The Construction And Analysis Of The Key CeRNA Network In Diabetic Nephropathy And The Study Of H2k2-miR-449a/b-Trim11 Regulating The Proliferation Of Mesangial Cells

Part ? : Analysis and verification of key ce RNA network for diabetic nephropathyObjective To detect differentially expressed m RNA and lncRNA in diabetic nephropathy mice,construct a key ce RNA interaction network for diabetic nephropathy,and screen for key ce RNA pairs that may be involved in cell proliferation of diabetic nephropathy.Methods High-throughput sequencing technology was used to obtain m RNA and lncRNA differentially expressed in kidney tissues of diabetic nephropathy mice;RNA not included in ENSEMBLE database was removed by Bio Mart;lncRNA-miRNA-m RNA was predicted by diabetic and nephropathy by Mi Randa and Star Base database The role of the network;the use of hypergeometric test to construct differential ce RNA network of diabetic nephropathy,KEGG and GO analysis of differential ce RNA network;analysis of central parameters(Degree and between),the construction of key ce RNA network of diabetic nephropathy;q RT-PCR detected the expression of key ce RNA network in DN tissue and high or low glucose cultured MCs.Results Using the RNA-seq,differentially expression m RNAs and lncRNAs were obtained,and the interaction with significant sharing relationship was retained by hypergeometric test.In the first 15% of degree and between,there were 3 lncRNA and 5 m RNA.The expression of m RNA Trim11 and lncRNA H2k2 in DN tissue and high glucose cultured MCs were consistent with RNA-seq.The expression of miR-449 a and miR-449 b shared by them is consistent with ce RNA principle.Conclusion H2k2/miR-449a/b/Trim11 in the key ce RNA interaction network of DN may be involved in cell proliferation fo MCs.Part ? : Differential expression of lncRNA H2k2 and the effect of targeting miR-449a/b on cell proliferation of MCsObjective To investigate the effect of lncRNA H2k2 on the proliferation of mesangial cells cultured in high and low glucose.Methods The in situ hybridization(FISH)technique and q RT-PCR were used to detect the subcellular distribution of H2k2 in MCs.q RT-PCR detected the efficiency of three si RNA of H2k2 or H2k2 overexpression plasmid in MCs cultured in high or low glucose;EDU detected effects of transfection of si H2k2 or H2k2(+)on cell proliferation of MCs;flow cytometry detected the effect of si H2k2 or H2k2(+)on cell cycle in MCs;Western Blot detected the expression of Cyclin D1 after transfection of si H2k2 or H2k2(+);The dual luciferase assay detected the relationship between H2k2 and miR-449a/b.Results lncRNA H2k2 was distributed mainly in the cytoplasm of MCs,.After transfection of expression plasmid H2k2(+)in low glucose cultured MCs,when H2k2up-regulated,cell proliferation of MCs increased;cell cycle was promoted;Cyclin D1 expression increased;Both si H2k2.2 and si H2k2.3 inhibited the expression of H2k2,in which si H2k2.3 had the best inhibition efficiency.In high glucose cultured MCs,the cell proliferation decreased after knockdown H2k2,cell cycle was prevented and Cyclin D1 decreased.Conclusion Overexpression of lncRNA H2k2 promotes the cell proliferation of MCs.H2k2 has a targeted binding with miR-449a/b;miR-449a/b regulates cell proliferation.Part ?: lncRNA H2k2 regulates the proliferation of mesangial cells cultured in high glucose by miR-449a/b and Trim11-Mek/Erk signaling pathways.Objective To investigate the effect of lncRNA H2k2 targeting miR-449 a or miR-449 b on cell proliferation of MCs by Trim11/Mek/Erk signaling pathway.Methods Bioinformatics predicted the binding sites of miR-449a/b to Trim11;dual luciferase assay verified the targeting of miR-449a/b with Trim11;q RT-PCR detected RNA expression after up-regulation of H2k2 or miR-449a/b;Western Blot detected Trim11,Cyclin D1,Mek,Eek signaling pathway expression after overexpression H2k2 or miR-449a/b;Ed U detected the effect of synergy between H2k2 and miR-449a/b on cell proliferation;flow cytometry detected the effect of miR-449a/b on cell cycle;Western blot and Ed U detcetede together with overexpression of H2k2,Mek inhibitor U0126 or knockdown trim11 on cell proliferation.Results miR-449a/b directly targeted to the Trim11 3'UTR region;when miR-449a/b overexpression,cell proliferation of MCs were decreased;cell cycle were promoted;Cyclin D1 were decreased,Trim11 protein expression were decreased,p-Mek1/2,p-Eek1/2 were decreased;when miR-449a/b knockdown,cells proliferati were increased,Cyclin D1 were increased,Trim11 protein expression were increased,p-Mek1/2,p-Eek1/2 were increased.Conclusion lncRNA H2k2 can inhibit cell proliferation of MCs through miR-449a/b,and its related to inhibited the expression of Mek and Erk by the target gene Trim11.