Analysis And Study Of CircRNA Expression Related To Skin Aging

Objective:Circular RNA(circRNA)is a class of non-coding RNA(ncRNA)formed by covalently closed-loop structure without 5'cap or a 3' polyadenylated tail.Circ RNA has just emerged in recent years.Since its discovery,the exploration of circRNA has started from the simple understanding of its structure and mechanism of action,and then related it to various diseases in various organ systems.Current research has shown that circRNA is associated with tumor,atherosclerosis,coronary heart disease,diabetes,nervous system diseases,depression and other diseases,among which the most popular research is on tumor diseases.The research of circRNA in skin related diseases mainly focuses on skin basal cell carcinoma,squamous cell carcinoma,melanoma and psoriasis,and skin wound healing.The functions of circRNA in skin aging remain largely unknown and need further investigation.As the largest organ of the human body,skin is also the "gateway" for the human body to contact with the external environment.The skin aging process is a complex process,mainly influenced by two factors: one is the Intrinsic aging,also known as Chronologic aging,the other is the process of skin aging under the influence of environment,which is called Extrinsic skin aging.Eyes as the first of the five features is the center of appearance,eye aging is very prominent in facial aging.This study involved in the field of circRNA and skin aging,and explored the mechanisms related to aging.Through the verification of tissue and cell functional experiments,we could observe the effects and influences of genes or proteins on aging cells.Methods:Taking the eyelid skin as the research object of aging,using high-throughput transcriptome sequencing technology to detect the differential expression of circRNAs in the two groups of samples,young and old,explored the differential expression profile of circRNA related to skin aging.Gene Ontology(GO),biological pathways(Pathway)analysis,from a biological process,molecular function,signal pathways in comprehensive aging mechanism is discussed in this paper.Targeted micro RNA prediction analysis of differential circRNAs was conducted to construct circRNA-micro RNA regulatory network.According to the high-throughput sequencing results,10 differentially expressed circRNAs were selected at the histological level for RT-qPCR verification,and the final 2 circRNAs were selected for subsequent studies.Skin fibroblast HSF was obtained by tissue patch adherent-culture method,and the overexpression vectors of hsa?circ?0077605 and hsa?circ?0137613 were constructed to observe the effects on the function of HSF cells,via ?-gal staining assay,CCK8 proliferation assay and cell flow cycle assay.Bioinformatics analysis was used to predict the target micro RNA of hsa?circ?0077605,dual-luciferase reporter assays and fluorescence in situ hybridization were used to confirm that the downstream micro RNA of hsa?circ?0077605.Results:1.A total of 20552 circRNAs from eyelid tissues were screened by high-throughput sequencing,including 5432 from exons,9318 from introns,and 5802 from intergene regions.There were a total of 14,915 circRNAs in the eyelid tissues of the elderly group and the young group,among which 571 circRNAs differentially expressed.Among the571 circRNAs,348 were up-regulated and 223 were down-regulated.GO analysis and KEGG analysis indicated that the eyelid tissue was more prone to aging in terms of cellular biological processes,cellular components and molecular functions.Validation of the RNA-Seq data was provided by subjecting a set of 10 of the differentially expressed circRNAs to quantitative RT-PCR,including five up-regulated circRNAs(hsa?circ?0077605,hsa?circ?0002957,hsa?circ?0137613,hsa?circ?0008089,hsa?circ?0000205).And five circRNAs down-regulated(hsa?circ?0112861,hsa?circ?0003803,hsa?circ?0032813,hsa?circ?0113488,hsa?circ?0026497).Through RT-qPCR verification,it was found that most of the results were consistent with the sequencing data,and hsa?circ?0077605 and hsa?circ?0137613 were significantly up-regulated with obvious differential multiple expressions,which could be used as the object of subsequent research.2.HSF was obtained by tissue block adherent culture method.Because hsa?circ?0077605 has high homology with other genes,specific si RNA sequences cannot be designed.Two si RNAs were designed targeted to the back-spliced site of hsa?circ?0137613.HSF was transfected with si RNA-hsa?circ?0137613,and the level of hsa?circ?0137613 in HSF cells was detected by RT-qPCR.It was finally confirmed that the two circRNA molecules expressed at extremely low,so overexpression strategies should be employed.3.The selected 2 circRNA molecules were constructed and sequenced for overexpression vectors.hsa?circ?0077605 and hsa?circ?0137613 overexpression vectors were constructed by PCR amplification of the full-length sequence and double enzyme digestion into plasmid plc5-cir.The cells were divided into hsa?circ?0077605/ hsa?circ?0137613,NC(p LC5-cir)and Control groups.The 293 T cells were transfected with Lipofectamine2000,and the overexpression efficiency was detected by qPCR.Compared with the control group,the overexpression times of hsa?circ?0077605 was 3380,and the overexpression times of hsa?circ?0137613 group was 98748.The qPCR product sequencing showed that the cyclization sequence was correct.These results indicated that hsa?circ?0077605 and hsa?circ?0137613 could be expressed in eukaryotic cells.After overexpression of hsa?circ?0077605/ hsa?circ?0137613,HSF cell function was affected by?-gal staining test,CCK8 proliferation test and cell flow cycle test.The morphology of overexpressed HSF cells showed typical senescence.?-Gal staining showed that the cell morphology became wide and flat,the cytoplasm was transparent,the nucleus granules were increased,and the peri-senescent nucleus was stained blue.In vitro culture of HSF for 3 days,the proliferation activity of CCK8 cells was significantly reduced in the over-expression group.Flow cytometry was used to detect the cell cycle,which showed that the G1 phase of HSF increased significantly in the overexpression group,while the S/G2 phase decreased.HSF aging could not carry out DNA replication.4.Three miRNA prediction software miRanda,RNAhybrid and Target Scan are used to predict the miRNA bound by hsa?circ?0077605,and there are 20 miRNAs predicted by the three prediction software.The dual luciferase assay confirmed that hsa?circ?0077605could efficiently bind to hsa-miR-612.In order to further verify the binding of hsa?circ?0077605 and hsa-miR-612,hsa?circ?0077605 was mainly located in HSF cytoplasm and hsa-miR-612 was also mainly located in HSF cytoplasm under confocal microscopy by FISH detection.Co-localization of hsa?circ?0077605 and hsa-miR-612 could be observed in cytoplasm through co-localization of the two.According to the above experimental results,circRNA?0077605 can bind to hsa-miR-612,and the circRNA?0077605-micro RNA-612 axis is related to skin aging.Conclusions: 1.The differential expression of circRNAs during skin aging suggests that circRNAs may be involved in the gene regulation of skin aging.2.Low expression of hsa?circ?0077605 and hsa?circ?0137613 in HSF,followed by overexpression study.3.HSF aging was induced by overexpression of hsa?circ?0077605 and hsa?circ?0137613.4.FISH assay and dual luciferase assay confirmed the interaction between hsa?circ?0077605 and hsa-miR-612.circRNA?0077605-micro RNA-612 axis is associated with skin aging.