Transcriptome Analysis Of Primary Cells And The Role Of LncRNA-SNHG16 In Adamantinomatous Craniopharyngioma

Background:Craniopharyngioma(Craniopharyngioma,CP)is a rare congenital intracranial tumor growing in the sellar region.It accounts for 3-7%of children’s primary intracranial tumors,which accounts for more than 60%of the sellar tumors in children.Craniopharyngioma is considered as a benign tumor of grade I in the classification of WHO central nervous system tumors.However,high incidence of low endocrine function,hypothalamic dysfunction and visual impairment make the quality of life of patients often be greatly affected.Craniopharyngioma can be divided into two pathological subtypes:adamantinomatous craniopharyngioma(ACP)and papillary craniopharyngioma(PCP).The detection rate of BRAF p.V600E mutation in PCP is over 95%,and there is no other gene mutation.Therefore,targeted drugs,such as Verofini and Dalafenib,have entered clinical trials.But the progress of new treatment for ACP is slow.In the genetic background,mutations in exon 3 of the CTNNB1 gene are present in almost all ACPs,resulting in the inability of β-catenin to be degraded and accumulating in the nucleus,leading to activation of the WNT pathway,which is considered to be the most important mechanism of the occurrence and development of ACP.Previous basic research on ACP has mainly focused on tumor tissue samples and genetically engineered mice.Recently,the research on Hesxl Cre/+;Ctnnb1 lox(ex3)/+ and Sox2 CreERT2/+;Ctnnb1 lox(ex3)/+ transgenic mice has made great progress.However,it is still undeniable that the mouse model can not completely replace the human tumor.The primary tumor cells are still an important research platform.With the development of high-throughput sequencing,more and more new long non-coding RNAs(lncRNAs)have been discovered and identified.Meanwhile,lncRNA has been found to play an important role in regulating biological processes at all levels through various mechanisms.However,there is no report about the lncRNA sequencing research of ACP,so it is urgent to find such a large RNA regulation mechanism in ACP.Objection:1.Optimize the primary cell culture method for adamantinomatous craniopharyngioma,identify the characteristics of the primary cell lineage,and make clear whether the primary cell can represent the tumor tissue as a stable research platform.2.To clarify the biological characteristics of the adamantinomatous craniopharyngioma system and find new regulatory mechanisms for the key biological behaviors of adamantinomatous craniopharyngiomas.3.Exploration of key regulation mechanisms of adamantinomatous craniopharyngiomas from the perspective of non-coding RNA.Methods and materials:1.Primary cells were cultured using a modified method.Immunofluorescence was used to complete the lineage identification.High-throughput sequencing was used to complete the characterization of the primary cell transcriptome.The similarities and differences between primary cells and tumor tissues were clarified by gene set enrichment analysis.2.Deep excavation of the open access adamantinomatous craniopharyngioma transcriptional microarray data.The core biological processes and key regulatory mechanisms of adamantinomatous craniopharyngioma were discovered by using GSEA and PPI network analysis tool.The mechanism of discovery was verified by immunohistochemistry of tissue specimens and functional experiments of primary cells.3.Using high throughput sequencing technology to obtain lncRNA structure,expression and other information.The key regulation mechanism was found by WGCNA and ceRNA mechanism prediction.The regulatory mechanism of lncRNA was verified by RT-qPCR,cell function test,Western blot and double luciferase reporter assay.Results:1.Primary cell phenotype identification suggests that it conforms to the lineage characteristics of epithelial cells.The results of the transcriptome sequencing showed that the primary cells and tumor tissues were basically consistent with the characteristic gene and gene sets level.2.The data of transcriptional microarray of human adamantinomatous craniopharyngioma showed that the abnormality was mainly centered on macro-development and micro-development,and contained many biological processes,including lipid metabolism,calcification and inflammatory response.TGF-β pathway activation in adamantinomatous craniopharyngioma was detected by GSEA and PPI network analysis.It was confirmed by immunohistochemistry and cell function tests that it promoted the occurrence of EMT and the expression of microRNA-132.3.LncRNA high throughput sequencing results found 3116 new lncRNA,and WGCNA and ceRNA predicted the key regulatory lncRNA-SNHG16.It is further verified that the abnormal high expression of SNHG16 promotes the continuous activation of TGF-β pathway by adsorbing miR-132.Conclusion:1.The modified primary cell culture method can be used to establish primary cell lines with limited passages,and it is proved that primary cells can partly represent tumor tissue as a stable research platform.2.Systematic biological analysis suggests the core biological processes of adamantinomatous craniopharyngioma and provides directions for the study of typical manifestations.Network prediction and experiment have proved that TGF-βpathway activation promotes the occurrence of EMT in the adamantinomatous craniopharyngioma and suggests that miR-132 plays an important role in it.3.High throughput sequencing found new transcriptional transcripts and predicted that the key regulation of lncRNA was SNHG16.It is suggested that the activation of classical WNT pathway promotes the abnormal high expression of lncRNA-SNHG16,and it can adsorb miR-132 through the mechanism of ceRNA,further promote the TGF-β pathway continuous activation.This leads to tumor penetrated pia mater and further embedded into the surrounding structure.