Changes in the Small Noncoding RNAome During M1 and M2 Macrophage Polarization

Abstract

Macrophages belong to a special phagocytic subgroup of human leukocytes and are one of the important cells of the human immune system. Small noncoding RNAs are a group of small RNA molecules that can be transcribed without the ability to encode proteins but could play a specific function in cells. SncRNAs mainly include microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs) and repeat RNAs. We used high-throughput sequencing analysis and qPCR to detect the expression changes of the small noncoding RNAome during macrophage polarization. Our results showed that 84 miRNAs and 47 miRNAs with were downregulated during M1 macrophage polarization and that 11 miRNAs were upregulated and 19 miRNAs were downregulated during M2 macrophage polarization. MiR-novel-3-nature and miR-27b-5p could promote expression of TNF-α which was marker gene of M1 macrophages. The piRNA analysis results showed that 69 piRNAs were upregulated and 61 piRNAs were downregulated during M1 macrophage polarization and that 3 piRNAs were upregulated and 10 piRNAs were downregulated during M2 macrophage polarization. DQ551351 and DQ551308 could promote the mRNA expression of TNF-α and DQ551351overexpression promoted the antitumor activity of M1 macrophages. SnoRNA results showed that 62 snoRNAs were upregulated and 59 snoRNAs were downregulated during M1 macrophage polarization, whereas 6 snoRNAs were upregulated and 10 snoRNAs were downregulated during M2 macrophage polarization. Overexpression of snoRNA ENSMUST00000158683.2 could inhibit expression of TNF-α. For snRNA, we found that 12 snRNAs were upregulated and 15 snRNAs were downregulated during M1 macrophage polarization and that 2 snRNAs were upregulated during M2 macrophage polarization. ENSMUSG00000096786 could promote expression of IL-1 and iNOS and ENSMUSG00000096786 overexpression promoted the antitumor activity of M1 macrophages. Analysis of repeat RNAs showed that 7 repeat RNAs were upregulated and 9 repeat RNAs were downregulated during M1 macrophage polarization and that 2 repeat RNAs were downregulated during M2 macrophage polarization. We first reported the expression changes of piRNA, snoRNA, snRNA and repeat RNA during macrophage polarization, and preliminarily confirmed that piRNA, snoRNA and snRNA can regulate the function of macrophages.

Keywords: macrophage polarization; miRNA; piRNA; repeat RNA; snRNA; snoRNA.

Figure 1

Changes in miRNA expression during macrophage polarization. Hotmap (A), MA (B) and volcano plot (C) show the small RNA sequencing results of the expression of miRNA in M0 and M1 macrophages. (D) The expression of miRNAs upregulated in M1 macrophages detected by qPCR. (E, F) The expression of miRNAs downregulated in M1 macrophages detected by qPCR. (G) The expression of miRNA in M2 macrophages detected by qPCR. Hotmap (H), MA (I) and volcano plot (J) show the small RNA sequencing results of the expression of miRNA in M0 and M2 macrophages. (K) The expression of miRNA in M2 macrophages detected by qPCR. (L) The expression of miRNA in M1 macrophages detected by qPCR. (M) PCA of miRNA expression in M0, M1 and M2 macrophages. * indicates p < 0.05 compared to M0, and ** indicates p < 0.01 compared to M0. (N) The expression of miR-novel-3-mature in RAW264.7 cells transfected with miRNA mimics NC or miR-novel-3-mature mimics were detected by qPCR. (O) The expression of miR-27b-5p in RAW264.7 cells transfected with miRNA mimics NC or miR-27b-5p mimics were detected by qPCR. (P) The expression of TNF-α in RAW264.7 cells were detected by qPCR. * indicates p < 0.05 and ** indicates p < 0.01 compared to M1+ miRNA mimics NC group.

