Establishment and analysis of the detection method for the target gene profile of miR-320 in Qingfei Paidu Decoction

WU Di-yao; LEI Jin-yue; ZHANG Xin-you; XU Tie-long; LI Long-xue

doi:10.20251/j.cnki.1003-3734.2026.01.013

Abstract：Objective:To establish a method for detecting target gene profiles of herbal-derived microRNAs(miRNAs)in mice lung tissue and to apply this method to analyze the target gene profile and biological functions of miR-320 contained in Qingfei Paidu Decoction(QFPD),thereby providing miR-level mechanistic insights into its therapeutic effects against viral pneumonia.Methods:Second-generation high-throughput sequencing was used to quantify the expression of the target miRNA(miR-320)in QFPD.The"Capturing and Sequencing of miRNA-target Complex Technology(CSCT)"was employed to preliminarily identify its target gene profile in mice lung tissue,followed by validation using AlphaFold3.The overlapping results were identified as the definitive target gene profile of miR-320.Functional enrichment analysis was performed to elucidate the regulatory role of QFPD mediated by miR-320 in mice lung tissue.The detection results were compared with TargetScan predictions to evaluate the advantages of the proposed sequential detecntion method(CSCT+AlphaFold3).Results:High-throughput sequencing revealed abundant miR-320 in QFPD,with its expression level ranked among the top 50 miRNAs.In mice lung tissues,miR-320 targeted 26 gene classes,including 19 annotated genes(18 mRNAs and 1 transcriptional enhancer).Through canonical miRNA-mRNA recognition,miR-320 exhibits strong base complementarity with all 26 targets,with minimum free energy values ranging from-35.8 to-21.8 kcal·mol-1.AlphaFold3 predictions yielded a minimum combined ipTM+pTM score of 1.1(within the high-confidence range),providing complete validation(100%)of the CSCT(Computational Small RNA Target Identification)results.This confirms these 26 gene classes as the functional spectrum of miR-320.Compared with TargetScan,the"CSCT+AlphaFold3 Sequential Detection Method"developed in this study demonstrated superior accuracy.Functional analysis revealed that the target genes are mainly enriched in interleukin mediated immune signaling pathways,involved in antigen processing and presentation,immune factor/cell mediated apoptosis,and lymphocyte proliferation/activation.Conclusion:miR-320 from QFPD predominantly regulates immune-related pathways in mice lung tissue,which may represent one of the miRNA mediated mechanisms underlying QFPD's efficacy against viral pneumonia.The developed"CSCT+AlphaFold3"method exhibits high reliability and accuracy for detecting miRNA-target interaction profiles and can serve as a robust technique for studying miRNA mediated regulation in traditional Chinese medicine decoctions.