Effect And Mechanism Of Circ-RBM33 Mediated Extracellular Matrix Remodeling On Abdominal Aortic Aneurysm

Background and objective:Abdominal aortic aneurysm(AAA)is a vascular disease involving the abdominal aorta with a rumen-like dilatation,more than 1.5 times the normal diameter.AAA occurs primarily in the male population over 65 years of age and is an important cause of death in the elderly population.At present,the treatment of larger diameter tumors and ruptured AAA is still based on surgical treatment,and no effective therapeutic agents have been found.The pathogenesis of AAA is complex,and it is important to deeply explore the pathogenesis of AAA and find new diagnostic markers and potential therapeutic targets to regulate the occurrence and development of AAA.There are a series of non-coding RNAs in the genome,including circ RNAs,long non-coding RNA,and mi RNAs,which are considered to be key regulators of many biological processes and human diseases.Among these RNAs,circ RNA is a circular closed non-coding RNA that plays a key role in cardiovascular diseases and can prognosis as a novel tumor marker.Various circ RNA molecules such as circ Fox O1,circ-IQGAP1,and circ RNA010383 have been shown to play a role in extracellular matrix remodeling in diseases such as diabetic nephropathy,myopia,and osteoarthritis.Extracellular matrix(ECM)remodeling is one of the pathogenesis of AAA,of which the changes of proteolytic enzymes such as metalloproteinases MMP and its inhibitor TIMP are important pathological changes.However,the role of circ RNAs in AAA-associated ECM remodeling is not clear,and the specific effector mechanisms have yet to be further explored.In this study,we analyzed whole transcriptome expression profiles in AAA and adjacent arterial control tissues by high-throughput sequencing technology and identified differentially expressed circ RNAs,lnc RNAs,and m RNAs.In addition,the functions and mechanisms of key circ RNAs in the development of AAA were studied,so as to provide a new perspective on the mechanism of the role of circ RNAs in the development of AAA and provide new ideas for the prevention and treatment of AAA.Part 1 Sequencing and analysis of the whole transcriptome of human abdominal aortic aneurysm tissueObjective:The changes of the expressions of m RNA,circRNA and lncRNA in AAA tissues and adjacent tissues were identified,and the regulatory networks of key RNAs in AAA were discovered.Methods:In this study,the whole transcriptome sequencing analysis was performed on the AAA tissues and adjacent tissues(n=3)and differentially expressed m RNA,circ RNAs and lnc RNAs were identified.The target genes of differentially expressed circ RNAs and lnc RNAs were predicted.The functions and signaling pathways of differentially expressed circ RNAs and lnc RNAs were analyzed by GO and KEGG analysis.Results:1.There were 4814 differentially expressed m RNAs in AAA and normal abdominal aorta control tissue tissues.Compared with normal abdominal aorta control tissues,2085 m RNAs were down-regulated and 2729 m RNAs were up-regulated in AAA tissues.Differentially expressed m RNAs were mainly enriched in GO entries such as immune response,cell adhesion,and extracellular matrix composition.KEGG analysis showed that differentially expressed m RNAs were mainly involved in signaling pathways such as cell adhesion molecules,NF-?B signaling pathway,and ECM-receptor interaction.2.There were 1231 differentially expressed lnc RNAs in AAA and normal abdominal aorta control tissue tissues,of which 529 lnc RNAs were down-regulated and 702 lnc RNAs were up-regulated in AAA tissues.GO analysis showed that the target genes of differentially expressed lnc RNAs were mainly involved in positive regulation of angiogenesis,ECM composition,and negative regulation of NF-?B transcription factor activity.KEGG analysis showed that the target genes of differentially expressed lnc RNAs were mainly involved in TNF signaling pathway,NF-?B signaling pathway,and NOD-like receptor signaling pathway.3.There were 65 differentially expressed circRNAs in AAA and normal abdominal aorta control tissue tissues,of