| Objective:To investigate the differential lncRNA and mRNA genes in Alzheimer’s Disease(AD)and mild cognitive impairment(MCI)in the elderly population of Xinjiang.High-throughput genome-wide sequencing,GO and KEGG enrichment analysis were used to screened the differentially expressed lncRNA genes in the differential expression profile of lncRNAs in AD and MCI patients.These differential genes were further verified in the replication population.Through bioinformatics analysis,the target gene of LncRNA AL133415.1 was predicted,and the mechanism of between LncRNA AL133415.1 and VIM was explored.Methods:Part 1:The plasma samples of 5 AD patients,5 MCI patients,and 5 matched healthy controls were randomly selected for expression levels of LncRNAs using the TruSeq RNA Sample Prep Kit(Illumina).High-throughput genome-wide sequencing,GO and KEGG enrichment analysis were used to screened the differentially expressed lncRNA genes.The target genes and biological functions were predicted by bioinformatics analysis.Part Ⅱ:According the differentially expressed lncRNA genes,combined with the related literature report,the candidate lncRNA genes were identifited.In this study,6 differential lncRNA genes were screened in the AD population.In the MCI population,5 lncRNA genes were identified.The real-time quantitative PCR method was used for verification of differentially expressed lncRNAs in 40 AD patients,40 MCI patients,and 40 control patients.Part Ⅲ:The AD cell model was established to explored the effect of LncRNA AL133415.1 and VIM gene expression by qRT-PCR,Western blot technology,cell transfection,plasmid construction and other technologies.Results:(1)①A total of 1013 significantly dysregulated genes were identified between AD group and control group,in which 514 were up-regulated genes and 499 were down-regulated genes.MSTRG.13771.2 was the most up-regulated gene,and MSTRG.3939.4 was the largest down-regulated gene.In the mRNA data,a total of 447 differentially expressed genes were identified,in which 206 were up-regulated and 241 were down-regulated.The MKL2 gene was the most up-regulated,and the ENST00000652454 gene was the most down-regulated.A total of 874 significantly dysregulated genes were identified between the MCI group and the control group,of which 413 were up-regulated gene and 461 were down-regulated gene.The MSTRG.5005.3 gene was the most up-regulated gene,and the MSTRG.12118.6 gene was the largest down-regulated gene.In the mRNA data,a total of 4813 differentially expressed genes were screened,of which 2307 were up-regulated gene and 2506 were down-regulated gene.The most up-regulated fold was MKL2 gene,and the largest down-regulated fold was SCFD2 gene.②According to the RNA-sequencing data,GO and KEGG enrichment analysis were used.6 differential LncRNAs(ENST00000654948,ASMTL-AS1,AC005730.3,AP001363.1,AL133415.1,AC020916.1)were screened between AD group and control group.4 differential LncRNAs(AP003068.2,AL355075.4,ENST00000668018,AC008750.2)were screened between MCI group snd control group.③GO enrichment analysis of differential genes revealed that the data of differential gene functional enrichment analysis results showed that differential genes are involved in biological processes in terms of the development of the nervous system,morphogenesis of cellular components,and regulation of cell cycle processes.The main processes involved in cellular localization are:dendritic spines,axons,neuronal spines,neuronal cytosol,synapses between neurons,major axons,axon initiation,axon membrane,presynapses,dendritic spine heads,axonal parts,nuclei,neuronal parts.Among the molecular functions performed,the main ones include ATP binding,enzyme binding,oxidoreductase activity,and brain-derived neurotrophic factor binding.KEGG enrichment analysis revealed that the signaling pathways in which the differential genes may be involved include:Alzheimer’s disease,regulation of tryptophan channels by inflammatory mediators,homologous recombination,and glycine,serine,and threonine metabolism.(2)①Through the verification of 6 differential LncRNAs(ENST00000654948,ASMTL-AS1,AC005730.3,AP001363.1,AL133415.1,AC020916.1)in the AD group of 80 independent samples,We found that the LncRNAs AL133415.1,AC020916.1,ENST00000654948,ASMTL-AS,AC005730.3,AP001363.1 levels in the plasma of the AD patients were significantly lower compared with the control group(Z=-4.061,P<0.001;Z=-2.054,P=0.039;Z=-2.723,P=0.006;Z=-6.197,P<0.001;Z=-3.416,P<0.001;Z=-4.176,P<0.001,respectively).howeve,The expression of LncRNA AL355075.4,AP003068.2.in the MCI group was higher than that in the control group,and the difference was statistically significant(Z=-2.261,P=0.023;Z=-2.312,P=0.020,respectively).