Transcriptional Expression Profiling Of MOTS-c Regulated Mature Adipocytes Based On RNA-Seq Technology

Objective: Mitochondrial ORF of the 12 S rRNA Type-C(MOTS-c)is a hormone peptide found in recent years that regulates the balance of cell energy metabolism,induces hormone secretion in vivo,and regulates oxidative stress response.Previous studies have shown that MOTS-c can promote the absorption and utilization of glucose,reduce insulin resistance and improve the metabolic function of cells by activating the AMPK signal pathway.On the other hand,MOTS-c can enter the nucleus,regulate gene expression,and play a biological role in reducing obesity.However,the current research on MOTS-c mainly focuses on animal experiments and the physiological function of MOTS-c through AMPK pathway.It is not clear about the downstream target genes of MOTS-c regulation.Based on the above reasons,this study uses RNA next-generation sequencing technology to find the downstream target gene of MOTS-c regulation in human adipocytes,and verifies the correlation between MOTS-c and downstream target gene expression in overweight people.The completion of the project provide theoretical basis for the prevention and treatment of obesity,and providing experimental basis for MOTS-c as a targeted therapeutic polypeptide drug for obesity.Methods:(1)Human adipose-derived mesenchymal stem cells were induced to differentiate into mature adipocytes by stem cell in vitro induction culture technology,and mature adipocytes were identified by oil red O staining;(2)By transfecting MOTS-c with FITC fluorescent to active or repress the genes regulated by MOTS-c,a human adipocyte model of MOTS-c overexpression was established.The control was cells with non-transfected MOTS-c.(3)By extracting RNA from human adipocytes,we analyzed the gene transcription profile expression of the over-expressed human adipocyte model and the control using RNA-Seq technology.The results showed that compared with the control group,there were 552 up-regulated genes and 572down-regulated genes in the MOTS-c overexpression adipocyte model.Nine genes we selected were verified by qPCR.(4)By collecting peripheral blood samples and information from 40 overweight people(body mass index>24.9)and 40 normal people(body mass index≥18.49),we use enzyme-linked immunosorbent assay to determine the expression of MOTS-c in their serum,and analyze the correlation between MOTS-c and overweight people.(5)Human whole blood RNA was extracted,and the correlation between the expression of MOTS-c downstream gene and overweight was analyzed by qPCR.Results:(1)Oil red staining showed that adult adipose mesenchymal stem cells were successfully induced to differentiate into mature adipocytes.(2)The results of fluorescent labeling showed that the exogenous MOTS-c with FITC was distributed in the nucleus and cytoplasm of adipocytes after being introduced into adipocytes for 36 hours.(3)The results of RNA-Seq analysis showed that compared with the control group,the 552 up-regulated genes and572 down-regulated genes were identified in MOTS-c overexpression adipocyte model.The results of qPCR showed that the expression of the 9 genes with the most significant differential expression(AHNAK、RAB31、EEF1A1、PCDHGC3、PCDHGC5、 TIMP2 、MYC、PGRMC1、RPS27A)was consistent with the results of RNA-Seq.(4)The results of enzyme-linked immunosorbent assay showed that the expression level of serum MOTS-c in the control group with normal body mass index was significantly higher than that in the overweight obese group(P<0.05),and there was a correlation between MOTS-c level and body mass index.(5)The results of qPCR of blood samples showed that four genes AHNAK、 EEF1A1、 PGRMC1 and RPS27 A had significant differences between overweight people and normal people.Conclusion:(1)MOTS-c can enter the nucleus of human adipocytes,activate or inhibit the expression of target genes,to participate in related physiological processes.(2)7 genes are downstream target genes of MOTS-c in human adipocytes.(3)The expression level of MOTS-c is related to obesity and significantly decreased in overweight and obese people.(4)The expression of AHNAK、EEF1A1、PGRMC1 and RPS27 A genes is associated with overweight and obesity,suggesting that these four genes are regulated by MOTS-c and participate in the occurrence of obesity.