The Study Of LncRNA/mRNA Expression In Type 2 Diabetes Mellitus With Kidney-yin Deficiency

ObjectiveSubjects in the study werepatients with type 2 diabetes mellitus(T2DM)with kidney-yin deficiency,patients with T2 DM without kidney deficiency,and normal subjects.The study used lncRNAs microarray to screen out the differential lncRNAs and mRNAs related to kidney-yin deficiency and type 2 diabetes mellitus.Meanwhile,bioinformatics analysis would be performed,the possible mechanism of the different lncRNAs in type 2 diabetes mellitus with kidney-yin deficiency would be discussed,and the molecular markers in type 2 diabetes mellitus with kidney-yin deficiency would be screened out.Methods1.The lncRNAs and mRNAs microarray was used in the study in order to get the expression of mRNAs and lncRNAs in patients with T2 DM with kidney-yin deficiency,patients with T2 DM without kidney deficiency and normal subjects.The data was compared to obtain the differentially expressed mRNAs and lncRNAs.According to the differential expression profile data,one differential lncRNA and five mRNAs were screened for RT-PCR validation.2.According to the data in the part one,GO and KEGG analysis were performed on the differential mRNAs.The target genes of differential lncRNAs were predicted.Meanwhile,GO and KEGG analysis were performed on the target genes.Finally,the differential lncRNA-mRNA co-expression network map would be constructed.Results1.Expression of mRNAs and lncRNAs: In comparison of kidney-yin deficiency T2 DM group and T2 DM without kidney deficiency group,117 differential mRNAs and208 differential lncRNAs were found.Among these mRNAs,the expression of 37 mRNAs was up-regulated,and 80 mRNAs was down-regulated.Among these lncRNAs,the expression of 64 lncRNAs was up-regulated,and 144 lncRNAs was down-regulated.In comparison of kidney-yin deficiency T2 DM group and normal subject group,107 differential mRNAs and 275 differential lncRNAs were found.Among these mRNAs,the expression of 88 mRNAs was up-regulated,and 19 mRNAs was down-regulated.Among these lncRNAs,the expression of 217 lncRNAs was up-regulated,and 58 lncRNAs was down-regulated.In comparison of T2 DM without kidney-yin deficiency group and normal subject group,89 differential mRNAs and 309 differential lncRNAs were found.Among these mRNAs,the expression 67 of mRNAs was up-regulated,and 22 mRNAs was down-regulated.Among these lncRNAs,the expression of 100 lncRNAs was upregulated,and 209 lncRNAs was down-regulated.One lncRNA and five mRNAs were selected for RT-PCR in the original samples,and the change trend was consistent with the results of microarray.2.Bioinformatics analysis:In comparison of kidney-yin deficiency T2 DM group and T2 DM without kidney deficiency,differential mRNAs were enriched in 164 significant GO classes such as immune response and GTPase activator activity,involving 30 signaling pathways such as MAPK signaling pathway,osteoclast differentiation,and NFkappa B signaling pathway.The target genes of differential lncRNAs predicted by cis were enriched in 275 significant GO classes such as lymphocyte migration and regulation,MAPK,involving 49 signaling pathways such as pantothenic acid and coenzyme A biosynthesis,regulation of adipocyte lipolysis,and pentose phosphate pathway.The target genes predicted by trans were enriched in 76 significant GO classes such as multicellular tissue homeostasis and GTPase activity,involving 22 signaling pathways such as pantothenic acid and coenzyme A biosynthesis,unsaturated fatty acid biosynthesis,and AMPK signaling pathway.In the co-expression network,there were 275 differential lncRNAs-mRNAs co-expression pairs.Among them,lncRNAs such as HMlinc RNA1030,uc010 atu and DA674418,mRNAs such as SLC11A1,SIRPB2 and HSPA6 were in the center of association.In comparison of kidney-yin deficiency T2 DM group and normal subject group,differential mRNAs were enriched in 97 significant GO classes such as hydrogen peroxide metabolic process and oxidoreductase activity,involving 25 signaling pathways such as apoptosis,AMPK signaling pathway,and pancreatic cancer.The target genes of differential lncRNAs predicted by cis were enriched in 282 significant GO classes such as TOR signal and carboxylic acid binding,involving 43 signaling pathways such as insulin signaling pathway,PPAR signaling pathway,and adipocytokine signaling pathway.The target genes predicted by trans were enriched in 59 significant GO classes such as carbohydrate biosynthesis and exogenous apoptotic signaling pathways,involving 17 signaling pathways such as insulin signaling pathway,cholesterol metabolism,and AMPK signaling pathway.In the co-expression network,there were 273 differential lncRNAs-mRNAs co-expression pairs.Among them,lncRNAs such asuc009 yax,ENST00000447729 and HMlinc RNA-881,mRNAs such as CSDA,MKRN1 and SLC4A1 were in the center of association.In comparison of T2 DM without kidney deficiency group and normal subject group,differential mRNAs were enriched in 90 significant GO classes such as antigen processing and presentation of exogenous peptide antigen via MHC class I,hydrolase activity,involving 27 signaling pathways such as neuroactive ligand receptor interaction,Wnt signaling pathway,and osteoclast differentiation.The target genes of differential lncRNAs predicted by cis were enriched in 434 significant GO classes such as Thelper cell differentiation,T cell differentiation and immune response,involving 56 signaling pathways such as regulation of lipolysis of fat cells,reabsorption of water regulated by vasopressin,and type 2 diabetes.The target genes predicted by trans were enriched in 27 significant GO classes such as alcohol metabolism and RNA catabolism,involving 18 signaling pathways such as fatty acid degradation,PPAR signaling pathway,and glucagon signaling pathway.In the co-expression network,there were 268 differential lncRNAs-mRNAs co-expression pairs.Among them,lncRNAs such as BC038432,ENST00000450783 and ENST00000499653,mRNAs such as SLC19A1,LOC100506301 and RBM47 were in the center of association.ConclusionIt is the first time to use lncRNAs and mRNAs microarray in kidney-yin deficiency T2 DM group,T2 DM without kidney deficiency,and normal subject group.A lot of abnormal expression of lncRNAs and mRNA between groups were found.Further bioinformatics analysis suggested that differential lncRNAs related to kidney-yin deficiency might be involved in immune response,energy metabolism and apoptosis,involving signaling pathways such as pantothenate and Co A biosynthesis,NF-? B signaling pathway and apoptosis.It provided strong scientific support for traditional Chinese medicine(TCM)syndromes on gene level.Some lncRNAs such as u NR?034173.1,AK024373,AF116618,n407257,HMlinc RNA1030,uc010 atu and DA674418 would be the molecular markers in type 2 diabetes mellitus with kidney-yin deficiency.It was helpful to the objectification and standardization of TCM syndrome,beneficial to reveal the essence of syndrome on gene level,and provided powerful scientific evidence for clinical treatmen of TCM.