which 35 circ RNAs were down-regulated and 30 circ RNAs were up-regulated in AAA tissues.GO analysis showed that the target genes of differential circ RNAs mainly participated in immune system processes,cell adhesion,and ECM remodeling.KEGG analysis showed that the target genes of differentially expressed circ RNAs were mainly enriched in cell adhesion molecules,interactions between cytokines,and NF-?B signaling pathways.Part 2 The function of circ-RBM33 in regulating the degradation of extracellular matrixObjective:The biological function of circ-RBM33 in ECM degradation of VSMC were explored.Methods:Five circRNAs with the most significant differences were detected by qRT-PCR,and circ-RBM33 was identified as the object of the further study.The expressions of circ-RBM33 in 20 pairs of AAA tissues and adjacent tissues were observed using q RT-PCR,and circ-RBM33 was identified by sanger sequencing.In vitro experiments,Ang II was used to stimulate VSMC to establish an injury model.The expression of circ-RBM33 in VSMC was detected by q RT-PCR.The cells were transfected with circ-RBM33 overexpression vector.The expression of ECM remodeling related proteins MMP-9 and TIMP-1 was detected by immunofluorescence and western blotting.Results:1.q RT-PCR results showed that the expression trend of the five candidate circ RNAs in AAA tissues was the same as that in sequencing data,indicating that the sequencing results were credible.Of these,circ-RBM33 expression was significantly elevated in AAA tissues.Therefore,circ-RBM33 was used as a research target for subsequent experiments.2.PCR amplification and Sanger sequencing confirmed the circular structure of circ-RBM33.3.The expression of circ-RBM33 was upregulated in VSMC stimulated by AngII.Overexpression of circ-RBM33 in VSMC promoted MMP-2 expression,and inhibited TIMP-1 expression,indicating that circ-RBM33 promoted ECM degradation in vitro.Part 3 The regulatory mechanism of circ-RBM33 in vascular smooth muscle cellsObjective:The mi RNA adsorbed by circ-RBM33 and its downstream target genes were identified and the mechanism of its regulation of ECM degradation was clarified.Methods:The mi RNA that circ-RBM33 may bind to and the target genes of mi RNA were screened by bioinformatics analysis.The expression of mi R-4268 was detected in VSMC with circ-RBM33 overexpression using qRT-PCR.The interaction of circ-RBM33 and mi R-4268 was verified by dual luciferase reporter assay.The expression of SOCS3 and EPHB2 in VSMC with mi R-4268 overexpression was detected by q RT-PCR,and EPHB2 was identified as the target gene of mi R-4268.Finally,the recovery experiment confirmed that circ-RBM33 regulated the downstream target gene Eph B2 through mi R-4268,thus regulating the ECM degradation.Results:1.mi R-4268,which has a complementary sequence to circ-RBM33 selected by bioinformatics methods.The overexpression of circ-RBM33 in VSMC significantly down-regulated the expression of mi R-4268.Luciferase reporter assay showed that mi R-4268 significantly activated the fluorescence activity of the wild-type circ-RBM33,but had no effect on the circ-RBM33 mutant.2.Bioinformatics combined with literature reports screened mi R-4268 downstream target genes.q RT-PCR confirmed that the overexpression of mi R-4268 in VSMC significantly down-regulated the expression of target gene Eph B2.3.Recovery experiments showed that circ-RBM33 could reverse the upregulated effect of EPHB2 and the inhibition of TIMP-1 expression in VSMC by circ-RBM33,suggesting that circ-RBM33 could promote ECM degradation via the mi R-4268/EPHB2 axis.ConclusionsIn this study,the circ RNA expression profiles of AAA were obtained by the whole transcriptome sequencing.It was revealed that circ-RBM33 was up-regulated in AAA tissues and Ang II-stimulated vascular smooth muscle cells.Circ-RBM33 promoted extracellular matrix degradation by regulating the mi R-4268/Eph B2 axis,thereby promoting the progression of AAA.Our results unraveled the specific regulatory mechanism of the occurrence of AAA promoted by circ-RBM33,and circ-RBM33 may be a novel biomarker and therapeutic target for patients with AAA.