Screening Of Differentially Expressed CircRNAs In The Liver Tissue Of Obese Mice And Construction Of The CeRNA Network

Obesity is a multifactorial metabolic disorder,among which obesityrelated lipid metabolism disorder has been a hot spot in clinical and basic research for many years,and it is also one of the important driving factors of many diseases.There is a lack of biomarkers for early diagnosis and prognosis,due to the complex pathogenesis of obesity and the mutual influence between different diseases,limited clinical treatment methods.Thus,it is of great significance for the research and treatment of obesityrelated diseases to further explore the pathogenesis of obesity,discover novel diagnostic biomarkers for obesity,and explore its potential functions.At present,most studies on obesity-related diseases biomarkers mainly focus on messenger RNA(m RNA)and non-coding RNA(nc RNA).In recent years,studies have discovered a new class of nc RNA,circular RNA(circ RNA),which has attracted great attention as an emerging field in obesity-related diseases research.Circ RNA is a single-stranded closed circular non-coding RNA produced by pre-m RNA splicing through various mechanisms,also known as a new generation of regulatory RNA,which is widely used in the diagnosis of diseases and the development of nucleic acid drugs.Existing literature has shown that circ RNA can participate in the regulation of lipid metabolism through a competing endogenous RNA(ce RNA)mechanism,including lipogenesis,transport,oxidative decomposition,proliferation and differentiation of adipocytes and so on.However,the study of circ RNA in obesity-related diseases is limited to some star molecules,so screening new circ RNA biomarkers is of great value for studying obesity-related metabolic diseases.In addition,whether the circ RNA expression profiles of liver tissue induced by high-fat diet in obese mice has changed,whether the corresponding changes are related to lipid metabolism disorders caused by obesity,and the role of ce RNA regulation mechanism of related circ RNA in obesity-related diseases deserves future study.For this reason,this study constructed a high-fat diet induced obese mouse model,and performed deep RNA sequencing(RNA-seq)on linerdepleted RNA extracted from mouse liver tissue to explore the circ RNA expression profiles changes in the mouse liver tissue.Bioinformatics technology was devoted to screen differentially expressed(DE)circ RNA,construct and further analyze its ce RNA network,and explore potential biomarkers of obesity-related diseases,in order to provide reference for future clinical and basic research.In this study,a high-fat diet induced obese mouse model was first established,and 30 SPF male 4 week old C57BL/6J mice were selected and divided into two groups.The high-fat diet(HFD)group was fed a high-fat diet(60% fat),the control diet(CD)group was fed with low-fat control diet(10% fat).When the average body weight of mice in the HFD group exceeded that of the CD group by 20%,the serum was separated to measure total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),fasting blood glucose(FBG),alanine transaminase(ALT),liver and adipose tissue of mice were collected for pathological tissue section analysis.In addition,glucose tolerance test(GTT)and insulin tolerance test(ITT)were used to assist the evaluation of mice metabolism.After the obese mouse model was successfully established,3 mice from each group were randomly selected to extract RNA from the liver for RNAseq.DE circ RNA molecules were screened out from the circ RNA expression profiles obtained by sequencing,bioinformatics analysis of DE circ RNA was carried out using cluster analysis,target prediction,functional annotation,and so on.Real-time quantitative polymerase chain reaction(RT-q PCR)and Sanger sequencing were used to verify the expression level and cyclic sites of circ RNA to ensure the reliability of the sequencing results.Bioinformatics was used to predict and construct obesity-related lipid metabolism ce RNA regulatory network,and key circ RNA molecules regulating lipid metabolism were selected.Based on the matching principle of mi RNA and circ RNA,the interaction binding sites of circ RNA-mi RNA were predicted.At the same time,the functional enrichment analysis of ce RNA network was conducted to determine the main mi RNA affected by key circ RNA and the biological functions and pathways they affected.The results showed that the average body weight of mice in the HFD group was 20% higher than in the CD group at the age of 12 weeks(P <0.01),and serum TC,FBG,ALT levels in the HFD group significantly increased(P < 0.05),TG was higher than in the CD group(P > 0.05),HDLC was lower than in the CD group(P > 0.05).GTT and ITT results showed impaired glucose tolerance and insulin resistance in the HFD group.The results of pathological tissue sections showed that inflammatory cell infiltration and fat vacuole appeared in the liver tissue of mice in the HFD group.The adipocyte area in the three adipose(epididymis,inguinal,subcutaneous)tissue was significantly larger than in the CD group(P < 0.01),all above results proved that the obese mouse model was successfully constructed.A total of 7,469 circ RNAs were detected by RNA-seq,which contains21 DE circ RNAs.The GO and KEGG analysis suggested that DE circ RNA may play an important role in obesity-related lipid metabolism.Based on circ RNA expression level,the number of back-spling junction(BSJ)sites and the functional annotation results,6 circ RNAs related to lipid metabolism(circ RNA4842,circ RNA1657,circ RNA4837,circ RNA601,circ RNA4550,circ RNA1709)were selected for verification.The RT-q PCR results showed that the expression pattern of the 6 circ RNAs was consistent with the RNAseq,indicating that the RNA-seq results were credible,and the circ RNA expression profiles were altered in liver tissue of obese mice.The ce RNA network of DE circ RNA was constructed by bioinformatics technology,and it was found that the network was widely involved in phosphatidylinositol 3 kinase-protein kinase B(PI3K-AKT),mitogenactivated protein kinase(MAPK)pathway.Circ RNA 4842 was generated by cyclization of exons 3,4,and 5 of its host gene phosphatase and tensin homologue(PTEN)and its length was 328 bp.Circ RNA4842 was identified with the correct cyclization site by comparison with circ RNA database,Sanger sequencing and divergent primer validation,circ RNA4842 was confirmed to be a newly discovered circ RNA molecule.Bioinformatics prediction results indicated that circ RNA4842 may bind mmu-mi R-106a-3p and mmu-mi R-204-5p to promote PTEN transcription,and participate in the regulation of lipid metabolism.Taken together,this study confirmed that there were significant changes in circ RNA transcripts in high-fat diet induced obese mouse model,and a systematic circ RNA lipid metabolism-related ce RNA network was constructed.Circ RNA4842,newly discovered in this study,is positively correlated with the expression of its host gene PTEN,which may regulate lipogenesis through the circ RNA4842–mmu-mi R-106a-3p/mmu-mi R-204-5p–PTEN axis.These findings contribute to a better understanding of the pathogenesis of obesity and provide novel potential diagnostic markers for obesity-related diseases..