Study On Differentially Expressed LncRNA And Their Functions In Peripheral Blood Of Patients With Allergic Rhinitis

The incidence of allergic rhinitis(AR)in the population was as high as 10-30%,which depressed the life quality of patients seriously.Present study has indicated that long non-coding RNA(lncRNA)was closely associated with the development of airway inflammatory diseases.However,its specific effect on AR and the corresponding mechanism were still unclear,which needed further study.Purpose:1.To clarify the difference of the lncRNA gene pool in the peripheral blood between AR patients and healthy people,and to further analyze the possible role of related mRNA and transcription factors in AR;2.The Tentative Study on the impact of lncRNA HIT000095414＿04 on the expression of the transcription factor Oct-1,which provided the foundation for successive study on the mechanism of lncRNA regulating the differentiation of CD4~+T cells and the regulatory effect of Oct-1 on the function and differentiation of CD4~+T cells.Methods:To analyze the expression profiles of lncRNA and mRNA in the blood samples of 3 AR patients and 3 healthy controls by microarray analysis,and to evaluate the reliability of microarray analysis by qRT-PCR,which was used to verify the most significant differential expression of lncRNA in the peripheral blood of 16 AR patients and 18 controls.By analyzing the lncRNA-mRNA co-expression network,we found out the mRNAs related to the significantly differentially expressed lncRNAs,detected their enrichment in gene annotation enrichment analysis,and analyzed the transcription factors related to the significantly differentially expressed lncRNAs to explore the synergistic effects of these lncRNAs and their related mRNAs in the pathogenesis and pathological changes of AR.After extracting CD4~+T cells and non-CD4~+T cells from the peripheral blood of 2 patients,the expression of differentially expressed lncRNAs between the two was verified by qRT-PCR.The adenovirus,which knocked down lncRNA HIT000095414＿04,was constructed to transfect 293 cells,the expression of the knockdown gene and its effect on Oct-1 were verified by qRT-PCR,and the effect of this gene on Oct-1 was further verified by Western blot experiment.Results:The result of microarray analysis showed that 31 lncRNAs(26 up-regulated and 5 down-regulated)and 152 mRNAs(150 up-regulated and 2 down-regulated)were significantly differentially expressed in peripheral blood between AR group and control group(FC≥2.0,P<0.05).We founded that 4(HIT000095414＿04,ENST00000445003.1,ENST00000456563.1 and ENST00000609268.1)of the 5 most differentially expressed lncRNAs(FC>3.0,P<0.05)had significantly higher levels in AR patients than in control subjects by qRT-PCR analysis,and the final consistency rate was 80.0%.The co-expression network analysis of lncRNA-mRNA showed that there were 111 network nodes,including 31 lncRNAs and 61 mRNAs,of which 104relationship pairs were positive correlation,and 7 relationship pairs were negative correlation.At the same time,16 pairs of positive correlations between the 4 significantly different lncRNAs and their co-expressed mRNAs were detected.The results of annotation enrichment analysis showed that 7 of the mRNAs,which co-expressed with those 4 lncRNAs,were also enriched in items related to immune response and allergic diseases.The prediction of transcription factor detected a total of 302 network nodes in 28lncRNAs and 52 transcription factors,and 4 of the 10 transcription factors(Oct-1,AP-1,NF-kappa B and c-Rel),which related to the above 4 lncRNAs,might play a key role in the pathogenesis of lncRNA-mediated AR.By comparing the expression of lncRNAs in CD4~+T cells and non-CD4~+T cells by qRT-PCR,it showed that the expression levels of those 4 lncRNAs were higher in CD4~+T cells.When lncRNA HIT000095414＿04 was knocked down in 293 cells,the results of qRT-PCR and western blot showed that the expression of transcriptional Oct-1 was significantly reduced.Conclusion:Our research was the first to detect the differences in the expression profile of lncRNA between AR patients and healthy people from human peripheral blood,analyzed the molecular mechanisms and biological functions of the significantly different lncRNAs,and found that these lncRNAs mainly expressed in CD4~+T cells.LncRNA HIT000095414＿04 can affect the expression of Oct-1,which involved in the differentiation of CD4~+T cells.Our results would provide reliability for further research on pathogenesis and seek new therapeutic targets of AR.