Integrated analyses of miRNA and mRNA profiles in leukocytes and serums in traditional Chinese medicine (TCM)-defined Pi-qi-deficiency syndrome and Pi-wei damp-heat syndrome resulting from chronic atrophic gastritis

Abstract

Background: To investigate the microRNA (miRNA)-gene interactions underlying leukocyte functions and characteristics, especially the potential serum biomarkers, implicated in the traditional Chinese medicine (TCM)-defined Pi-qi-deficiency syndrome (PQDS) and Pi-wei damp-heat syndrome (PDHS) resulting from chronic atrophic gastritis (CAG).

Methods: Using RNA/miRNA-sequencing approach, compared with healthy control population, we identified the PDHS- or PQDS-specific miRNAs and genes in leukocytes or serums, especially the Zheng (syndrome)-specific miRNA-gene interactions, and further decoded their functions and pathways.

Results: Despite being the TCM-defined Zhengs resulting from the same disease of CAG, the Zheng-specific genes and miRNAs were not same. The PDHS-specific leukocyte genes were mainly involved in defense and immune responses, including NOD-like receptor signaling and several synapses-related pathways. The expression upregulation of PDHS-specific genes enriched in the neutrophil degranulation pathway, indicated the enhanced leukocyte degranulation activation. The PQDS-specific genes in leukocytes were implicated in inflammatory response, extracellular matrix (ECM) organization and collagen catabolism. They could be enriched in MAPK and IL17 signaling and helper T cell differentiation pathways, especially the pathways associated with cell-to-cell adhesion/junction and communication such as cell adhesion molecules, ECM organization and ECM-receptor interaction, probably contributing to the characteristics and functions of leukocytes. Also, the experimentally-supported miRNA-gene interactions, concerned with COL4A2, COL26A1, SPP1 and PROCR, were implicated in the regulation of pathways related to cell-to-cell adhesion/junction and communication, suggesting the potential roles of the PQDS-specific miRNA-gene interactions for the characteristic and functional changes of leukocytes. Interestingly, the PQDS-specific miRNAs in the serums and the corresponding leukocytes, seemed to have the common roles in contributing to the characteristics and functions of leukocytes. Importantly, the hsa-miR-122-5p could be a potential biomarker, capable of being contained and carried in plasma exosomes and much higher expression in both the leukocytes and corresponding serums in the CAG patients with PQDS rather than PDHS.

Conclusions: These results may provide new insights into the characteristic and functional changes of leukocytes in the two Zhengs, PDHS and PQDS, especially the miRNA-mediated gene regulation underlying leukocyte characteristics and functions, with potential leukocyte and serum biomarkers for future application in integrative medicine. Trial registration ClinicalTrials.gov, NCT02915393. Registered on September 17, 2016.

Keywords: Chronic atrophic gastritis; Leukocyte; Pi-qi-deficiency syndrome; Pi-wei damp-heat syndrome; Serum; miRNA biomarker.

Conflict of interest statement

The authors declare no conflict of interests regarding the publication of this paper.

Figures

Fig. 1

Expression pattern clustering analyses of the differential genes and miRNAs identified in the PDHS and PQDS populations. a Veen diagrams detailing the differential genes and miRNAs found in the leukocytes and serums. b hierarchical clustering (HCL) analyses of expression profiles of the differential miRNAs in leukocytes. c HCL analyses of expression profiles of the differential genes in leukocytes. d HCL analyses of expression profiles of the differential miRNAs in the serums. Control healthy individuals, PQDS chronic atrophic gastritis patients with Pi-qi-deficiency syndrome, PDHS chronic atrophic gastritis patients with Pi-wei damp-heat syndrome

Fig. 2

Gene ontology (GO) function enrichment analyses of the Zheng-specific genes and the targets of the Zheng-specific miRNAs discovered in the different case populations. a Bubble diagrams showing the enriched GO function terms of PDHS-specific genes (left) and PQDS-specific genes (right) in leukocytes. b Heatmaps indicating the GO function enrichment results of the validated targets of PDHS-specific miRNAs (left) and PQDS-specific miRNAs (right) in leukocytes. The common GO terms between the two Zhengs were shown in bold font. c Heatmaps displaying the enriched GO function terms of the validated targets of PDHS-specific miRNAs (left) and PQDS-specific miRNAs (right) in serums. The common GO terms between the two Zhengs were shown in bold font. The pink highlighted GO terms indicate the commonly enriched GO terms of the validated targets of the PDHS-miRNAs in the leukocytes and corresponding serums. The indigo highlighted GO terms mark the commonly enriched GO terms of the validated targets of the PQDS-miRNAs in the leukocytes and corresponding serums. PQDS chronic atrophic gastritis patients with Pi-qi-deficiency syndrome, PDHS chronic atrophic gastritis patients with Pi-wei damp-heat syndrome