②The Receiver operating characteristic curve(ROC)was used to evaluate the relative expression of LncRNAs(ENST00000654948,ASMTL-AS1,AC005730.3,AP001363.1,AL133415.1,AC020916.1)in peripheral blood to AD diagnostic value.ROC curve analysis revealed that the area under the curves(AUC)of AL133415.1 was 0.635(95%CI:0.507-0.763,P=0.036).the AUC of ASMTL-AS1 was 0.658(95%CI:0.513-0.785,P=0.015),the AUC of AC005730.3 was 0.627(95%CI:0.498-0.756,P=0.049),the AUC of AP001363.1 was 0.708(95%CI:0.595-0.822,P=0.001).In the MCI group,the AUC of AL355075.4 and AP003068.2 were statistically significant.(3)The vitro experiment results showed that the LncRNA AL133415.1 silence group was able to increase cell activity compared to the control,while the LncRNA AL133415.1 overexpression group was able to decrease cell activity compared to the control,and the differences were both statistically significant(t=-0.65,P=0.005;t=0.074,P=0.001,respectively).miR-138-5p suppression group was able to increase cell activity compared to the control group,while miR-138-5p overexpression group inhibited cell activity,and the differences were statistically significant(t=-0.118,P<0.001;t=-0.141,P<0.001,respectively).LncRNA AL133415.1 silence group was able to decrease apoptosis compared to blank control,while LncRNA AL133415.1 overexpression group was able to increase apoptosis compared to blank control,and the differences were statistically significant(χ2=9.280,P=0.002;χ2=24.175,P<0.001,respectively).miR-138-5p silence group was able to decrease apoptosis compared to the blank control,while miR-138-5p overexpression group promoted apoptosis compared to the control group,both differences were statistically significant(x2=5.555,P=0.018;x2=30.862,P<0.001,respectively).The expression of VIM in the LncRNA AL133415.1 silence group was higher than that in the Aβ1-42 control group and NC control group,with statistically significant differences(t=0.159,P<0.001;t=-0.187,P<0.001,respectively).And in the LncRNA AL133415.1 overexpression group,VIM expression was found to be lower than that of NC control and Aβ1-42 control group,with statistically significant differences(t=0.141,P<0.001;t=-0.165,P<0.001,respectively).After inhibition of MiR-138-5p expression,VIM expression was higher than that of Aβ1-42 control and NC control,and the difference was statistically significant(t=-0.337,P<0.001;t=-0.350,P<0.001,respectively).And in the MiR-138-5p overexpression group,VIM expression was found to be significantly lower than that of NC controls and Aβ1-42 controls,with statistically significant differences(t=-0.288,P<0.001;t=-0.828,P<0.001,respectively).Conclusion:(1)A total of 10 differential LncRNAs were identified in this study,including 6 in the AD group and 4 in the MCI group,which may be potential biomarkers for the diseases.GO terms including neuronal,synaptic,axonal,and microtubule cytoskeleton biological functions,KEGG pathways including Alzheimer’s disease pathway.(2)The expression of six differential LncRNAs in the AD group was much lower than the control group,and the expression of two differential LncRNAs in the MCI group were higher than the control group,which indicated that the expression levels of the differential LncRNAs were lowly in the AD group and highly expression in the MCI group.(3)The ROC results show that LncRNAAL 133415.1,ASMTL-AS1,AC005730.3 and AP001363.1 gene had predictive value for disease diagnosis in the AD.LncRNAAL355075.4 and AP003068.2 genes were found with high predictive value for MCI.It is suggested that the those LncRNAs would be used as potential biomarkers for early diagnosis of diseases for AD and MCI.(4)Inhibition of expression of LncRNA AL133415.1 and miR-138-5p would promote the expression of VIM,while overexpression of LncRNA AL133415.1 and miR-138-5p can inhibit the expression of VIM.VIM can also increase intracellular amyloid production,which is a pathogenic risk factor for AD,and thus can indirectly regulate VIM expression and reduce the development of AD by regulating LncRNA AL133415.1 and miR-138-5p. |
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