Differential genes and miRNAs identified in leukocytes and serums
Compared with the control population (healthy individuals, n = 5), 186 and 196 differential genes, as well as 30 and 72 differential miRNAs, were respectively identified in the leukocytes from two case populations, including the CAG patients with PDHS (n = 5) and CAG patients with PQDS (n = 5) (Fig. 1A; Supplemental Table S2-5). Besides, total 52 and 99 differential miRNAs were respectively found in the serums of the CAG patients with PDHS and PQDS (Fig. 1A; Supplemental Table S6 and 7). We particularly performed hierarchical clustering (HCL) expression analyses of the differential genes and miRNAs which were discovered in the leukocytes and serums from the three populations (Fig. 1B-D). The HCL showed that the expression profiles of differential miRNAs in the leukocytes of individuals in the same population clustered well together, distinguished from those in other populations (Fig. 1B). Interestingly, the miRNA termed hsa-miR-122-5p, was observed to be the common differential miRNAs in the leukocytes and serums of the CAG patients with PQDS (Fig. 1B and D).
The Zheng-specific genes and miRNAs
The Zheng-specific genes in this study mean the differential genes and miRNAs which were observed in the individuals only with the TCM-defined PQDS or PDHS, excluding their common differential genes and miRNAs. Thus, the PQDS-specific genes and miRNAs were found only in the CAG patients with PQDS rather than PDHS. As indicated (Fig. 1A), 155 genes and 66 miRNAs (40 were novel), were PQDS-specific in the leukocytes, including the additional 84 PQDS-specific miRNAs (51 were novel) in the serums. Also, 145 genes and 24 miRNAs (12 were novel) were PDHS-specific in the leukocytes, and 37 PDHS-specific miRNAs (21 were novel) were discovered in the serums (Fig. 1A).
Gene ontology functions of the Zheng-specific genes and miRNAs
The gene ontology (GO) function-based enrichment analyses were performed to investigate the possible functions of the above-mentioned Zheng-specific genes and miRNAs. As revealed (rich factor ≥ 0.035 & count ≥ 3), the PDHS-specific leukocyte genes were associated with the biological processes including defense response to virus, immune and innate immune response (Fig. 2A left). The PQDS-specific genes were mainly related to the biological processes such as inflammatory response, collagen catabolism and extracellular matrix (ECM) organization (Fig. 2A right).
Moreover, for the biological processes of the validated targets of syndrome-specific leukocyte miRNAs, it was observed that the validated targets of the PDHS-specific and PQDS-specific miRNAs could be implicated in the common biological processes including response to stress, gene expression, mitotic cell cycle, cell death, catabolic process, cellular protein modification process and cellular nitrogen compound metabolic process (Fig. 2B). The targets of the PDHS-specific leukocyte miRNAs were also involved in negative regulation of apoptotic process (Fig. 2B left). Notably, the targets of the PQDS-specific leukocyte miRNAs were enriched in more additional biological processes such as mRNA metabolism, neurotrophin biosynthesis, neurotrophin tyrosine kinase (TRK) receptor signaling, macromolecular complex assembly, membrane organization, nucleobase-containing compound catabolism, cellular protein metabolic process, small molecule metabolic process (Fig. 2B right).
In particular, for the biological processes enriched by the validated targets of syndrome-specific serum miRNAs, we discovered that the targets of the PDHS-specific or PQDS-specific serum miRNAs were also associated with the above-mentioned processes which were enriched by the corresponding PDHS-specific or PQDS-specific leukocyte miRNAs (Fig. 2B and C), but the PDHS-specific serum miRNAs seemed to be involved in more additional biological processes than the PDHS-specific leukocyte miRNAs (Fig. 2B and C left). These results suggested the potential common roles of syndrome-specific miRNAs in the leukocytes or serums in contributing to the characteristics and functions of leukocytes in the TCM-defined syndrome of PDHS and PQDS.
Enriched pathways of the Zheng-specific genes and miRNAs
The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based enrichment analyses were performed to decode the potential pathways of the Zheng-specific genes and miRNAs. The results showed that the PDHS-specific genes in the leukocytes were enriched in the pathways related to serotonergic, glutamatergic and dopaminergic synapse, including the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway (Fig. 3A left; Supplemental Fig. S1-4). The PQDS-specific genes in the leukocytes were mainly involved in the pathways containing ECM-receptor interaction, cell adhesion molecules (CAMs), helper T (Th)1 and Th2 cell differentiation, Th17 cell differentiation, Interleukine (IL)17 signaling, cytokine-cytokine receptor interaction, mitogen-activated protein kinase (MAPK) signaling, arachidonic acid metabolism and protein digestion and absorption (Fig. 3A right; Supplemental Fig. S5-12).
In addition, concerning the enriched pathways of the validated targets of Zheng-specific miRNAs in the leukocytes, we found that the targets of the PDHS-specific and PQDS-specific miRNAs were both implicated in ECM-receptor interaction pathway (Fig. 3B), but the targets of the PQDS-specific miRNAs were involved in the additional pathways such as fatty acid biosynthesis, chronic myeloid leukemia, hippo signaling, proteoglycans in cancer and central carbon metabolism in cancer (Fig. 3B right).
