conclusion:KLF15 can enhance the SUMOylation of P53 by regulating the expression of SUMO1,inhibiting the proliferation of mesangial cells. |
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