Figure 1. The canonical microRNA biogenesis pathway. This figure is modified from Figure 1 in Daugaard and Hansen et al. 2017. 13

Figure 2. The selection of the mature miRNA strand and the fate of its passenger strand. This figure is modified from Figure 1 in Guo and Lu et al. 2010. 14

Figure 3. MicroRNA plays a crucial role in the developmental and pathological processes. 15

Figure 4. Schematic representation of canonical miRNA response element (MRE) types. This figure is modified from Figure 1 in Steinkraus et al. 2016. 17

Figure 5. The computational approach for the prediction of microRNA-mRNA interactions. 19

Figure 6. Expression-based miRNA target prediction. 20

Figure 7. Experimental validation confirms condition-specific microRNA-mRNA interactions. 21

Figure 8. Origins and classification of adipocyte cells. 24

Figure 9. Three types of adipocyte cells. This figure is modified from Figure 2 in Paul et al. 2018. 25

Figure 10. (A) Location of brown, white, beige adipocytes in mice. This figure is modified from Figure 3 in Bartelt and Heeren et al. 2014. (B) Structure of white adipose tissue.... 26

Figure 11. (A) Schematic of IL-4 induced priming of APs. (B) The cell proliferation rate of APs cultured in IL-4 containing media (n＝3). (C) The beige adipogenic markers (Pgc1-... 36

Figure 12. (A) The mRNA levels of Ucp1, Ap2, and Pgc1-α genes were measured by quantitative PCR (qPCR) in differentiated adipocytes (n＝3). (B) The protein levels of... 37

Figure 13. (A) Principal component analysis (PCA) of three replicates for control and IL- 4-stimulated adipocyte precursors. (B) Correlation matrix of all the samples. 39

Figure 14. Transcriptomic analysis of control and IL-4–treated APs (four-day sample). The mRNA-seq results are presented as a volcano plot (Total 18345 genes). The genes of... 40

Figure 15. KEGG pathway analysis of upregulated (log2 fold change〉 1, p.adj 〈0.05, upper panel, red) and downregulated (log2 fold change 〈-1, p.adj 〈0.05, lower panel, blue)... 41

Figure 16. (A) The analysis of differentially expressed miRNAs using the small RNA sequencing data of control and IL-4–treated APs (two-day sample). Differentially... 43

Figure 17. (A) The primary transcripts of miR-503, miR-351, and miR-542 were examined by qPCR for the expression levels in APs incubated with IL-4 for 0, 2, 4, and 6 days (n＝3).... 44

Figure 18. (A) Transcription factor enrichment analysis for the promoter region of H19X- encoded miRNAs. Top-ranked transcription factors are presented in the table. (B) Klf4... 46

Figure 19. KEGG pathway analysis in shared target genes of H19X-encoded miRNAs. 49

Figure 20. (A) Analysis of shared target genes of H19X-encoded miRNAs using TargetScan. The numbers of overlapping target genes are presented in the table. (B) KEGG... 50

Figure 21. (A) The gene structures of Ccnd1 is shown with binding sites for H19X- encoded miRNAs in the 3′ UTR. (B) The CCND1 protein levels decreased with miR-322-... 52

Figure 22. (A) The gene structures of Fzd6 is shown with binding sites for H19X-encoded miRNAs in the 3′ UTR. Data are presented as mean ± SEM. *** p 〈0.005, ** p 〈0.01, * p 〈0.05. 53

Figure 23. (A) The positions and sequences of miRNA binding sites in the 3′ UTR of Ccnd1 is presented. The seed region of miRNA is highlighted in red and is base paired with... 54

Figure 24. (A) The positions and sequences of miRNA binding sites in the 3′ UTR of Fzd6 is presented. The seed region of miRNA is highlighted in red and is base paired with the... 55

Figure 25. (A) H19X-encoded mature miRNAs expression levels were measured in APs after induction of LiCl treatment. miR-322-5p, miR-351-5p, and miR-542-3p were... 58

