Insight Into The Impact Of Circ₇22 On Aging Process And Age-related Diseases And Its Potential Underlying Mechanisms

Chapter 1 Expression of circ₇22 in the long-lived individuals from Hongshuihe River BasinBackground: Aging is a universal biological process determined by the interaction of genetic and environmental factors whose underlying mechanism remains largely unknown.Circular RNA has been a research hotspot currently due to its increasing evidence of participating in biological functions.Very recently,the serum expression of hsa_circ₀000722,a newly defined circRNA?also known as circ₇22 thereafter?,was noted to be elevated in elderlies as compared to yonger controls and was implicated in the pathogenesis of coronary heart disease?CHD?.Nevertheless,no investigation on the role it may play in aging process has been reported.Objectives: The aim of current study is to explore the association between serum level of circ₇22 and human aging and longevity.Methods:Three groups of participants were enrolled in the current study,i.e.,long-lived group?LG,aged 85 or above,mean age 93.25±3.09 yrs,n = 132?,elderly group?EG,aged 65-84,mean age 75.25±4.02 yrs,n = 67?and yonger control group?CG,aged less than 65,mean age 49.20±8.94 yrs?.Blood pressure was measured and blood sample was collected for all paticipants to determine serum levels of total cholesterol?TC?,triglyceride?TG?,LDL-C,HDL-C,fasting blood glucose?FBG?.The serum expression of circ₇22 was evaluated by RT-PCR.Wilcoxon and Spearman test were used to compare the levels of circ₇22 among groups and to evaluate the association of circ₇22 and metabolic parameters,respectively.Results:?1?The circ₇22 level of LG was significantly lower than that of EG [0.73?1.02?vs 1.13?1.57?,P < 0.05] and CG [0.73?1.02?vs 0.85?1.27?,P <0.05];the circ₇22 level of EG tended to be higher than that of CG,but the difference did not reach statistical significance [1.13?1.57?vs 0.85?1.27?,P >0.05];?2?When stratified by sex,the circ₇22 level of EG males was the highest,being remarkably higher than that of LG and CG [1.51?2.00?vs 0.74?1.16?,1.51?2.00?vs 0.74?1.16?,respectively,both P < 0.05],while the circ₇22 level of LG females was lower than that of CG [0.73?0.99?vs 1.06?1.37?,P < 0.05];?3?The expression of circ₇22 was negatively linked to serum TG?r =-0.1142,P =0.0470?while positively related to HDL-C?r = 0.1583,P = 0.0058?and FBG?r= 0.1390,P = 0.0142?.Conclusion:circ₇22 tends to express less in long-lived cohort which may partially interpret their better survivorship due to its lower risk in cardiovascular diseases.Chapter 2 The molecular mechanisms underlying the alteration of circ₇22 expressionBackground: circRNA is derived from the back splicing of RNA.There are several styles for its formation,in which the cyclization by the pair driving of introns is the most common forming pathway,whose cyclization is usually driven by the flank Alu elements.Objectives: To determine the association of Alu element polymorphism of circ₇22 flanks with longevity and age-related diseases and the impact of polymorphism on circ₇22 expression.Methods:?1?The genome DNA was extracted from white blood cells from participants of LG,EG and CG?sample information is the same as described in Chapter 1?.The SNPs of the flank Alu elements of circ₇22 were determined by PCR-Sanger sequencing.Chi-square test was employed to compare genotype,allele and haplotype frequency of every SNP among the three groups;?2?Wilcoxon test was used to analyze the relationship between SNPs and serum circ₇22 level and several cerebro/cardiavascular risk parameters.?3?The SNPs of Alu7 elements of circ₇22 were defined for patients with hypertension?age60.32 ± 5.81 yrs,n = 99?and healthy controls?59.98±6.01,n = 172?.And the frequencies of genotype,allele and haplotype of every SNP were compared between hypertension group and controls;?4?The Alu7 of 239 T cell line was knocked out by CRISPR/Cas9 gene editing and circ₇22 expression was determined by RT-PCR.Results:?1?Seven Alu elements were detected in both flanks of circ₇22.Small sample size?n = 40?revealed no SNPs in Alu 1 to 6.While six SNPs werefound in Alu7,i.e.,rs35297506,rs6539973,rs6539974,rs8056654,rs8056655 and rs113042029;?2?Six SNPs of Alu7 are in aggrement with Hardy-Weinberg equilibrium in both cohorts?longevity and hypertension?;?3?Singnificant differences were found on the genotypic and allelic frequency of Alu7rs6539973 between long-lived and non-long-lived group?P < 0.008?;?4?Singnificant differences were found on the genotypic and allelic frequency of Alu7 rs8056654 between hypertensive and control group?P < 0.008?;?5?The serum expression of circ₇22 and several lipid levels were found markedly different among genotypes of several Alu7 SNPs;?6?The expression of circ₇22was markedly reduced after the Alu7 element was knocked out in 293 T cell