Fig. 4

The microprotein TPM3P9 regulates the oncogenic RNA splicing of TCF7L2. a GSEA of ccRCC proteomic data and TCGA data showing that TPM3P9 is involved in the spliceosome pathway. b Enrichment analysis suggested that the proteins co-expressed with TPM3P9 were related to RNA splicing. c Pie chart based on RNA sequencing showing the distribution of alternative splicing changes following TPM3P9 overexpression in ACHN and 786-O cells. d Schematic representation of TCF7L2 alternative splicing and its splice variants. e RT-PCR validation of the increased expression of the TCF7L2-L variant in cells overexpressing the TPM3P9 protein. f The results of qRT-PCR using a specific primer pair confirmed the increase in the expression of the TCF7L2-L variant upon overexpression of the TPM3P9 protein.***p < 0.001.  g Preparation of an antibody specific to the TCF7L2-L variant. h Western blot validation of the successful preparation of the anti-TCF7L2-L antibody and exogenous overexpression of the TCF7L2-L and TCF7L2-S variants. The HA-tag was fused with TCF7L2-L or TCF7L2-S. i Western blot showing the upregulation of the TCF7L2-L variant in cells ectopically expressing TPM3P9-Flag (TPM3P9) but not in those ectopically expressing TPM3P9-mut-Flag (Mut). j, k Knockdown of TCF7L2-L inhibited the ability of TPM3P9 to promote ccRCC cell proliferation. The expression of TCF7L2-L was measured by qRT-PCR (j), and the cell growth ability was confirmed by a CCK-8 assay (k). *p < 0.05; ***p < 0.001. Bars, SEMs. l, m Overexpression of TCF7L2-L reversed the inhibitory effect of TPM3P9 silencing on ccRCC cell growth. The expression of TCF7L2-L was measured by qRT-PCR (l), and the cell growth ability was confirmed by a CCK-8 assay (m). **p < 0.01; ***p < 0.001. Bars, SEMs. n–p Mouse xenograft model showing that overexpression of TCF7L2-L reversed TPM3P9 silencing-mediated inhibition of proliferation in ACHN cells. Tumor volume (n), tumor weight (o), and the increase in tumor volume over time (p) in nude mice. **p < 0.01; ns, nonsignificant. Bars, SD. q The body weight of the nude mice was monitored. ns, nonsignificant