Figure 26. The proliferation of APs was analyzed after treatment with LiCl and miR-322- 5p/miR-503-5p and miR-450b-5p/miR-542-3p mimics. 59

Figure 27. IL-4-induced cell proliferation was analyzed after transfection of miR-322- 5p/miR-503-5p and miR-450b-5p/miR-542-3p mimics. Data are presented as mean ± SEM.... 60

Figure 28. The proliferation of APs was analyzed by flow cytometry. The percentage of Ki67-positive cells measured after six-days incubation in IL-4 containing medium with... 61

Figure 29. Total cell number analysis of adipocyte precursors. Adipocyte precursors were counted for each condition (control, IL-4, IL-4 + miR-322-5p/miR-503-5p)(n＝2). Data are... 62

Figure 30. The proliferation of APs was analyzed by immunocytochemistry. The percentage of Ki67-positive cells increased after six-days incubation in IL-4 containing... 63

Figure 31. The mRNA expression levels of beige adipogenic markers were measured by qPCR (n＝3) after induction of IL-4 and LiCl in APs. Data are presented as mean ± SEM.... 64

Figure 32. H19X-encoded mature miRNAs, miR-322-5p and miR-542-3p and adipogenic marker levels were measured after cells were cultured in a differentiation medium for beige... 66

Figure 33. The primary transcripts of miR-503 and miR-542 levels and adipogenic markers were measured after induction of adipocytes differentiation. The let-7b primary transcript... 67

Figure 34. mRNA expression levels of IL-4, IL-4Rα, and Ap2 were measured via qPCR in scWAT samples of 1, 2, and 3 week postnatal mice (n＝3). IL-4 mRNA level was below... 68

Figure 35. APs were transiently transfected with miRNA mimic mix (miR-322-5p/miR- 503-5p/miR-450b-5p/miR-542-3p) for 24 h and stimulated with adipogenic cocktail for the... 69

Figure 36. The mRNA levels of Ucp1 and Ap2 were analyzed during beige adipocyte differentiation and the pretreatment of miRNA mimic mix (miR-322-5p/miR-503-5p/miR-... 70

Figure 37. The protein levels of CCND1 and PLIN1 were examined by western blotting during beige adipocyte differentiation. Data are presented as mean ± SEM. *** p 〈0.005,... 71

Figure 38. A proposed model of H19X-encoded miRNAs-mediated regulation of adipocyte precursors under IL-4 stimulation. 72

Figure 39. Model representing the nine hallmarks of aging. 76

Figure 40. Comparative analysis of the most commonly used model systems in aging research. This figure is modified from Figure 1 in Taormina et al. 2019. 77

Figure 41. The schematic diagram illustrates the experimental scheme. (Created with Biorender.com) 86

Figure 42. Pearson's correlation coefficients between gene expressions in each young and aged mouse brain hypothalamus and hippocampus samples are presented as a heat map.... 87

Figure 43. Volcano plot of mRNA sequencing analysis of young and aged mouse brain from (A) hypothalamic and hippocampal (B) tissue. Differentially expressed genes (cut-off... 88

Figure 44. Bar graph showing the number of total detected genes, and specifically detected genes (dark gray) in only hypothalamic or hippocampal tissue. Among the genes detected... 89

Figure 45. Scatter-plot of the expression level changes for a gene set that are both detected in the hypothalamic and hippocampal tissues. Significantly upregulated/downregulated... 90

Figure 46. Top 5 KEGG signaling pathways associated with differentially upregulated (red) and downregulated (blue) genes in hypothalamic tissue and hippocampal tissue. 91

Figure 47. Pie chart shows total genes expressed in bulk mRNA sequencing and the proportion of genes detected by snRNA sequencing from hypothalamic (A) and... 92

Figure 48. Violin plot of a gene set that are analyzed both in bulk mRNA seq and snRNA- seq data, showing the distribution of module scores for astrocytes, ependymocytes,... 93