lines?0.96±0.12 vs 0.03±0.01,P = 0.000?.Conclusion: The SNPs in the Alu7 element of circ₇22 influence the expression of circ₇22 and are associated with aging and hypertension,which may underpin the genetic basis of age-related pathology and human longevity.Chapter 3 The influence of circ₇22 on cellular biological functionsBackground: Disorders,including neurodegenerative disease,diabetes and cardio-and cerebral vascular diseases may develop during aging process,in which available data have indicated that circRNAs play critical roles.Findings from past chapters have displayed that circ₇22 expresses lower in long-lived individuals and is linked to lipid levels.Furthermore,the expression of circ₇22is influenced by the variants in its Alu7,the SNPs of Alu7 presented significant difference in long-lived individuals and hypertensive patients versus counterparts.We thus hypothesize that circ₇22 may involve in the development of cardioand cerebral vascular diseases.Objectives: To seek the function of circ₇22 in endothelial cells and relevant mechanisms.Methods:?1?To overexpress and to silence circ₇22 respectively in a human umbilical vein endothelial cell line with lentivirus transfection technique by using infected empty virus and lentivirus free cell line,EA.hy926,as controls.CCK8,flow cytometry and transwell were used to test cell proliferation,cell cycle and apotosis and cell migration respectively to evaluate the alteration of aged-related parameters;?2?Fluorescence in situ hybridization?FISH?was employed to determine the localization of circ₇22 in EA.hy926 cells and confer its potential molecular mechanism.Results:?1?Both overexpresion and silence of circ₇22 did not change the proliferation of EA.hy926 cells;?2?Both overexpresion and silence of circ₇22did not affect the cell cycle of EA.hy926;?3?Overexpresion of circ₇22increased the early and late apotosis of EA.hy926;while silence of circ₇22 did not influnce apotosis;?4?Overexpresion of circ₇22 increased while silence of circ₇22 decreased the migration of EA.hy926;?5?Overexpresion of circ₇22increased the rate of aging cells and the level of MDA,but did not change the activity of SOD;?6?FISH indicated circ₇22 distributes widely in cytoplasm.Conclusion: The upregualtion of circ₇22 facilitate the apotosis and aging of EA.hy926 cells,probably through the pathway of competing endogenous RNAs?ceRNA?.Chapter 4 Construction of the regulating newwork of circ₇22-miRNA-mRNABackground: High throughput DNA sequencing has allowed researchers to quickly access transcritional information in cellular level thus to further understand a variety of molecular pathways.In the past chapter above,circ₇22was found to affect the functions of HUVEC in vitro,but relevant molecular mechanism remains unexplored.We spaculate its main functions may involve in ceRNA according to the localization of circ₇22 in cytoplasm by FISH technique.The knocking out of the Alu7 element of circ₇22 in 293 T greatly downregulate its expression?about 30 fold lower than that without knockout?,indicating that 293 T cell line is an ideal cell model for studying circ₇22.Objectives: In this chapter,the potential molecular function of circ₇22 is explored.Methods:?1?To evaluate the alteration of circ₇22 in transcriptional level after its Alu7 is knocked out by RNA-seq technique,with wildtype 293 T being a control;?2?To prodict the miRNA and its targeting mRNA which may combine with circ₇22 in order to understand the potential regulating genes of circ₇22by using bioinformatics;?3?To take the interaction of downregulated mRNA and potential circ₇22 regulating mRNA as the target genes of circ₇22 after its Alu7 is knocked out;?4?To construct the regulating newwork of circ₇22-miRNA-mRNA by Cytoscape 3.7.0;?5?To analyse the potential functions of target genes by GO?Gene Ontology?and KEGG Pathway.Results:?1?After downregulating circ₇22,2037 genes were found to express differently,1422 and 615 of which are downregulated and upregulated,respectively;?2?Bioinformatics predicts that five miRNAs,i.e.,hsa-miR-324-5p,hsa-miR-326,hsa-miR-330-5p,hsa-miR-652 and hsa-miR-661,can combine with circ₇22.1541 potential target genes are predicted to be associated with circ₇22 according to the functions of these miRNAs;?3?After interaction analyses,189 of the target genes were found to potentially interact with the genes downregulated by RNA-seq.GO analyses showed that these genes were mainly linked to signal transduction in nerve system;while KEGG pathway analyses indicated that these genes involve in pathways of signaling and transcriptional regulation.Conclusion:circ₇22 may influence the expression of several genes through ceRNA pathway and play critical roles in nerve system.Study on the potential roles of circ₇22 in the pathogenesis of age-related diseases are thus of interest for researcher in this field.