miR-150-5p showed decreased expression in NPC patients and was associated with poor prognosis
To examine the miRNA expression profile in NPC, we performed analysis of five NPC datasets in GEO, GSE32960, GSE36682, GSE43039, GSE70970 and GSE118613.Two microRNAs, miR-150-5p and miR-342-3p, were consistently down-regulated in the NPC group compared with the non-NPC group in all five GEO datasets (Fig. 1A–B, Fig. S1A). Decreased expression of miR-150-5p and miR-342-3p was associated with poor overall survival (OS) in NPC patients (Fig. 1C, Fig. S1B). Decreased miR-150-5p expression was also associated with poor metastasis-free survival in NPC patients (Fig. 1D). miR-342-3p had no effect on the metastasis-free survival in NPC patients (Fig. S1C).
Gene expression profile associated with miR-150-5p
We next explored the potential mechanism by which miR-150-5p affects NPC patient survival using TCGA HNSC database. miR-150-5p was decreased in HNSC tissues compared with paired non-tumor tissues (Fig. 2A). Decreased expression of miR-150-5p associated with poor OS and progression-free survival (PFS) in HNSC patients (Fig. 2B–C). miR-150-5p was also an independent prognostic factor of OS and PFS in HNSC (Fig. 2D).
We next compared the gene expression profiles of HNSC with high miR-150-5p expression and those with low miR-150-5p expression (Fig. 2E–G). GO enrichment analysis of the differentially expressed genes were mainly associated with immune-related terms, such as lymphocyte differentiation, T cell activation, B cell activation, lymphocyte-mediated immunity, humoral immune response, antigen-receptor mediated signal pathway, B cell–mediated immunity, B cell proliferation, and B cell receptor signal pathway (Fig. 2H and Supplementary table 1). KEGG pathway analysis revealed that the differentially expressed genes were also mainly involved in immune-related pathways, such as natural killer cell–mediated cytotoxicity, primary immunodeficiency, T cell receptor pathway, antigen processing and presentation, Th17 cell differentiation, B cell receptor signal pathway, graft versus host disease, intestinal immune network for IgA production, and allograft rejection (Fig. 2I and Supplementary table 1).
GSEA analysis showed increased activated pathways in B cell activation, B cell–mediated immunity, leukocyte-mediated immunity, activation of immunity, immune response to tumor cell, allograft rejection, graft versus host disease, B cell receptor signal, antigen processing and presentation, T cell receptor signal, complement cascade and adaptive immune system in HNSC with high expression of miR-150. While the activation of pathways in MET signal, cell junction organization, non-integrin membrane–ECM interactions, hypoxia, glycolysis, angiogenesis and epithelial mesenchymal transition were decreased in HNSC with high expression of miR-150. (Fig. 2J–N and Supplementary table 2). These results suggested that the expression of miR-150-5p was associated with immune-related cells and the immune reaction in HNSC patients.
miR-150-5p was associated with tumor-infiltrated B lymphocytes
The Extracellular Vesicles miRNA Database (EVmiRNA) contains 462 small non-coding RNA sequencing datasets of extracellular vesicles from 17 tissues/diseases. In the EVmiRNA database, miR-150-5p was found in exosomes or microvesicles secreted by some cell types in the TME, such as lymphocytes, fibroblasts, endothelial cells, mast cells and mesenchymal stem cells[16–19] (Fig. 3A). Exosomes function as messengers of cell-cell communication and are released into paracellular and body fluids, such as plasma, salvia, urine, and breast milk[20, 21]. The cross-talk between tumor cells and stromal cells via exosomes in the TME regulates TME remodeling, invasion, metastasis, angiogenesis, immune suppression and drug resistance[22, 23]. Therefore, we investigate the correlation of miR-150-5p and TME.
The proportions of TME-infiltrating cells in HNSC were calculated using Timer, quanTIseq, MCP-Counter, EPIC, xCell and CIBERSORT. B cells, CD4+T cells, CD8+T cells, neutrophils, macrophages, myeloid-dendritic cells, NK cells, monocytes, mast cells and endothelial cells were upregulated while cancer-associated fibroblasts (CAFs) were decreased in the high miR-150-5p expression group (Fig. 3B–G). Correlation analysis revealed that B lymphocytes were correlated with the expression of miR-150-5p throughout all five predicting algorithms (Fig. 3H–L and Supplementary table 3). This suggested that decrease of miR-150-5p may result from the decrease of B lymphocytes. Survival analysis showed that a higher abundance of B lymphocytes indicated better survival in HNSC patients (Fig. 3M and Supplementary table 4).
CIBERSORT and xCell were used to calculate the abundance of the subtypes of B lymphocytes. Naïve B cells, memory B cells and plasma B cells were all positively correlated with miR-150-5p expression (Supplementary table 3). Higher level of plasma B cells was also correlated with longer survival of HNSC patients (Fig. 3N and Supplementary table 4). These results suggested that the impaired survival of HNSC patients may be related to the decrease of B lymphocyte in the TME.
miR-150-5p was regulated by AC073103.1
As miR-150-5p was found down-regulated in NPC and TCGA HNSC compared with non-tumor tissues, we next investigate the potential cause of decreased miR-150-5p. To explore potential ceRNAs that may regulate miR-150-5p, we examined lncRNA expressions in HNSC with high miR-150-5p expression and those with low miR-150-5p expression (Fig. 4A–C). Subsequent analysis identified 89 potential lncRNAs that regulate miR-150-5p and 6 mRNAs that may be regulated by miR-150-5p (Fig. 4D, E). The ceRNA network associated with miR-150-5p is shown in Fig. 4F.
The exoRBase database is a repository of circular RNA (circRNA), lncRNA and mRNA derived from RNA-seq data analyses of human blood exosomes. The lncRNA AC073103.1 was one of the candidate regulators of miR-150-5p identified above and was found in blood exosomes in the exoRBase database (Fig. 4G). AC073103.1 was increased in cancer patients compared with normal person (Fig. 4G). In TCGA HNSC, the expression of AC073103.1 was increased in tumor tissues compared with paired non-tumor tissues (Fig. 4H). High expression of AC073103.1 was associated with poor survival in HNSC. Furthermore, high expression of AC073103.1 was associated with low enrichment of B cells (Fig. 4J–L). These data indicated that tumor cells may down-regulated immune reaction pathways and decrease the enrichment of immune-related cells in the TME by increasing the expression of AC073103.1(Fig. 4M) .