Figure 49. The bar graph shows the number of genes detected both in bulk RNA-seq and snRNA-seq data in hypothalamic (A) and hippocampal tissue (B). 94

Figure 50. Pearson's correlation of Log2FC for upregulated and downregulated genes in bulk mRNA sequencing (cut-off value: fold change〉 1.5, padj 〈0.05) and snRNA... 96

Figure 51. Pearson's correlation of Log2FC for upregulated and downregulated genes in bulk mRNA sequencing (cut-off value: fold change〉 1.5, padj 〈0.05) and snRNA... 97

Figure 52. Strip chart of differentially expressed genes from bulk RNA-seq (cut-off value indicated as gray dash lines: fold change〉 1.5, padj 〈0.05) and snRNA-seq data (cut-off... 99

Figure 53. TPM log10 of bulk mRNA-seq and TP10k log10 of snRNA-seq for aging- dependent upregulated genes (A) and downregulated gene (B) in hypothalamus. 100

Figure 54. TPM log10 of bulk mRNA-seq and TP10k log10 of snRNA-seq for aging- dependent upregulated genes (A) and downregulated gene (B) in hippocampus. 101

Figure 55. Pearson's correlation coefficients of log2(Count+1) values for all miRNAs expression between each young and aged mouse brain from hypothalamic and hippocampal... 103

Figure 56. (A). The bar graph shows the number of total detected miRNAs and differentially upregulated, downregulated miRNAs (cut off value: fold change〉 1.5, padj... 104

Figure 57. Scatter-plot of miRNA expression level changes in the hippocampal and hypothalamic tissues during aging. Significantly upregulated/downregulated genes (padj 〈... 105

Figure 58. (A) Volcano plot of miRNA sequencing analysis of young and aged mouse brain from hypothalamic tissue. Differentially expressed genes (cut-off value: fold change... 106

Figure 59. (A) Volcano plot of miRNA sequencing analysis of young and aged mouse brain from hippocampal tissue. Differentially expressed genes (cut-off value: fold change... 107

Figure 60. Heatmap of differentially downregulated miRNAs predicted to be target differentially upregulated mRNAs in hypothalamus of mice. 108

Figure 61. Heatmap of differentially upregulated miRNAs predicted to be target differentially downregulated mRNAs in hypothalamus of mice. 109

Figure 62. Heatmap of differentially downregulated miRNAs predicted to be target differentially upregulated mRNAs in hippocampus of mice. 110

Figure 63. Heatmap of differentially upregulated miRNAs predicted to be target differentially downregulated mRNAs in hippocampus of mice. 111

Figure 64. (A) Protein-protein interaction (PPI) analysis of upregulated genes targeted by downregulated miRNAs from hypothalamus. (B) Protein-protein interaction (PPI) analysis... 113

Figure 65. (A) Protein-protein interaction (PPI) analysis of upregulated genes targeted by downregulated miRNAs from hippocampus. (B) Protein-protein interaction (PPI) analysis... 114

Figure 66. KEGG signaling pathways visualized in a network of genes. Dot size for each gene indicates the number of differentially expressed miRNAs expected to target that gene.... 116

Figure 67. KEGG signaling pathways visualized in a network of genes. Dot size for each gene indicates the number of differentially expressed miRNAs expected to target that gene.... 117

Figure 68. KEGG signaling pathways visualized in a network of genes. Dot size for each gene indicates the number of differentially expressed miRNAs expected to target that gene.... 118

Figure 69. KEGG signaling pathways visualized in a network of genes. Dot size for each gene indicates the number of differentially expressed miRNAs expected to target that gene.... 119

Figure 70. The miRNA- target gene network commonly detected in the hypothalamus and hippocampus. Node sizes for target genes ranked by degree method. The network of... 121

Figure 71. Quantification of coagulation factor III levels. (A) Representative images of immunoblot for Coagulation factor III and 110 soluble proteins in the brain from young... 122