tRNA-Derived Small Non-Coding RNAs as Novel Epigenetic Molecules Regulating Adipogenesis

Abstract

tRNA-derived fragments (tRFs), a novel type of non-coding RNA derived from tRNAs, play an important part in governing gene expressions at a post-transcriptional level. To date, the regulatory mechanism of tRFs governing fat deposition and adipogenesis is completely unknown. In this study, high fat diet was employed to induce an obese rat model, and tRFs transcriptome sequencing was conducted to identify differentially expressed tRFs that response to obesity. We found out that tRF^GluTTC, which promoted preadipocyte proliferation by increasing expressions of cell cycle regulatory factors, had the highest fold change in the 296 differentially expressed tRFs. Moreover, tRF^GluTTC also suppressed preadipocyte differentiation by reducing triglyceride content and lipid accumulation, and by decreasing expressions of genes that related to fatty acid synthesis. According to results of luciferase activity analysis, tRF^GluTTC directly targeted Kruppel-like factor (KLF) 9, KLF11, and KLF12, thus significantly suppressing mRNA expressions of these target genes. Moreover, tRF^GluTTC suppressed adipogenesis, accompanying by suppressing expressions of adipogenic transcription factors (aP2, PPARγ, and C/EBPα). In conclusion, these results imply that tRF^GluTTC may act as a novel epigenetic molecule regulating adipogenesis and could provide a new strategy for the intervention treatment of obesity.

Keywords: 3T3-L1; KLF family; adipogenesis; tRFs; transcriptome.

Figure 1

High fat diet induces obese rat model. (A) The body weight of Strague Dawley (SD) rats that were fed with normal chow and high fat diet, n = 8. (B) The ratio of perirenal fat to body weight in the low fat diet (LFD) and the high fat diet (HFD) groups, n = 8. (C,D) The concentration of serum cholesterol (TC) and triglyceride (TG) in the LFD and HFD group, n = 8. (E) HE staining of perirenal fat from normal and obese SD rats, n = 3. Scale bar = 100 μm. (F) The average adipocyte area of perirenal fat from normal and obese SD rats, n = 3. (G) The blood glucose concentration of rats after an intravenous glucose tolerance test (i.v. test of glucose tolerance (GTT)), n = 8. All data were presented as means ± standard deviation. * p < 0.05.

Figure 2

tRNA derived fragments (tRFs) transcriptome sequencing and target gene prediction. (A) The average length of identified tRFs from 6 libraries. (B) Heat map diagram of differentially expressed tRFs between normal and obese rats. (C) The list of top 10 differentially expressed tRFs between normal and obese rats. (D) tRF78576 cleaved from tRNA-Glu-TTC, which occupied 67.43% of fragments derived from tRNA-Glu-TTC (E) tRF^GluTTC has complementary sequences with 3’UTR of Kruppel-like factor (KLF) 12, KLF12, KLF9, and KLF11. (F) The inhibitory effect of tRF^GluTTC on target genes measured by luciferase assays. All data were presented as means ± standard deviation. * p < 0.05.

Figure 3

The role of tRF^GluTTC in proliferation and differentiation of preadipocytes. (A) The proliferation ability of 3T3-L1 preadipocyte was measured by CCK-8. (B,C) 3T3-L1 preadipocyte proliferation was measured by EdU staining. (D) The relative mRNA expression levels of cell cycle regulatory factors (CDK4, Cyclin E, and Cyclin D1) by RT-PCR. (E,F) Oil Red O staining and triglyceride content of terminally differentiated (Day 8) adipocytes that transfected with tRF^GluTTC mimics and inhibitors. (G) The relative expression of KLF family (KLF9, KLF11, KLF12, and KLF13) by RT-PCR. (H) The relative expression of adipogenic transcription factors (aP2, peroxisome proliferator-activated receptor gamma (PPARγ), and CCAAT enhancer binding protein alpha (C/EBPα)) by RT-PCR. (I) The relative expression of genes related to fatty acid synthesis and oxidation by RT-PCR. All data were presented as means ± standard deviation. * p < 0.05.