Especially, regarding the potential pathways of the validated targets of Zheng-specific miRNAs in the serums, the targets of the PDHS-specific and PQDS-specific miRNAs were both associated with the pathways including fatty acid biosynthesis, lysine degradation and proteoglycans in cancer (Fig. 3C). The targets of PDHS-specific miRNAs were also involved in other pathways related to adherent junction and cancer (Fig. 3C left). The targets of PQDS-specific miRNAs were specially implicated in several additional pathways such as ECM-receptor interaction, fatty acid metabolism, forkhead box protein O (foxO) signaling, chronic myeloid leukemia, cell cycle, p53 signaling, central carbon metabolism in cancer, colorectal cancer and transcriptional misregulation in cancer (Fig. 3C right). Interestingly, the targets of the PQDS-specific serum miRNAs were enriched in more pathways, covering almost all the enriched pathways of the targets of PQDS-specific leukocyte miRNAs (Fig. 3B and C right), which suggested the possible common roles of these miRNAs in contributing to the characteristics and functions of leukocytes in the CAG patients with PQDS.
Interaction networks of Zheng-specific genes
The interaction networks of the syndrome-specific leukocyte genes were carefully created (Fig. 4). The generated networks not only detail the possible interactions between each node gene (edge thickness indicates interaction strength of data support), but also visually present the expression pattern of each node gene (node shade of green or red relies on degree of down-regulation or up-regulation of gene expression). Node size depends on the number of miRNAs which were experimentally validated targeting to the corresponding node gene, and the number is particularly displayed in the corresponding gene node. Especially, several enriched pathways were highlighted and annotated in the resultant networks. As shown (Fig. 4A; Supplemental Fig. S13; Supplemental Table S8), several pathways, including neutrophil degranulation (reactome), NOD-like receptor signaling, serotonergic synapse, dopaminergic synapse and glutamatergic synapse, were specially marked in the interaction network of the PDHS-specific genes. Obviously, 15 up-regulated genes were enriched in neutrophil degranulation pathway, indicating the active and enhanced neutrophil degranulation in the CAG patients with TCM-defined PDHS. Each of the genes, NBEA (neurobeachin), MTCL1 (microtubule cross-linking factor 1) and TRIB1 (TRIBbles homolog 1), kept a corresponding PDHS-specific miRNA regulator.
In addition, in the interaction network of the PQDS-specific genes (Fig. 4B; Supplemental Fig. S14; Supplemental Table S9), several genes were related to arachidonic acid metabolism pathway. In particular, the genes keeping more complex interaction relationships with each other, were specially enriched in MAPK signaling pathway and Th cell differentiation pathway, especially the pathways implicated in cell-to-cell adhesion/junction and communication such as CAMs, ECM-receptor interaction, ECM-organization and cell surface interactions at the vascular wall. Notably, the genes for the cross-talking between these pathways, including COL4A2 (collagen, type IV, alpha 2), COL26A1 (collagen, type XXVI, alpha 1), SPP1 (secreted phosphoprotein 1), FOS (proto-oncogene c-Fos) and PROCR, underwent regulation of the PQDS-specific leukocyte miRNAs. These results suggested that the PQDS-specific miRNAs had potential roles in the regulation of cell-to-cell adhesion/junction and communication, contributing to the characteristics and functions of leukocytes in the CAG patients with TCM-defined PQDS.
The Zheng-specific miRNA-gene interactions
Based on the Zheng-specific genes and miRNAs discovered in this work (Fig. 1A), especially the experimentally-supported miRNA-gene interactions, the interaction networks were particularly generated to detail the Zheng-specific miRNA-gene interaction pairs and visually presented the expression patterns of miRNA and gene in each interaction pair (Fig. 5A). As shown, three miRNA-gene interaction pairs were contained in the PDHS-specific miRNA-gene interaction network (Fig. 5A right; Supplemental Table S10), but there were 21 miRNA-gene interaction pairs for the PQDS-specific miRNA-gene interaction network (Fig. 5A left; Supplemental Table S11). Based on the gene functional information from the GeneCards human gene database (https://www.genecards.org), we found that the PQDS-specific miRNA-gene pairs seemed to be implicated in immunity, cancer, development and mentalism (Fig. 5A left). The further pathway enrichment analyses also indicated that these PQDS-specific miRNA-gene interactions could link with the pathways related to cancer and immunity, and notably there was multiple crosstalk mediated by the common targets among these pathways (Fig. 5B). The enriched pathways included the immune pathways, such as Th1 and Th2 cell differentiation, toll-like receptor signaling, and PI3K (phosphatidylinositol 3-kinase)-Akt (serine/threonine-protein kinase) signaling. Especially, the focal adhesion and ECM-receptor interaction pathways were involved in cell-to-cell adhesion/junction and communication, probably contributing to the characteristics and functions of leukocytes in the CAG patients with TCM-defined PQDS.
In addition, for the miRNAs belonging to the miRNA-gene interaction pairs, two heatmaps were specially generated to profile their expression in the leukocytes and serums of individuals from different populations. Particularly, using the ExoCarta exosome database , we analyzed weather these miRNAs could be contained and carried in the plasma exosome (Fig. 5C). As indicated, although the syndrome-specific leukocyte miRNAs kept high levels in the leukocytes, they almost couldn’t be found in the corresponding serums. Interestingly, hsa-miRNA-122-5p, a PQDS-specific miRNA discovered both in leukocytes and serums (Fig. 1A), kept much higher expression both in leukocytes and the corresponding serums (Fig. 5C). It could target to the PQDS-specific leukocyte genes in the above-mentioned immune pathways, including the focal adhesion and ECM-receptor interaction pathways related to cell-to-cell adhesion/junction and communication (Fig. 5B). Notably, there were additional experimental evidences supporting that it could be encapsulated and carried in the plasma exosomes, suggesting it could function as a regulator of genes in the far away recipient cells throughout the body.