Comprehensive Analysis Of RNA Sequencing And CircRNA Regulated CeRNA Network In Invasive Pituitary Adenoma

Objective:Pituitary adenoma is a kind of the most common intracranial tumors in clinic.Generally speaking,most pituitary adenomas are benign tumors,but the biological characteristics of some invasive pituitary adenomas are similar to those of malignant tumors.In recent years,nc RNAs including miRNA,lnc RNA and circ RNA have been confirmed to be related to the formation of invasive characteristics of pituitary adenomas.In this studym,high-throughput sequencing technique was used to nc RNAs related to invasive pituitary adenomas,and to construct a differentially expressed circ RNA-regulated ce RNA network to explore potential biomarkers related to the invasiveness of pituitary adenomas.This study is to provide a theoretical basis for the study of biological function and specific regulatory mechanism closely related to the invasive characteristics of pituitary adenomas.Methods:Fifty pathological specimens of pituitary adenomas treated by transnasal and sphenoidal approach in the Department of Neurosurgery,Affiliated Hospital of Hebei University from January 2019 to September 2020 were collected.According to the imaging results,pathological results,intraoperative conditions and other criteria,the patients were divided into invasive pituitary adenoma case group and non-invasive pituitary adenoma case group.3cases of non-invasive pituitary adenomas and 3 cases of invasive pituitary adenomas were randomly selected,and the age,sex and comorbidity diseases were collected.Total RNA was extracted from 6 tumor tissue specimens and then a c DNA library was constructed.The full transcriptome was sequenced using Illumina platform to screen the differentially expressed circ RNA,miRNA and mRNA related to the invasiveness of pituitary adenomas.The functional enrichment analysis of the screened differential genes was carried out.Cytoscape software was used to construct specific ce RNA regulatory networks for circ RNA,miRNA and mRNA obtained by difference analysis,and the key circ RNA and its ce RNA networks were screened out according to the number of Degree.The functional enrichment analysis of ce RNA regulatory network is analyzed.The remaining 44 tumor tissue samples were selected for qRT-PCR verification of the screened circ RNA.Results:1.The results of high-throughput sequencing showed that there were 14 differentially expressed circ RNA including 7 up-regulated and 7 down-regulated related to the invasiveness of pituitary adenomas,and their functions were mainly concentrated in the process of transcriptional regulation,the binding of nuclei and organelles,and the binding of DNA molecules,but no significantly enriched signal pathways were obtained.2.There were 18 differentially expressed miRNA including 11 up-regulated and 7down-regulated,and their main functions were related to transcriptional regulation,nervous system development and phosphorylation,nuclear and molecular binding,and are significantly related to the famous c AMP,MAPK and Rap1 signal pathways.3.There are 85 differentially expressed mRNA including 40 up-regulated and 45down-regulated,its function is mainly concentrated in growth and development,cytoplasm,organelle,protein molecular binding,and the signal pathway was mainly related to PI3K-Akt signal pathway.4.Bioinformatics techniques were used to screen the miRNA,that can bind to differentially expressed circ RNAs and the target mRNA,of miRNAs and compared with the results of high-throughput sequencing.Finally,the differentially expressed circ RNA-miRNA,was screened to construct a ce RNA network regulated by specific circ RNA associated with invasive pituitary adenomas.The circ RNA in ce RNA net is screened by Cytoscape software,and the two circ RNA,with the highest number of Degree are selected as hsa＿circ＿0036795and hsa＿circ＿0068067 respectively.The related ce RNA networks of the two circ RNA are visualized with a total of 17719 edges and 10215 nodes.Functional analysis of ce RNA network showed that it was mainly enriched in transcriptional process,GTP enzyme activity,intracellular components,neuronal projection and protein binding,while KEGG signal pathway analysis showed that ce RNA network was significantly related to c AMP and Ras signaling pathways and pathways in cancer.5.Real-time quantitative PCR showed that the expression of hsa＿circ＿0036795 and hsa＿circ＿0068067 in invasive pituitary adenomas was significantly lower than that in non-invasive pituitary adenomas,which verified the accuracy of RNA-Seq.6.Furthermore,the miRNA roles of these two circ RNAs were analyzed,and it was found that hsa-miR-124-3p and hsa-miR-1304-3p could play an interregulatory role with multiple genes in the TGF-β/Smad signaling pathway.Conclusion:1.In this study,we found that invasive pituitary adenomas have specific circ RNAs,miRNAs and mRNAs.We use bioinformatics techniques to predict their respective molecular functions and biological processes.These results provide new ideas for the pathogenesis of invasive pituitary adenomas and provide a reference basis for subsequent research on the mechanism of pituitary adenoma invasion.2.In this study,we found that hsa＿circ＿0036795 and hsa＿circ＿0068067 play an important role in the formation of invasive characteristics of pituitary adenomas and are expected to become new diagnostic markers and therapeutic targets for invasive pituitary adenomas.The possible mechanisms are that hsa＿circ＿0036795 and hsa＿circ＿0068067 affect the TGF-β/Smad signal pathway through respectively sponging with miR-124-3p and miR-1304-3p,which participates in the formation of invasiveness of pituitary adenomas.