Transcriptome analysis reveals a potential regulatory mechanism of the lnc-5423.6/IGFBP5 axis in the early stages of mouse thymic involution

Figures

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  Figure 1 Morphological and histological observation of age-related thymic involution (A) Thymus tissues in 1-month-old, 3-month-old, and 6-month-old mice. (B) Thymus weight. (C) Body weight. (D) Thymus index. Data are expressed as the mean±standard deviation (SD). n=5. *** P<0.001.

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  Figure 2 Identification of DEGs between different ages during thymic involution (A–D) Volcano plots of DEGs in males and females. Venn and heatmaps of overlapping DEGs in males (E,F) and females (G,H), respectively. |log 2(fold change)|>1 and P<0.05 represent a significant difference.

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  Figure 3 GO and KEGG pathway analysis of DEGs during age-related thymic involution GO analysis of DEGs in males (A) and females (B). KEGG pathway analysis of DEGs in males (C) and females (D). P<0.05 represents a significant difference.

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  Figure 4 Identification of DELs between different ages during thymic involution (A–D) Volcano plots of DELs in males and females. Venn diagram and heatmap of overlapping DELs in males (E,F) and females (G,H), respectively. Venn diagram and heatmap of DELs overlapping in the thymic development and involution stages of males and females (I,J).

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  Figure 5 Verification of DELs (A) and DEGs (B) by qRT-PCR The thymus of F3 and F1 mice was used for validation of lnc-1882.1 and lnc-177363. The thymus of M3 and M1 mice was used for validation of lnc-115107, lnc-195025, lnc-201493, lnc-5459.3, and lnc-4777.3. The thymus of F6 and F1 mice was used for validation of Car3, Jchain, Tnxb, Vldlr, Casp1, Scd2, and E2f3. The thymus of M6 and M1 mice was used for validation of lnc-1674.1, lnc-219154, lnc-194215, Scd1, Igkc, and Igkv1-135. Log 2 of fold change was expressed as the mean±SD, n=3.

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  Figure 6 The novel lncRNA-miRNA-mRNA triple regulatory network associated with mouse age-related thymic involution (A) lncRNA-miRNA-mRNA network. (B) The betweenness centrality of lncRNAs, miRNAs and mRNAs. (C) Closeness centrality. (D) The degree centrality. The green rectangle represents lncRNA; the brown diamond represents miRNA; the purple circle represents mRNA.

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  Figure 7 IGFBP5 inhibits the proliferation and promotes the apoptosis of MTEC1 cells (A) qRT-PCR was used to verify the transfection efficiency of the IGFBP5 overexpression plasmid and si- IGFBP5 in MTEC1 cells ( n=3). (B) CCK-8 assay was used to detect the effect of IGFBP5 on the viability of MTEC1 cells ( n=6). (C,D) The effect of IGFBP5 on the proliferation of MTEC1 cells was evaluated by EdU incorporation assay ( n=3). (E) Western blot analysis was used to verify the effect of IGFBP5 on apoptosis-related genes in MTEC1 cells ( n=3). (F) qRT-PCR was used to verify the effect of IGFBP5 on apoptosis-related genes. (G,H) The effect of IGFBP5 on MTEC1 cell apoptosis ( n=3). Data are shown as the mean±SD. * P<0.05,** P<0.01, *** P<0.001.

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  Figure 8 miR-193b-3p alleviates MTEC1 cell apoptosis by targeting IGFBP5 (A) qRT-PCR was used to verify the transfection efficiency of miR-193b-3p in MTEC1 cells ( n=3). (B) The results of the dual-luciferase reporter assay. (C) CCK-8 assay was used to detect the effect of miR-193b-3p on the viability of MTEC1 cells ( n=6). (D, E) The effect of miR-193b-3p on the proliferation of MTEC1 cells was evaluated by EdU incorporation assay ( n=3). (F, G) The effect of miR-193b-3p on MTEC1 cell apoptosis ( n=3). Data are shown as the mean±SD. ** P<0.01, *** P<0.001.

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  Figure 9 lnc-5423.6 promotes the apoptosis of MTEC1 cells by upregulating IGFBP5 (A) The results of the dual-luciferase reporter assay. (B) CCK-8 assay was used to detect the effect of lnc-5423.6 on the viability of MTEC1 cells ( n=6). (C,D) The effect of lnc-5423.6 on MTEC1 cell apoptosis ( n=3). (E,F) The effect of lnc-5423.6 on the proliferation of MTEC1 cells was evaluated by EdU incorporation assay ( n=3). (G,H) Western blot analysis was used to verify the effect of lnc-5423.6 on IGFBP5 in MTEC1 cells ( n=3). ** P<0.01, *** P<0.001.