A Preliminary Study Of RNA Binding Protein GRN Regulating Gene Expression In Primary Progressive Aphasia

Objective:In this study,transcriptome sequencing(RNA-Seq)technique was used.To investigate the effect of GRN knockdown on the expression of genes related to primary progressive aphasia(PPA)in PC12(rat adrenal pheochromocytoma cells)cells,and to analyze the genes whose expression level and splice level changed after GRN knockdown.The molecular mechanism of GRN in PPA was preliminarily discussed,so as to better understand the pathogenesis of PPA.Methods:(1)Passage culture and grouping of PC12 cells.Two groups were set up: control group(si Control group)and silent group(si GRN group).(2)Cells were transfected and real-time fluorescence quantitative PCR(RT-q PCR)was performed to verify the knockdown effect.(3)Transcriptome sequencing: Illumina Novaseq 6000 sequencing platform was used to obtain high quality transcriptome data and analyze differential expressed genes and differentially variable splicing genes.(4)Bioinformatics analysis: Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases were used for enrichment analysis.Results:(1)The expression of 197 genes was significantly differentially expressed after GRN knockdown(the expression change factor was higher than 1.5 times,p-value < 0.01),among which 59 genes were significantly up-regulated and 138 genes were significantly down-regulated.(2)237 significant variable splicing events were detected after GRN knockdown.GRN could significantly regulate the variable splicing of NFRKB and promote the formation of a new variant of NFRKB variable splicing(NFRKB-b).(3)Although no genes enriched in PPA related functional pathways were screened out in GO and KEGG analysis,SLC30A3,NFRKB,TTLL9 and other genes were associated with PPA related neurodegenerative diseases.Conclusion:(1)NFRKB may play a role in PPA.We speculate that the protein translated by NFRKB-B may be involved in the occurrence and development of primary progressive aphasia,or become a new direction of aphasia research in the future.Both TTLL9 and SLC30A3 are associated with neurodegenerative diseases,but more verification of the mechanism of action between these two genes and PPA is needed.(2)Although these genes did not have significant PPA related functional pathways enriched in GO and KEGG analysis,the role of Nocth signaling pathway,Wnt signaling pathway,G protein-coupled receptor and other pathways in neurodegenerative diseases in the enrichment pathway is worth further exploration.