Exploring The Differential Expression And Correlation Of MiRNA-93-5p In Papillary Thyroid Cancer Based On High-throughput Sequencing

ObjectivePapillary thyroid carcinoma（PTC）is the most common malignant tumor of the endocrine system.Micro RNA,as a non-coding protein RNA,is widely involved in a variety of biological processes.This study is based on second-generation high-throughput sequencing analysis to find out the differential miRNAs related to the occurrence and development of PTC,explore the diagnostic value and correlation of miRNA-93-5p in PTC,and provide biomarkers for the early diagnosis of PTC.Methods1.Sample collection:Peripheral blood samples were collected from January 2022 to September 2022 from patients with thyroid nodules who attended our nail and breast surgery department to be treated surgically,and patients with pathological findings of PTC and benign thyroid masses will be enrolled in this study2.High-throughput sequencing and bioinformatics analysis:A total of 8 peripheral whole blood samples from patients with PTC and benign thyroid tumor were collected and divided into TC group（4 patients with PTC）and TN group（4 patients with benign thyroid tumor）.Eight peripheral blood samples were used for second-generation high-throughput sequencing analysis,and the peripheral blood samples of PTC and benign thyroid tumor were sequenced using transcriptome sequencing technology（RNA-seq）,DESeq（Ver Sion 1.18.0 and Ander Huber W2010）was used to screen differentially expressed miRNA molecules through differential expression multiple（|log₂Fold Change|>1）and significance of expression difference（P-value<0.05）,and bioinformatics was used to explore the important role played in biological development stage,molecular biological mechanism,protein composition and signal pathway,etc.Through GO enrichment analysis（gene ontology analysis）KEGG pathway enrichment analysis（Kyoto Encyclopedia of Genes and Genomes）and miRNA-m RNA gene interaction study to determine the important role of differentially expressed miRNAs in organisms by regulating m RNA.3.Clinical study:A total of 60 peripheral blood samples were collected from patients with PTC and patients with thyroid benign tumor,and were divided into PTC group（40patients with PTC）and control group（20 patients with thyroid benign tumor）.The differential expression of miRNA-93-5p was verified by real-time fluorescent quantitative polymerase chain reaction（q RT-PCR）,According to the cycle threshold（CT value）,calculate 2^-ΔΔCt as the relative expression of miRNA-93-5p,analyze the expression level of miRNA-93-5p in the peripheral blood of patients with PTC and patients with thyroid benign tumor,study the diagnostic value of miRNA-93-5p in PTC,and explore the relationship between miRNA-93-5p and clinical parameters.4.Statistical Methods:Use SPSS.26.0 software to adopt different statistical methods according to different types of data.Spearman correlation coefficient is used to analyze the correlation between miRNA-93-5p and clinical features.ROC curve is used to evaluate the diagnostic value of miRNA-93-5p for PTC.Chi-square tes is used to analyze the correlation between the pathological data of PTC and the expression level of miRNA-93-5p.The difference is statistically significant with P<0.05.Results1.Combined with RNA-seq data,with|log₂Fold Change|>1 and P<0.05 as the cutoff criteria,a total of 89 up-regulated miRNAs and 20 down-regulated miRNAs were screened,of which the differential expression multiple of miRNA-93-5p was the largest and the expression of miRNA-93-5p was significantly up-regulated in PTC.2.According to the GO enrichment analysis of the target genes of differentially expressed miRNA,it is concluded that the molecular function may be related to protein binding,organic matter binding,nucleotide binding and ion binding;In the process of biological development,it may be related to the positive regulation of cell function,biological process,molecular function,cell communication,signal transduction and cell metabolism;In the layer of cell group,it may be related to organelles,membrane-bound organelles,plasma membrane components and plasma membrane components.The KEGG pathway enrichment analysis of target genes with differentially expressed miRNA showed that they might be related to tumor signal pathway,human herpesvirus 1 infection,regulation of cell adhesion,calcium signal pathway,miRNA in cancer,chemokine signal pathway,etc.3.Based on bioinformatics analysis,prediction of target gene of miRNA-93-5p and KEGG enrichment analysis showed that miRNA-93-5p can regulate PI3K-AKT signal pathway and FOXO signal pathway by targeting CCND2 and ERBB3.4.According to the q RT-PCR experimental data,the relative expression of miRNA-93-5p in the peripheral blood of 40 patients with PTC was 2.10±0.42,and the relative expression of miRNA-93-5p in the peripheral blood of 20 patients with benign thyroid tumor was 1.03±0.16.The expression of miRNA-93-5p in PTC was significantly up-regulated（P<0.05）.The difference was statistically significant.5.The diagnostic value of miRNA-93-5p was 0.846（95%CI:0.727-0.925）,the sensitivity and specificity were 80%and 88%respectively,which indicated that miRNA-93-5p had certain diagnostic efficacy for PTC.6.Spearman correlation analysis showed that miRNA-93-5p was positively correlated with CEA（r=0.132,P=0.043<0.05）,while miRNA-93-5p was not significantly correlated with TSH,Tg,Tg Ab,TPOAb and CT（P>0.05）.Combined with the clinicopathologic parameters of PTC patients,the expression of miRNA-93-5p in the peripheral blood of PTC patients had a significant positive correlation with the TNM stage,lymph node metastasis,capsule or vascular invasion of PTC patients（P<0.05）,but had no significant correlation with the sex,age and tumor tissue size of PTC patients（P>0.05）.Conclusion:1.The differentially expressed miRNAs in the peripheral blood of PTC were obtained through the second-generation high-throughput sequencing,and the expression of miRNA-93-5p was significantly up-regulated.2.Through bioinformatics analysis,the target gene of miRNA-93-5p and its regulated signal pathway may be involved in the occurrence and development of PTC.3.Through q RT-PCR test,the expression level of miRNA-93-5p in peripheral blood of patients with PTC was significantly higher than that of benign thyroid tumor（P<0.05）,which could be used as a molecular marker for PTC diagnosis.4.The expression of miRNA-93-5p has a significant positive correlation with the TNM stage,lymph node metastasis,capsule or vascular invasion of PTC patients（P<0.05）,suggesting the potential value of miRNA-93-5p in predicting the severity of PTC.