Systematic Analysis of tRNA-Derived Small RNAs Discloses New Therapeutic Targets of Caloric Restriction in Myocardial Ischemic Rats

Abstract

Caloric restriction (CR) is a novel dietary therapy that has a protective effect on myocardial ischemia. However, the mechanisms underlying the therapeutic effect of CR remain unclear. Transfer RNA-derived small RNAs (tsRNAs) are a novel type of short non-coding RNAs that have potential regulatory functions in various physiological and pathological processes. In this study, we explored new therapeutic targets of CR through tsRNA sequencing. Rats were randomly divided into three groups: a normal control group (norm group), isoproterenol (ISO)-induced myocardial ischemic group (MI group), and CR pretreatment plus ISO-induced myocardial ischemic group (CR + MI group). Triphenyl tetrazolium chloride staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, serum creatine kinase (CK) and lactic acid dehydrogenase activity detection kits, and creatine kinase isoenzyme 1 levels were used to measure the degree of myocardial ischemic injury. These indicators of myocardial ischemia were significantly improved in the CR + MI group compared with those in the MI group. In the ischemic myocardial tissue of the MI group, a total of 708 precisely matched tsRNAs were identified, and 302 tsRNAs (fold change >1.5, P < 0.05) were significantly changed when compared with those in the norm group. Furthermore, 55 tsRNAs were significantly regulated by CR pretreatment, among which five tsRNAs (tiRNA-His-GTG-004, tRF-Gly-TCC-018, tRF-Cys-GCA-022, tRF-Lys-CTT-026, tRF-Met-CAT-008) were randomly selected and verified by quantitative real-time polymerase chain reaction. In addition, predictions of target genes and bioinformatics analysis indicated that these tsRNAs may play a therapeutic role through the regulation of macromolecular metabolism. In conclusion, our findings reveal that tsRNAs are potential therapeutic targets for CR pre-pretreatment to improve myocardial ischemic injury. This study provides new ideas for future research on elucidating the mechanisms of CR pretreatment in ameliorating myocardial ischemic injury.

Keywords: bioinformatics analysis; caloric restriction; myocardial ischemia; sequencing; tsRNA.

FIGURE 1

Study design. Norm, normal control group; MI, isoproterenol-induced myocardial ischemic group; CR, caloric restriction; CR + MI, CR pretreatment plus ISO-induced myocardial ischemic group.

FIGURE 2

Effects of CR pretreatment on myocardial ischemic injury. (A) Representative morphological images of myocardial tissue in norm, MI, and CR + MI groups after TTC staining. (B) TUNEL assay of myocardial tissues in norm, MI, and CR + MI groups. Brown staining of the nucleus indicates apoptosis. (C) The activities of serum CK were determined by CK assay kit in norm, MI, and CR + MI groups. (D) The activities of serum LDH were examined by LDH activity detection kit in norm, MI, and CR + MI groups. (E) The levels of serum CK-MB1 were measured by CK-MB1 ELISA kit in norm, MI, and CR + MI groups. The data are presented as the mean ± SEM (n = 6). *P < 0.05, **P < 0.01 compared with the MI group.

FIGURE 3

Altered expression profiles of tsRNAs following CR pretreatment. (A) Venn diagram indicating the total number of exact-matched tsRNAs in the myocardial tissues of norm, MI, and CR + MI groups. (B) PCA plot illustrating the clustering of three repeats in each group and evaluation of the corresponding variability and repeatability (FC > 1.5 and P < 0.05). The blue dots represent the norm group, the green dots represent the MI group, and the orange dots represent the CR + MI group. (C) Histogram showing the expression levels of each subtype of tsRNA in norm, MI, and CR + MI groups. (D) Volcano plot showing 302 significantly altered tsRNAs between the MI and norm groups (FC > 1.5 and P < 0.05). (E) Volcano plot showing 136 significantly changed tsRNAs between MI and CR + MI groups (FC > 1.5 and P < 0.05). Red indicates upregulated tsRNAs, and green denotes downregulated tsRNAs. The data are presented as the mean ± SEM (n = 5). *P < 0.05, **P < 0.01 compared with the MI group.

FIGURE 4

Significantly altered CR-related tsRNAs and their verification via qRT-PCR. (A) Significantly altered CR-related tsRNAs are shown in the heat map. The colors in the panel indicate the relative expression levels (log₂ transformation). The color bar graph on the top panel shows the sample group at the top, and the color bar graph on the right side of the panel represents the partitioning performed using K-means. (B) qRT-PCR confirmation of the CR-related tsRNAs, tiRNA-His-GTG-004, and tRF-Gly-TCC-018, among the three groups. (C) qRT-PCR confirmation of CR-related tRF-Met-CAT-008, tRF-His-GTG-016, tRF-Cys-GCA-022, and tRF-Lys-CTT-026 among the three groups. The data are presented as the mean ± SEM (n = 6). *P < 0.05, **P < 0.01 compared with the MI group.

FIGURE 5

Target genes of CR-related tsRNAs and their verification. (A) Venn diagram showing 982 mRNA targets of five CR pretreatment–related tsRNAs that were predicted by two prediction algorithms. (B) The target of each tsRNA is shown separately. (C) The binding region and seed sequence of five randomly selected mRNA transcripts (each corresponding to a tsRNA sequence). (D) Verification of target predictions of Mastl and Gpx8 via qRT-PCR. (E) Verification of target predictions of Casp2, Rbfox1, and Stk39 via qRT-PCR. The data are presented as the mean ± SEM (n = 6). *P < 0.05, **P < 0.01 compared with the MI group.

FIGURE 6

mRNA levels of H9c2 cells transfected with tsRNA mimics. (A) The qRT-PCR results of relative mRNA levels in H9c2 cells transfected with rno-tiRNA-His-004 mimics. (B) The qRT-PCR results of relative mRNA levels in H9c2 cells transfected with rno-tRF-Cys-022 mimics. (C) The qRT-PCR results of relative mRNA levels in H9c2 cells transfected with rno-tRF-Lys-026 mimics. (D) The qRT-PCR results of relative mRNA levels in H9c2 cells transfected with rno-tRF-Met-008 mimics. The data are presented as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01 compared with NC group.

FIGURE 7

Target mRNAs regulated by four candidate tsRNAs enriched in macromolecular metabolic process and their verification via qRT-PCR. (A) The general GO annotations for biological processes, cellular components, and molecular functions of the target mRNAs regulated by the four candidate tsRNAs. (B) The relative mRNA levels enriched in macromolecular metabolic process in H9c2 cells transfected with rno-tiRNA-His-004 mimics. (C) The relative mRNA levels enriched in macromolecular metabolic process in H9c2 cells transfected with rno-tRF-Cys-022 mimics. (D) The relative mRNA levels enriched in macromolecular metabolic process in H9c2 cells transfected with rno-tRF-Lys-026 mimics. (E) The relative mRNA levels enriched in macromolecular metabolic process in H9c2 cells transfected with rno-tRF-Met-008 mimics. The data are presented as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01 compared with NC group.