Figure 2

Changes in piRNA expression during macrophage polarization. Hotmap (A), MA (B) and volcano plot (C) show the small RNA sequencing results of the expression of piRNA in M0 and M1 macrophages. (D) The expression of piRNA upregulated in M1 macrophages as detected by qPCR. (E) The expression of piRNA was downregulated in M1 macrophages as detected by qPCR. (F) The expression of piRNA in M2 macrophages as detected by qPCR. Hotmap (G), MA (H) and volcano plot (I) show the small RNA sequencing results of the expression of miRNA in M0 and M2 macrophages. (J) The expression of piRNA in M2 macrophages as detected by qPCR. (K) The expression of piRNA in M1 macrophages as detected by qPCR. (L) PCA of piRNA expression in M0, M1 and M2 macrophages. * indicates p < 0.05 compared to M0, and ** indicates p < 0.01 compared to M0. (M) The expression of piRNA DQ551351 in RAW264.7 cells transfected with piRNA mimics NC or piRNA DQ551351 mimics were detected by qPCR. (N) The expression of piRNA DQ551308 in RAW264.7 cells transfected with piRNA mimics NC or piRNA DQ551308 mimics were detected by qPCR. (O) The expression of TNF-α in RAW264.7 cells were detected by qPCR, * indicates p < 0.05 and ** indicates p < 0.01 compared to M1+ miRNA mimics NC group. (P) Photographs showed the size of the tumor in mouse injected with RAW264.7 cells with piRNA mimics NC or piRNA DQ551308 mimics. (Q) Tumor weight in mouse injected with RAW264.7 cells with piRNA mimics NC or piRNA DQ551308 mimics, ** indicates p < 0.01 compared to piRNA mimics NC group.

Figure 3

Changes in snoRNA expression during macrophage polarization. Hotmap (A), MA (B) and volcano plot (C) show the small RNA sequencing results of the expression of snoRNA in M0 and M1 macrophages. (D) The expression of snoRNA upregulated in M1 macrophages as detected by qPCR. (E, F) The expression of snoRNA was downregulated in M1 macrophages as detected by qPCR. (G) The expression of snoRNA in M2 macrophages as detected by qPCR. Hotmap (H), MA (I) and volcano plot (J) show the small RNA sequencing results of the expression of snoRNA in M0 and M2 macrophages. (K) The expression of snoRNA in M2 macrophages as detected by qPCR. (L) The expression of piRNA in M1 macrophages as detected by qPCR. (M) PCA of snoRNA expression in M0, M1 and M2 macrophages. * indicates p < 0.05 compared to M0, and ** indicates p < 0.01 compared to M0. (N) The expression of snoRNA ENSMUST00000158683.2 in RAW264.7 cells transfected with pLVX-Vector or pLVX-ENSMUST00000158683.2 were detected by qPCR. (O) The expression of TNF-α, IL-6, TLR2, CD86 and CD16 in RAW264.7 cells were detected by qPCR, * indicates p < 0.05 and ** indicates p < 0.01 compared to M1+ pLVX-Vector group.

Figure 4

Changes in snRNA expression during macrophage polarization. Hotmap (A), MA (B) and volcano plot (C) show the small RNA sequencing results of the expression of snRNA in M0 and M1 macrophages. (D) The expression of snRNA in M1 macrophages as detected by qPCR. (E) The expression of snRNA in M2 macrophages as detected by qPCR. MA (F) and volcano (G) show the small RNA sequencing results of the expression of snRNA in M0 and M2 macrophages. (H) PCA of snRNA expression in M0, M1 and M2 macrophages. * indicates p < 0.05 compared to M0, and ** indicates p < 0.01 compared to M0. (I) The expression of snRNA ENSMUST00000157129.4 in RAW264.7 cells transfected with pLVX-Vector or pLVX- ENSMUST00000157129.4 were detected by qPCR. (J) The expression of snRNA ENSMUSG00000096786 in RAW264.7 cells transfected with pLVX-Vector or pLVX- ENSMUSG00000096786 were detected by qPCR. (K) The expression of IL-1 and iNOS in RAW264.7 cells were detected by qPCR, * indicates p < 0.05 and ** indicates p < 0.01 compared to M1+ pLVX-Vector group. (L) Photographs showed the size of the tumor in mouse injected with RAW264.7 cells with pLVX-Vector or pLVX- ENSMUSG00000096786. (M) Tumor weight in mouse injected with RAW264.7 cells with pLVX-Vector or pLVX- ENSMUSG00000096786, ** indicates p < 0.01 compared to pLVX-Vector group.

Figure 5

Changes in repeat RNA expression during macrophage polarization. Hotmap (A), MA (B) and volcano plot (C) show the small RNA sequencing results for the expression of repeat RNA in M0 and M1 macrophages. MA (D) and volcano (E) show the small RNA sequencing results for the expression of repeat RNA in M0 and M2 macrophages. (F) PCA of repeat RNA expression in the M0, M1 and M2 macrophages.