Long Non‐Coding RNA IGFRIL Couples with PTBP1 to Destabilize IGFBP3 mRNA to Promote the IGF1R‐AKT‐mTOR Axis and Hepatocellular Carcinoma

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  Figure 1. Identification of IGFRIL as an HCC‐associated lncRNA. A) Heatmap showing the differentially expressed lncRNAs in HCC tumors compared to non‐tumor liver tissues (normal liver [NL], inflammatory liver [IL], and cirrhotic liver [CL]; GSE54238). The dysregulated lncRNAs were ranked according to their fold‐changes of HCC versus (NL+ IL + CL). LncRNAs with a gradually increasing or decreasing expression pattern during tumor progression are highlighted in red or blue, respectively. B) IGFRIL expression levels in tumor and adjacent non‐tumor tissues (ANT) from three HCC validation cohorts (VALI1‐3), as determined by qRT‐PCR. C–E) Kaplan–Meier survival curves showing the overall survival (OS) and disease‐free survival (DFS) of HCC patients in the VALI1, VALI2, and VALI3 cohorts, stratified by IGFRIL expression levels (log‐rank test). F) IGFRIL expression levels in tumor and adjacent non‐tumor tissues from gastric cancer (GC) and colorectal cancer (CRC) cohorts (VALI4 and VALI5), as determined by qRT‐PCR. G) Kaplan–Meier survival curves showing the OS and DFS of GC patients in the VALI4 cohort, stratified by IGFRIL expression levels. H) Schematic representation of the IGFRIL locus,…

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  Figure 2. IGFRIL plays an oncogenic role in HCC in vitro and in vivo. A) Confirmation of the knockdown effect on the expression of IGFRIL by using qRT‐PCR assays. B–D) IGFRIL knockdown suppresses cell proliferation, colony formation, migration (24 h), and invasion (48 h) in HepG2 cells. E) Confirmation of the overexpression effect on the expression of IGFRIL by using qRT‐PCR assays. F–H) IGFRIL overexpression promotes cell proliferation, colony formation, migration, and invasion in HepG2 cells. I,J) IGFRIL knockdown inhibits HepG2 cell‐derived subcutaneous tumor growth and reduces Ki‐67 staining in tumor tissues (n = 6). K,L) IGFRIL overexpression promotes HepG2 cell‐derived subcutaneous tumor growth (n = 6). M–O) IGFRIL knockdown inhibits MHCC97H cell‐derived tumor metastasis and prolongs the survival of mice. Luciferase‐labeled IGFRIL‐knockdown or control MHCC97H cells were injected via the tail vein into the nude mice (n = 10). P–R) IGFRIL overexpression promotes HepG2 cell‐derived tumor metastasis (n = 10). Scale bars in (J,L,O,R), 100 µm. Data are shown as mean ± SEM, ** P < 0.01, *** P < 0.001 by 1‐way ANOVA (B–D,M), Student's t‐test (F–I,K,P), Wilcoxon signed‐rank test (J,L,O,R) or…

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  Figure 3. IGFRIL interacts with PTBP1 and relies on PTBP1 to play oncogenic roles in HCC cells. A) Silver staining of IGFRIL‐associated proteins after RNA pull‐down, with the identified PTBP proteins indicated. B) Immunoblotting (IB) assays of PTBP1, PTBP2 and PTBP3 pulled down by IGFRIL. C) RNA immunoprecipitation (RIP) assay validating the IGFRIL‐PTBP1 interaction. D) Mapping the PTBP1‐binding region on IGFRIL using truncated IGFRIL mutants. E) CLIP‐seq analysis showing PTBP1 binding to the 5′ end of IGFRIL. F) Mapping the IGFRIL‐binding region on PTBP1 using truncated PTBP1 mutants. WT, wild‐type. G) Colocalization of IGFRIL and PTBP1 in HepG2 cells, as shown by RNA FISH and immunofluorescence. Scale bar, 20 µm. H,I) The tumorigenic role of PTPB1 in HepG2 cells. J) PTBP1 knockdown abolishes the tumor‐promoting effects of IGFRIL overexpression on HepG2 cells. K) Re‐introduction of full‐length IGFRIL, but not the IGFRIL△380 mutant, rescues the decreased malignant phenotypes by IGFRIL knockdown in HepG2 cells. Data are shown as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 by 1‐way ANOVA (H,J,K) or Student's t‐test (C,I). n.s., not significant.

