The Expression Profile And Functional Analysis Of Circular RNAs In Infantile Hemangiomas

Background and objectives:Infantile hemangioma?IH?is the most common soft tissue tumor in infancy,with an increasing incidence of 3-5%.Although most of IHs are self-limiting with a rapid proliferating period and a subsequent involuting stage,some IHs can still cause serious complications such as ulceration,bleeding,disfiguration and even life-threatening complications.The pathological features of proliferative infantile hemangiomas include dense endothelial cell masses and disordered microvasculature,which are gradually replaced by fibrous adipose tissue during the involuting phase.The pathogenesis of infantile hemangioma is still unclear.The extrinsic factors,such as hypoxia and acidosis and intrinsic factors,such as hypoxia-inducible factor 1 alpha?HIF1-??,vascular endothelial growth factors?VEGFs?,and other angiogenic factors have been suggested to contribute to the development of IH.Non-coding RNAs?ncRNAs?such as long non-coding RNAs?LncRNAs?and microRNAs?miRNAs?have been proved to play a critical roel in the development of infantile hemangiomas.Some miRNAs can serve as potential diagnostic markers and prognostic indicators for infantile hemangiomas.Circular RNA?circular RNA?is a special type of ncRNA,which is a closed circular structure formed by alternative splicing of pre-mRNAs.In recent years,with the rapid development of high-throughput sequencing technology,circRNAs that were previously mistaken for“splicing errors" have been discovered extensively and have become a research priority of ncRNAs.Substantial evidence have proved that circRNA expression is specific in many diseases such as cancer,cardiovascular disease,and nervous system diseases,and can affect disease progression through various mechanisms.The high abundance,high stability,sequence conservation,and tissue and cell specificity of circRNA suggest that it plays an important regulatory role in the occurrence and development of diseases and is expected to become a new biomarker and therapeutic target.Based on current research,the function mechanism of circRNA mainly includes the following four aspects:inhibition of miRNA activity through competitive endogenous RNA?ceRNA?mechanisms or miRNA sponges,which in turn regulates related mRNA expression;competition with linear splicing or regulation of transcription to regulate parental gene expression;interacts with important proteins such as RNA-binding protein?RBP?to regulate gene expression;recent studies have found that some circRNAs also function as encoded proteins.The important role of CircRNA in tumorigenesis and vascular diseases and other related fields suggests that it may also play a critical role in the occurrence and development of infantile hemangiomas.The expression and function of circRNA in infantile hemangiomas still remains unknown.Exploration of the expression and function of circRNA in proliferative infantile hemangiomas is significant for ncRNA regulatory network in infantile hemangioma,and may provide new clues for pathogenesis of infantile hemangioma.Materials and methods:1.Specimen Collection:The specimens of proliferating hemangiomas from June 2015 to May 2017 were collected,washed in PBS,placed in RNALater overnight at 4? and transferred to-20? for microarray and PCR assay;a small number of specimens were put in 4%paraformaldehyde for pathological diagnosis.HE staining and GLUT 1,CD31 immunohistochemical staining was performed to determine the diagnosis and pathological stages of 1Hs.2.Microarray and data analysis:Arraystar Human circRNA V2 Array was used in 4 pairs of proliferative hemangioma specimens and tumor adjacent tissues.TheTrizol method was used to separate and extract RNA,the ultraviolet spectrophotometer was used to det ermine the purity and concentration of RNA samples,and the integrity of the samples was detected by denaturing agarose gel electrophoresis.Rnase R digestion removes linear RNA and enriches circular RNA.Enriched circRNAs were amplified using random primers and transcribed into fluorescent cRNAs.The labeled cRNA was hybridized to Array star Human circRNA V2 Array?8x15K,Arraystar?.The array is scanned by the Agilent Scanner G2505C.Agilent Feature Extraction software was used for array images analysis.R limma package was used for data processing.The t-test?FC>2.0 and P<0.05?was performed for statistics.Differentially expressed circular RNA is shown by volcano maps and cluster maps.3.Validation of qRT-PCR:The expression of circRNA in IHs was verified by qRT-PCR including six significantly defferentially expressed circRNAs?circRNA₁00709,circRNA₁021 16,circRNA₀51239,cricRNA₁00933 and circRNA₁02039 and circRNA₁04310?.CircRNA diverse primers were designed using Primer 5.0.?-actin was selected as an internal reference.4.CircRNA Functional Analysis:A variety of bioinformatics techniques were used to analyze the potential function of differentially expressed circRNAs.CircRN A/miRNA interactions were analyzed by M RE target prediction software?based on TargetScan&miRanda?.M iRWalk 2.0 was used to predict the downstream target genes of miRNAs,combined with mRNA expression profile of proliferative infantile hemangiomas.Cytoscape was used to construct circRNA/miRNA/mRN A regulatory networks.Based on DAVID and KOBAS 3.0 online tool,GO and KEGG pathway enrichment analysis was used to explore the function of parent genes and target genesof circRNAs.5.Preliminary functional verification:we constructed two RNAi lentiviral vectors of circRNA 100933 and transfected them into umbilical vein endothelial cells;MTT,clone formation assay and FACS assay were used to detect the effect of circRNA₁00933 on cell proliferation and apoptosis;Dual luciferase reporter assays were performed to verify the binding sites of circRNA₁00933 and miR-137.QRT-PCR was used to detect the regulation of miR-137 by circRNA₁00933 and the expression of miR-137 in proliferative infantile hemangioma.The effect of circRNA₁00933 on the expression of VEGFA was detected by qRT-PCR and western blot..Results:1.Pathological features of proliferative infantile hemangiomas:GLUT1 and CD31 were both positive in