The Research On Expression Profiling Of Long Nocoding RNAs In Hashimoto’s Thyroiditis

Objective: In this study,the expression profile of Long non-coding RNA（Lnc RNA）in the peripheral blood of both HT patients and healthy volunteers was analyzed through next-generation sequencing（NGS）technology;Significant differentially expressed LncRNAs were screened and verified,and their relationship with patients’ autoantibody levels was analyzed,and then explore their diagnostic value;The functions of differentially expressed LncRNAs was analyzed by Bioinformatics to explore the biological processes in which they may participate and to predict the potential regulatory genes.The expression level of regulatory genes was detected and the relationship between LncRNAs and LncRNAs was analyzed to preliminarily explore the biological role of LncRNAs in the pathogenesis of HT,providing a new direction for the study of LncRNAs involvement in the pathogenesis of HT.Methods: 1、 Five primary HT patients and five healthy volunteers were selected as the experimental group and healthy control group,respectively,and total RNA in peripheral blood mononuclear cells（PBMCs）was extracted by Trizol method.After the RNA quality control was qualified,the sequencing library was constructed,and the 10-pm library was denaturation into single-stranded DNA molecules;After amplification,the sequencing was conducted by using PE mode on Illumina Hi Seq 4000 sequencer,and the biological and relevant statistical software was used for calculation and analysis,then the LncRNAs with different expressions between HT patients and healthy volunteers were obtained.2、 Peripheral blood samples of 30 clinically confirmed HT patients and 30 healthy volunteers were collected.Six LncRNAs with significant difference in expression （Fold change≥2.0,P≤0.05）between the HT group and the healthy control group were selected,and their relative expression were verified by the real-time fluorescent quantitative reverse transcription polymerase chain reaction（RT-q PCR）technology;LncRNAs with differential expression consistent with sequencing results were selected to analyze their correlation with autoantibodies of HT patients,and their diagnostic value for diseases was analyzed by ROC curve.3、 Bioinformatics methods were used to perform Gene Ontology（GO）functional analysis and KEGG Pathways analysis on the adjacent target genes of differentially expressed Lnc RNA,so as to speculate and annotate the signaling pathways in which these genes may participate and predict their biological functions.4、 By searching literatures,four potential target genes that might be correlated between verified LncRNAs and HT were selected,and their expression changes in HT were detected by PCR technology to analyze their correlation with the expression changes of LncRNAs.Results: 1、Through high-throughput sequencing technology,69,544 upregulated and 27,742 down-regulated LncRNAs were detected between HT patient group and the healthy control group.Of the 218 LncRNAs with significant difference,94 were up-regulated and 124 were down-regulated（Fold-change≥2.0,P-value 0.05）.2、The 218 significantly differentially expressed LncRNAs were analyzed,and six LncRNAs were selected and their expressions were detected by PCR according to the average distribution degree among sequencing samples,P value difference,multiple molecular weight and other factors,results showed that Lnc RNA-XLOC_l2₀06631 and Lnc RNA-LOC729737 were up-regulated,Lnc RNA-LOC100288778 and Lnc RNA-BC041964 were down-regulated,which were consistent with the sequencing results;Correlation analysis showed that the four LncRNAs were correlated with thyroid autoantibodies,suggesting that they may be involved in the occurrence and development of HT;ROC curve analysis indicated that the above four LncRNAs have certain diagnostic value（AUC:0.5-1.0,P<0.05）,of which Lnc RNA-XLOC_l2₀06631（AUC:0.926,95% CI 0.855-0.997,P<0.001）has the highest diagnostic value for diseases.3、Biological function prediction and analysis: GO analysis of the potential target genes of differentially expressed LncRNAs from high-throughput sequencing results showed that the three terms which had the highest enrichment degree in biological process,cell component and molecular function.Among the potential target genes of up-regulated LncRNAs were respectively “the assembly proton-transporting two-sector ATPase complex”,“catalytic domain and proton-transporting ATP synthase activity” and “rotational mechanism”;Among the potential target genes of down-regulated LncRNAs were respectively “embryonic viscerocranium morphogenesis”、“transcription export complex” and “acetylcholine binding”;Pathways analysis showed that there were 17 and 19 signaling pathways associated with adjacent target genes of up-regulated and down-regulated LncRNAs.4、The mRNA of RUNX3 and STAT3 were down-regulated,the mRNA of IL-21 R and MECP2 was up-regulated in the potential target genes of the above verified LncRNAs consistent with sequencing results.Correlation analysis showed that Lnc RNA-LOC729737 was positively correlated with mRNA expression of MECP2,Lnc RNA-LOC100288778 was positively correlated with mRNA expression of RUNX3,Lnc RNA-BC041964 was negatively correlated with mRNA expression of IL-21 R,and Lnc RNA-XLOC_l2₀06631 was negatively correlated with mRNA expression of STAT3.Conclusions: 1、This study used the second-generation high-throughput sequencing method to screen and construct the expression profiles of differentially expressed Lnc RNA and mRNA between HT patients and healthy volunteers.2、The results of validation of some significantly differentially expressed LncRNAs showed that the expression changes of Lnc RNA-LOC729737 、Lnc RNA-LOC100288778 、 Lnc RNA-BC041964 and Lnc RNA-XLOC_l2₀06631 were consistent with those of high-throughput sequencing.Correlation analysis showed that the four LncRNAs were correlated with thyroid autoantibodies,suggesting that they may be involved in the occurrence and development of HT.ROC curve analysis indicated that the above four LncRNAs have certain diagnostic value,of which Lnc RNA-XLOC_l2₀06631 has the highest diagnostic value for diseases.3、GO analysis and Pathway analysis suggested that LncRNAs were involved in biological processes related to HT,such as thyroid hormone signaling pathway.4、Potential target genes of Lnc-LOC729737、Lnc-LOC100288778、Lnc-BC041964 and Lnc-XLOC_l2₀06631 are MECP2、RUNX3 、IL-21 R and STAT3,which were abnormally expressed in HT patients and there were correlation between LncRNAs and potential target genes,suggesting that these four LncRNAs may participate in the pathogenesis of HT and provide a research basis for subsequent functional experiments.