Silencing Of Cyp4a14 Inhibits Proliferation And Fibrosis Of Mouse Mesangial Cells

Background: Diabetic nephropathy(DN)is a major chronic complication of diabetes.If it is not treated effectively,it will often progress irreversibly within a few years,eventually leading to end-stage renal disease.The pathogenesis of diabetic nephropathy is very complex,and there are a lot of comprehensive factors involved.Traditional blood glucose control and drug treatment can control the disease to some extent,but the effect is not satisfactory.In our previous study,we found that lncRNA CYP4B1-PS1-001 can effectively inhibit the proliferation and fibrosis of mouse mesangial cells(MMCs),and RNA microarray results also indicate that Cyp4a14 is the nearby mRNA of lncRNA CYP4B1-PS1-001.The Cyp4a14 gene belongs to the cytochrome P450(CYP450)family which encodes proteins involved in the synthesis of cholesterol,steroids,and other lipids.Some studies have reported that Cyp4a14 is related to renal hemodynamics,sugar and lipid metabolism,but the role of Cyp4a14 in diabetic nephropathy is still unclear.Objective: To detect the expression level of Cyp4a14 in kidney tissue and high glucose-cultured MMCs of diabetic nephropathy mice,and to explore its relationship between Cyp4a14 and lncRNA CYP4B1-PS1-001,then to investigate the the effect of Cyp4a14 on the proliferation and fibrosis of MMCs,and to explore its role in the occurrence and progression of DN.Methods: C57BL/Ks J background db/db mice,which is regarded as the model for type 2 diabetes,and control db/m mouse models were enrolled for the kidney tissues.RNA microarray and Gene Ontology(GO)analysis were used to screen the nearby mRNA of lncRNA CYP4B1-PS1-001 and select Cyp4a14 as the research target.Real-time quantitative PCR(RT-qPCR)were used to verify the gene expression in mouse kidney and mouse mesangial cells(MMCs).Subsequently,we obtained lncRNA CYP4B1-PS1-001 stably expressed cell line via Lentivirus packaging and infecting MMCs.We use RT-qPCR and Western blot to detect the expression level of Cyp4a14,respectively.To silence the expression of Cyp4a14,we transfect MMCs with the siRNA designed for Cyp4a14,then RT-qPCR and Western blot were performed to verify its silencing efficiency.A series of functional experiments were performed: Western blot and RT-qPCR to identificate the expression of PCNA,Cyclin D1,collagen I and fibronectin and other cell proliferation and fibrosis markers;CCK-8 assay to identificate cell proliferation activity;plate colony formation assay to detect the colony formation ability.Results:(1)According to the result of RNA microarray of renal tissue of diabetic nephropathy db/db mice and control db/m mice,the abnormally high expression gene Cyp4a14 in the kidney tissue of db/db mice was screened.The RT-qPCR results of the mice kidney tissue samples were consistent with the microarray results;(2)Cyp4a14 is a nearby mRNA of lncRNA CYP4B1-PS1-001.CYP4B1-PS1-001 is lowly expressed in renal tissues of diabetic nephropathy mice and can inhibit proliferation and fibrosis of mesangial cells;(3)The mouse mesangial cell line that stably expressing lncRNA CYP4B1-PS1-001 was constructed and collected for RT-qPCR and Western blot experiments.The results showed that overexpression of lncRNA CYP4B1-PS1-001 can downregulate the expression of Cyp4a14;(4)After silencing Cyp4a14 with specific siRNA transfected by MMCs,Western blot and RT-qPCR assay showed that the proliferation and fibrosis markers(PCNA,Cyclin D1,Collegn Ⅰ and fibronectin)expression levels were inhibited;CCK-8 experiment results The results showed that the cell proliferation activity decreased.The plate colony formation assay results showed that compared with the negative control group,the number of colonies formed in the MMCs transfected with siRNA-Cyp4a14 was significantly reduced.Conclusion: Cyp4a14 was highly expressed in MMCs cultured in high glucose environment,and the expression level was regulated by lncRNA CYP4B1-PS1-001;siRNA-Cyp4a14 could significantly inhibit the proliferation and fibrosis of mouse mesangial cells.