Fig. 3

Pathway enrichment analyses of the Zheng-specific genes and the targets of the Zheng-specific miRNAs identified in the different case populations. a Bubble diagrams showing the enriched KEGG pathways of PDHS-specific genes (left) and PQDS-specific genes (right) in leukocytes. b Heatmaps indicating the KEGG pathway enrichment results of the validated targets of PDHS-specific miRNAs (left) and PQDS-specific miRNAs (right) in leukocytes. The common pathway terms were specially shown in bold font. c Heatmaps displaying the enriched KEGG pathways of the validated targets of PDHS-specific miRNAs (left) and PQDS-specific miRNAs (right) in serums. The common pathway terms were specially shown in bold font. The pink highlighted pathway terms indicate the commonly enriched pathways of the validated targets of the PDHS-miRNAs in leukocytes and serums. The indigo highlighted pathway terms mark the commonly enriched pathways of the validated targets of the PQDS-miRNAs in leukocytes and serums. PQDS chronic atrophic gastritis patients with Pi-qi-deficiency syndrome, PDHS chronic atrophic gastritis patients with Pi-wei damp-heat syndrome

Fig. 4

Interaction network analyses of the PDHS-specific and PQDS-specific genes in leukocytes. a The network detailing the interactions of PDHS-specific genes. b The network generated to show interactions of PQDS-specific genes. The number marked in a node indicates the count of the PQDS-specific or PDHS-specific miRNAs which were validated targeting to the corresponding node gene. The node genes-enriched pathways were specially marked in the interaction networks. PQDS chronic atrophic gastritis patients with Pi-qi-deficiency syndrome, PDHS chronic atrophic gastritis patients with Pi-wei damp-heat syndrome

Fig. 5

Function and pathway analyses for the experimentally supported miRNA-gene interactions in the leukocytes from the PQDS and PDHS populations. a The network detailing the PQDS-specific and PDHS-specific miRNA-gene interactions in leukocytes. The target genes having similar functions were specially marked and grouped together. The expression levels of miRNAs and genes were visually presented in the form of colorful nodes showing the color change (green to red) in brightness and chromaticity. b The relationship network of the enriched pathways of genes belonging to the PQDS-specific miRNA-gene interaction pairs. The edge between two nodes indicate there are several common genes implicated in the nodes-labeled pathways, thus the edge thickness depends on the number of the existed common genes. The blue arrows denoted the immune pathways containing the validated targets of the has-miR-122-5p. c Clustering analyses of the expression profiles of the miRNAs belonging to the experimentally supported miRNA-gene interaction pairs in different populations. Two heatmaps were generated to profile the miRNAs expression in the leukocytes and serums of individuals from different populations. Especially, if there are additional experimental evidences supporting that a listed miRNA can be contained and carried in the plasma exosomes, it was thus specially marked with an orange circle. Control healthy individuals, PQDS chronic atrophic gastritis patients with Pi-qi-deficiency syndrome, PDHS chronic atrophic gastritis patients with Pi-wei damp-heat syndrome

Fig. 6

Function and pathway analyses of the validated targets of the exosome-carried hsa-miR-122-5p. a Boxplot graphs of expression levels of hsa-miR-122-5p in the leukocytes and serums from different populations. b Overview of the interaction network of targets of hsa-miR-122-5p. The invalidated targets of has-miR-122-5p were retrieved from the released “TarBase” collecting the experimentally supported miRNA-gene interactions, both in vivo and in vitro. Especially, the orange nodes show the validated targets that are also found in the PQDS-specific genes identified in the leukocytes, including JAG2, SH3RF1, MYOF, PCSK6 and COL4A2. c The relationship network of the enriched pathways of targets of hsa-miR-122-5p. A node labeled with a pathway term, indicates an enriched pathway of targets, and the node size depends on the number of edges linking the node. The edge between two nodes denotes that there exist several common genes involved in the two nodes-labeled pathways, so the edge thickness relies on the number of the existed common genes. d The bubble diagram generated to show the enriched pathways of the validated targets of hsa-miR-122-5p. TPM transcripts per million reads, Control healthy individuals, PQDS chronic atrophic gastritis patients with Pi-qi-deficiency syndrome, PDHS chronic atrophic gastritis patients with Pi-wei damp-heat syndrome