Patient characteristics and baseline measurements
Fifty patients with BC from January 2018 to September 2019 met the study criteria. We collected blood samples from all of the patients at BF; two samples were hemolyzed, and two samples were degraded. At AF, blood samples were not collected from 15 cases. There were an eventual 14 cases in the RNA-seq group: five in the IT group and nine in the OC group. There were 17 cases in the verification group: ten in the IT group and seven in the OC group. There were no significant differences between the OC and IT groups in terms of age, menstrual status, chemotherapy regimen, tumor stage, molecular typing, and blood routine test in the sequencing group and verification group (Table 2, 3).
Neutrophil identification
Diff-quik staining showed clear neutrophil granules and purple-red nuclei. CD44 and CD11b were the molecular markers of neutrophils. Anti-CD44-FITC was detected on the surface of 98.7% isolated neutrophils. Anti-CD11b-FITC was detected on the surface of 96.6% isolated neutrophils, which proved the high purity of neutrophils extracted from human peripheral blood (Fig 2).
DEG and functional analysis in OC and IT groups
PCA and HCA showed that each group had significant specificity (Fig. 3a, 3b). The DEG analysis showed that, after chemotherapy, there were 1092 DEGs in total between the OC_AF and OC_BF groups, 571 DEGs between the IT_AF and IT_BF groups, and 707 DEGs between the OC and IT groups (Fig. 3c).
In the OC_AF group as compared to the OC_BF group, the upregulated genes included CD177, PTX3, and AZU1, and the downregulated genes included ITGB1, TSC22D1, and ITGB3. GOBP and KEGG pathway analysis showed that the RNA transport, translation and metabolic pathways were significantly enriched (Fig. 3d).
The effect of moxibustion on the neutrophil transcriptome was analyzed to gain insight into the potential mechanism of moxibustion in the efficacy of CIN. In the IT group, the ANKFY1, ATP11A, and TJP2 genes were upregulated after moxibustion, whereas TK1, TRAPPC2L, and SNHG8 were downregulated. The translation, adaptive immune response, cell adhesion and metabolic pathways were significantly enriched by GOBP and KEGG pathway analysis (Fig. 3e).
DEG analysis related to moxibustion regulation of neutrophils and network analysis
To explore the molecular mechanisms of moxibustion regulation of neutrophils after chemotherapy, RNA-seq was performed to analyze the DEG profiles, gene function, and signaling pathways between the IT and OC groups after chemotherapy (OC_AF vs. IT_AF). Moxibustion after chemotherapy upregulated the expression of TSC22D1, ITGB3, and GFI1B, and downregulated PRKCZ and TK1. The signaling pathways, including cell adhesion, leukocyte migration, and cell cycle, were significantly enriched (Fig. 4a). The co-network revealed that most DEGs were regulated by TGFβ1 and TSC22D1. KEGG enrichment pathway analysis suggested a close correlation with the TGFβ1/TSC22D1-mediated cell adhesion molecules (CAMs) pathway, in which adhesion molecules such as ITGB1, ITGB3, JAM3, CLDN5, and ESAM were significantly up-regulated (Fig. 4b).
Transcription factors (TF) such as FOSL1, ZNF274, E2F4, PRRX2, ZNF284, HHEX, CEBPG, and ZBTB2 have regulatory effects on DEGs, of which FOSL1 has the most negative regulatory effect. In addition, LILRB3 and LILRB4 are jointly regulated by TF such as HHEX, ZNF284, and PRRX2 (Fig. 4c). To investigate the potential functional implication of the TF network, we performed functional enrichment analysis for all genes in the network based on GO terms. Based on the P-values, the enriched GO BP terms included blood coagulation, platelet degranulation, cell–matrix adhesion, leukocyte migration, and neutrophil homeostasis. The enriched KEGG included platelet activation, cell adhesion molecules (CAMs), vascular smooth muscle contraction, and antifolate resistance (Fig. 4d).
RT-qPCR verification
RNA-seq results suggested up-regulation of CD177 expression and down-regulation of ITGB1 and TSC22D1 in the OC_AF group compared to the OC_BF group. ANKFY1 and ATP11A were up-regulated and TK1was down-regulated in the IF_AF group compared to IT_BF group. In addition, TSC22D1 and ITGB3 were up-regulated and TK1 was down-regulated in the IT_AF group compared to OC_AF group. Therefore, we validated the gene expression of CD177, ITGB1, TSC22D1, ANKFY1, ATP11A, TK1 and ITGB3 by RT-qPCR.
Without moxibustion, CD177 gene expression in neutrophils after chemotherapy was significantly higher than that before chemotherapy (Fig. 5a), while the expression of TSC22D1 showed a downward trend (P = 0.094), and ITGB1 have no significant differences (Fig. 5b). Under the intervention of moxibustion, ANKFY1, TSC22D1 and ITGB3 expression was significantly upregulated (P < 0.05) (Fig. 5c-e). ATP11A and TK1 have no significant differences (Fig. 5f).