| Objective: Cutaneous squamous cell carcinoma(CSCC)is a non-melanoma skin cancer,and its incidence is increasing year by year.CircRNAs(circular RNAs)are a kind of newly discovered RNAs,formed by reverse splicing of m RNA precursors,and have a certain degree of stability and conservation.A large number of studies have shown that CircRNA is differentially expressed in the physiological and pathological processes of cancer,and affects the progress of cancer.However,the role of CircRNA in skin squamous cell carcinoma(CSCC)is unclear.This study aims to explore the role of CircRNA in the occurrence,development and response to different treatments of skin squamous cell carcinoma,and to evaluate their potential as diagnostic and prognostic biomarkers and new treatment methods.This study aims to identify the differentially expressed CircRNA(Has?circ?0002891)and its potential function in the occurrence and development of skin squamous cell carcinoma.Methods: In this study,human skin squamous carcinoma cell lines(A431,SCL-1),human keratinocytes(Ha Ca T)and human skin squamous cell carcinoma tissue were selected as the research objects.Among them,20 pairs of skin squamous cell carcinoma tissues and their adjacent non-tumor tissues were derived from skin squamous cell carcinomas(already diseased)that were excised in the operating room of the Dermatology and Venereology Department of the First Affiliated Hospital of XXXXXX from September 2017 to September 2018.Physiological diagnosis),the relevant experimental protocol was approved by the ethics committee of the First Affiliated Hospital of XXXXXX.In this study,the expression profiles of circRNAs in 3 pairs of skin squamous cell carcinoma and adjacent non-tumor tissues were detected by RNA-seq high-throughput sequencing technology,and differential circRNAs were screened according to the multiple of difference FC?2 and expression p<0.005.Then,q RT-PCR was used to verify the candidate circRNAs in another 17 pairs of tissue samples and skin squamous cell carcinoma cell lines A431 and SCL-1,and further screen possible key circRNAs.Then the screened out circRNA molecule hsa?circ?0002891 was further analyzed to determine its biological function effect on skin squamous cell carcinoma.Predict the regulatory axis of ce RNA mechanism through bioinformatics and literature research.Finally,the regulatory mechanism of hsa?circ?0002891 on skin squamous carcinoma is analyzed through experiments.Results: 1)Using high-throughput RNA-seq sequencing technology and bioinformatics methods,the expression of circRNAs in 3 pairs of CSCC and adjacent non-tumor tissues was analyzed,and circRNAs with significant differences among tissue samples from 6patients were mapped.The results showed that the expression of circRNA in CSCC and adjacent non-tumor tissues was significantly different.The five chromosomes enriched by differentially expressed circRNA were ChR13,ChR16,chr17,chr18 and chr19.GO functional analysis of differentially expressed circRNA mainly involves DNA replication,endosome transport,nucleic acid transport,RNA transport,positive regulation of cell morphogenesis,cytoplasmic transport,purine nucleoside diphosphate metabolism,retrograde transport,endosome to Golgi,cells-Substrate adhesion junctions,cytoplasmic stress particles,reverse polymerization complexes.The enrichment analysis of the KEGG pathway differentially expressing circRNA mainly involves autophagy,axon guidance,endocytosis,insulin resistance,epithelial mesenchymal changes,thyroid hormone signaling pathway,phosphatidylinositol signaling system,HIF-1 signaling pathway,and cells cycle,bacterial invasion of epithelial cells,degradation of valine,leucine and isoleucine.3)According to the sequencing results and bioinformatics analysis,10669 circRNAs with differential expression were screened out,among which395 were up-regulated circRNA and 55 were down-regulated circRNA(FC?2,p<0.005).According to the difference multiple and expression level,8 circRNAs(4 up-regulated hsa?circ?0002891,hsa?circ?0049398,sa?circ?0001821 and hsa?circ?0007976,4down-regulated: hsa?circ?0096920,hsa?circ?0005435,hsa?circ?0073762hsa?circ?0006628)were screened out for further study.The other 17 pairs of matched samples(20 pairs of tissue samples)and candidate circRNAs in the skin squamous cell carcinoma cell lines A431 and SCL-1 were verified by q RT-PCR.It was found that the sequencing results of 6 molecules were consistent with the q RT-PCR results in tissues and cell lines.Among them,the difference of hsa?circ?0002891 is the most significant and the expression level is higher,so hsa?circ?0002891 was selected for the next study.5)According to the expression of has?circ?0002891 in different squamous cell carcinoma cell lines(A431 and SCL-1),select the appropriate cell line(A431)for the next step of functional verification.The results show that after has?circ?0002891 is interfered,skin squamous cell carcinoma.The cell proliferation ability is significantly weakened,and the apoptotic ability was significantly enhanced,while the migration and invasion capabilities are weakened.6)In the skin squamous cell line A431,si RNA interference inhibited the expression of has?circ?0002891,while the expression of miR604 increased,and the expression of HNRNP F decreased.Conclusions: 1.The level of has?circ?0002891 in skin squamous cell carcinoma tissue was significantly increased.2.The high expression of has?circ?0002891 can induce carcinogenesis of skin squamous cells.3.Has?circ?0002891 may reduce the inhibitory effect of miR-604 on HNRNP F,leading to an increase in the level of HNRNP F,thereby enhancing the proliferation ability and epithelial-mesenchymal transition of skin squamous cell carcinoma,and ultimately promoting the occurrence of skin squamous cell carcinoma. |
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