infantile hemangiomas;HE staining showed proliferative hemangiomas were composed of dense endothelial cell mass and disordered microvascular structure.2.CircRNA expression profile of proliferative infantile hemangiomas:each sample was qualified for RNA quality testing and the cRNA labeling efficiency met the requirements.The box plot results showed that the circRNA expression levels in each sample were consistent.Cluster analysis showed that circRNA expression was significantly different between hemangiomas and para-neoplastictissues.Atotal of 234 up-regulated and 374 down-regulated circRNAs?FC>2.0,P<0.05?were identified through scatter plots and volcano plots.Exonic circRNAs occupy the largest proportion,followed by intronic circRNAs.Except chrY,circRNA localization was found in all the other chromosomes.3.Results of qRT-PCR validation:We selected six significantly differently expressed circRNAs for validation.Among them,cricRNA₁00933 and circRNA₁00709 were significantly up-regulated in infantile hemangiomas,and circRNA 104310 was significantly down-regulated in infantile hemangiomas,which was consistent with the microarray results.The difference was statistically significant.The otherthree circRNAs?circRNA₁02116,circRNA₀51239,circRNA₁02039?showed the same trend as the microarray results but lacked statistical significance..4.Functional analysis of differentially expressed circRNAs:MREtarget prediction software was used to predict the miRNA binding sites of circRNAs.A total of 1,248 potential miRNAs were identified.Some of these miRNAs were found to have important regulatory roles in infantile hemangiomas.The circRNA/miRNA network was constructed using Cytoscape.In addition,GO and KEGG pathway enrichment analysis of the parental genes was proved to be closely related to tumorigenesis,angiogenesis-related functions,and signaling pathways.Finally,the circRNA/miRNA/mRNA network construction was performed with significantly upregulated circRNA₁00933 and significantly downregulated circRNA₁04310.The MREs of circRNA₁00933 included miR-137,miR-1298-3p,miR-892b,miR-2113,miR-514a-5p;while the MREs of circRNA₁04310 included miR-499a-3p,miR-1271-3p,miR-216a-3p,miR-197-3p,miR-489-3p.By integrating the miRNA target gene prediction data and mRNA expression profile of proliferative infantile hemangiomas,100 target genes indirectly mediated by circRNA₁00933 and 94 target genes mediated by circRNA₁04310 were obtained.GO and KEGG pathway analysis showed that circRNA₁00933 mediated network mainly enriched in epithelial cell migration,blood vessel development and other biological processes,as well as VEGF signaling pathway,Rap 1 signaling pathway.The circRNA₁04310-mediated network was mainly enriched in biological processes such as lipid metabolism,intracellular signal transduction and other important signaling pathways such as HIF-1? pathway.5.Preliminary functional study of circRNA₁00933:circRNA₁00933 was selected for preliminary functional studies.The results showed that knockdown of circRNA₁00933 significantly inhibited umbilical vein endothelial cell proliferation and promoted apoptosis.Bioinformatics analysis revealed that circRNA₁00933 has a binding site with the important tumor suppressor miR-137.PCR detection showed that miR-137 was significantly down-regulated in infantile hemangiomas.Double luciferase reporter assays confirmed that circRNA₁00933 binds to miR-137 through the predicted site.Knockdown of circRNA₁00933 in cells significantly up-regulated miR-137 expression,suggesting that circRNA-100933 may have a miR-137 sponge effect.VEGFA is an important regulator in infantile hemangiomas.PCR detection showed that VEGFA was significantly upregulated in infantile hemangiomas.Knockdown of circRNA₁00933 in cells significantly down-regulates VEGFA mRNA and protein levels,suggesting that circRNA₁00933 may positively regulate VEGFA expression..Conclusion:1.In this study,we identified the circRNA profile in infantile hemangioma for the first time.A large number of differentially expressed circRNAs were found and six differentially expressed circRNAs were verified by qRT-PCR.The results suggest that circRNAs may play an important role in the occurrence and development of infantile hemangiomas.2.Bioinformatics analysis was used to perform functional analysis of differentially expressed circRNAs.A total of 1024 miRNAs with complementary binding sites to circRN A were found using the M RE target prediction soft ware,and circRNA/miRNA interaction networks were constructed.GO analysis and KEGG pathway analysis showed that the parental genes of circRN A participated in tumorigenesis and vascular development.The results jointly suggest that circRNA may play an important role in infantile hemangiomas through different mechanisms.In the following study,we preliminarily explored the miRNA sponge effect of target circRNA.However,whetherthe differentially expressed circRNA can compete with linear splcing or combine with important proteins that affect the expression of the parent genes remains to be further validated in the future.3.ThecircRNA₁00933/miRNA/mRNA and circRNA₁04310/miRNA/mRNA networks were constructed by integration of miRNA target gene prediction data and mRNA expression profiles.GO analysis and KEGG pathway enrichment analysis showed that the two interaction networks were enriched in several important pathways closely related to infantile hemangiomas.4.Based on the results of PCR validation and bio informatics analysis,we first conducted a preliminary exploration of circRNA₁00933 function.The results showed that knockdown of circRNA₁00933 significantly inhibited cell proliferation,promoted apoptosis,and inhibited the expression of the important factor VEGFA,suggesting that it may play a tumor-promotingrole in infantile hemangiomas.In addition,circRNA₁00933 can also bind to miR-137 and regulate its expression through complementary sites,suggesting that circRNA₁00933 may play a role through the miR-137 sponge mechanism.The preliminary experimental results suggest that circRNA₁00933 may have important research value in infantile hemangiomas.The next step still needs to be further verified by in vitro and in vivo experiments.