The Expression Profile of mRNA and tRNA Genes in Splenocytes and Neutrophils after In Vivo Delivery of Antitumor Short Hairpin RNA of Indoleamine 2,3- Dioxygenase

Abstract

RNA-based therapeutics are considered as novel treatments for human diseases. Our previous study demonstrated that treatment with short-hairpin RNA against Ido1 (IDO shRNA) suppresses tumor growth, detects Th1-bias immune responses, and elevates expression of tryptophan transfer RNA (tRNA^Trp) in total splenocytes. In addition, depletion of Ly6g⁺ neutrophils attenuates the effect of IDO shRNA. The aim of this study was to investigate the regulatory network and the expression profile of tRNAs and other non-coding RNAs in IDO shRNA-treated spleens. The total splenocytes and magnetic bead-enriched splenic neutrophils were collected from the lung tumor bearing mice, which were treated with IDO shRNA or scramble IDO shRNA, and the collected cells were subsequently subjected to RNA sequencing. The gene ontology analysis revealed the different enrichment pathways in total splenocytes and splenic neutrophils. Furthermore, the expression of tRNA genes was identified and validated. Six isoacceptors of tRNA, with different expression patterns between total splenocytes and splenic neutrophils, were observed. In summary, our findings not only revealed novel biological processes in IDO shRNA-treated total splenocytes and splenic neutrophils, but the identified tRNAs and other non-coding RNAs may contribute to developing a novel biomarker gene set for evaluating the clinical efficiency of RNA-based cancer immunotherapies.

Keywords: RNA sequencing; animal tumor model; biomarker; indoleamine 2,3-dioxygenase1 (IDO1); neutrophil; non-coding RNA; short-hairpin RNA (shRNA); transfer RNA (tRNA).

Figure 1

The effect of indoleamine 2,3-dioxygenase (IDO) short-hairpin RNA (shRNA) treatment on LLC1 tumor-bearing mice. (a) 2 × 10⁵ LLC1 cells were subcutaneously injected into female inbred C57BL/6JNarl mice. Then, 50 μg of Scramble IDO shRNA (Scr IDOsh) or IDO shRNA (IDO sh) was treated by intramuscular injection at 7 and 14 days after LLC1 injection. The total splenocytes at day 16 and magnetic bead-enriched neutrophils at day 18 were collected, and subsequently subjected to RNA sequencing. (b) Mean tumor volume at day 16 (n = 7). (c) The tumor-infiltrating Ly6g⁺ cells. The labels sample 1 and sample 2 indicate that the images were taken from two independent murine tumors of each group. (d) Quantification of Ly6g⁺ cells for immunohistochemistry analysis (n = 4).

Figure 2

The expression of inflammatory molecules. (a) The expression of inflammatory molecules of total splenocytes, determined by RNA sequencing and (b) by QPCR. To detect the secreted protein levels, splenocytes were collected at 16 days after LLC1 injection. After 24-h culture, these molecules were detected by magnetic fluorescence microsphere immunoassay (MAGPIX) analysis. The expression of (c) IFN-γ, (d) IL-10, (e) TNF-α, (f) IL-1α, (g) CCL5, and (h) S100A9 was shown. (i) The expression of inflammatory molecules of splenic neutrophils determined by RNA sequencing and (j) by QPCR. *, **, and *** A statistically significant difference when compared with scramble IDO shRNA group (* p < 0.05; ** p < 0.01; *** p < 0.001). n = 3–4 in each experiment, except to RNA sequencing data. IFN-γ, interferon-γ; IL-10, interlukein-10; TNF-α, tumor necrosis factor- α; TGF-β, tumor transforming growth factor-β; CCL5, CC chemokine ligand 5; S100A9, S100 calcium-binding protein A9; Arg1, arginase 1.

Figure 3

The IDO shRNA-mediated network of biological processes. The Scr IDO shRNA-treated splenocytes or neutrophils, and the identified genes, with differential expression in IDO shRNA-treated cells, were selected, and then the gene ontology analysis was performed via ClueGo and CluePedia. (a) The enriched biological processes in IDO shRNA-treated splenocytes (p value < 0.001). (b) The enriched biological processes in IDO shRNA-treated splenic neutrophils (p value < 0.01).

Figure 4

The expression of tRNA genes. (a) The heatmap shows the expression of each tRNA gene. Each column indicates a specific tRNA gene. The relative color scheme is based on minimum-maximum values (RPKM), per column. (b) The expression of some tRNA genes identified via RNA sequencing in total splenocytes and splenic neutrophils. (c) The expression of some tRNA genes identified via QPCR in total splenocytes, and (d) in splenic neutrophils. n = 3–4 in QPCR analysis. * A statistically significant difference when compared with the Scr IDO shRNA group (* p < 0.05).

Figure 5

The potential regulatory genes of tRNA transcription. (a) The expression of RNA polymerase III machinery, including the subunits of RNA polymerase III (RNA polIII), transcription factor for polymerase III B (TFIIIB), and TFIIIC. (b) The expression of aminoacyl-tRNA synthetase genes. The official gene symbol of each aminoacyl-tRNA synthetase gene is shown on the label of x-axis in this figure.

Figure 6

Venn diagrams indicating the shared miRNA genes between miRNet-predicted miRNAs and RNA sequencing-identified miRNAs. (a) Total splenocytes. (b) Splenic neutrophils.