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  Figure 4. IGFRIL couples with PTBP1 to enhance the activation of AKT‐mTOR signaling in HCC cells. A) Overlapping DEGs upon IGFRIL and PTBP1 knockdown in HepG2 cells. B) GO and KEGG pathway enrichment analyses of the overlapping differentially expressed genes (DEGs). C) Spearman's correlation of gene set enrichment scores between IGFRIL and PTBP1 knockdown. NES, normalized enrichment score. D) GSEA plots of “PI3K_EVENTS_IN_ERBB4_SIGNALING” in IGFRIL‐knocked‐down and PTBP1‐knocked‐down HepG2 cells. E) IB assays of key cascades of the receptor tyrosine kinases (RTK) and AKT‐mTOR signaling in HepG2 cells upon IGFRIL knockdown or overexpression. F) Knockdown of IGFRIL accelerates the autophagic flux in HepG2 cells. HepG2 cells stably expressing mRFP‐GFP‐LC3 were treated with DMSO, NVP‐AEW541 (2 µm), or Rapamycin (10 µm) for 12 h upon knockdown of IGFRIL, followed by staining with DAPI. Red puncta represent the autophagosomes; and yellow puncta in the merged picture represent the autolysosomes. Scale bars, 40 µm. G,H) Rapamycin attenuates the promoting effects of IGFRIL overexpression on cell malignant phenotypes and mTOR signaling. Data are shown as mean ± SEM, * P < 0.05, ** P < 0.01, *** P <…

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  Figure 5. IGFRIL couples with PTBP1 to reduce the stability of IGFBP3 mRNA. A) Distribution of PTBP1 binding sites across the transcriptome, showing altered binding patterns in HepG2 cells upon IGFRIL knockdown. TSS, transcriptional start site; TES, transcriptional end site. B) Pearson's correlation of the PTBP1 binding peak intensities determined by CLIP‐seq between groups. RPM, reads per million. C) Overlapping genes with altered PTBP1 binding and expression upon IGFRIL or PTBP1 knockdown. D) Heatmap showing changes in PTBP1 binding and gene expression for 16 candidate genes. E) IGFRIL knockdown affects the mRNA expression of 16 candidate genes, including IGFBP3. F) Gene tracks showing the PTBP1 CLIP occupancies across the gene body of IGFBP3 and the RNA abundance of IGFBP3. G) The binding of PTBP1 to the 3′ UTR of IGFBP3 mRNA by electrophoresis mobility shift assay (EMSA). P, probe. H) IGFRIL enhances the interaction between PTBP1 and IGFBP3 mRNA determined by RIP assays. I) PTBP1 knockdown abolishes the IGFRIL overexpression‐induced repressive effect on IGFBP3 mRNA. J) Re‐expression of full‐length IGFRIL, but not IGFRIL△380, reduces the promoting effect of IGFRIL knockdown on levels of…

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  Figure 6. IGFRIL activates the IGF1R‐AKT‐mTOR signaling and plays oncogenic roles dependent on IGFBP3. A) The effects of knockdown or overexpression of IGFBP3 on the activities of the IGF1R‐AKT‐mTOR axis. B) The effects of IGFBP3 overexpression on IGFRIL‐activated IGF1R‐AKT‐mTOR axis, and the effects of IGFBP3 knockdown on IGFRIL knockdown‐repressed IGF1R‐AKT‐mTOR axis. C,D) The effects of IGFBP3 knockdown or overexpression on the abilities of plate colony formation, migration, and invasion. E) The effects of IGFBP3 overexpression on the IGFRIL‐enhanced plate colony formation, migration, and invasion. F) The effects of IGFBP3 knockdown on the IGFRIL knockdown‐reduced plate colony formation, migration, and invasion. G) The effects of IGF1R inhibition with its inhibitor AEW541 (2 µM) or siRNAs against IGF1R on the IGFRIL‐activated IGF1R‐AKT‐mTOR axis. H,I) The effects of IGF1R inhibition on the IGFRIL‐enhanced plate colony formation, migration, and invasion. J) j Spearman's correlations between the levels of IGFRIL and levels of IGFBP3, p‐IGF1R, p‐AKT and p‐mTOR, respectively, in HCC tumor tissues (n = 83) from the VALI1 cohort. Data are shown as mean ± SEM, * P < 0.05, ** P < 0.01, *** P <…

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  Figure 7. Upregulation of IGFRIL sensitizes HCC cells to IGF1R and mTOR inhibitors. A) Spearman's correlations between the levels of IGFRIL and sensitivities to inhibitors against IGF1R (BMS‐754807) and mTOR (rapamycin) in eight types of HCC cell lines. The effects of BMS‐754807 or rapamycin on HCC cell growth were determined by CCK‐8 assays. B) The effects of IGFRIL knockdown on the sensitivity to BMS‐754807 or rapamycin in HepG2 cells. C) The effects of IGFRIL overexpression on the sensitivity to BMS‐754807 or rapamycin in JHH‐2 cells. D) The effects of BMS‐754807 or rapamycin treatment on tumor growth in patient‐derived xenografts (PDX) mouse models. The mice were treated with control (DMSO), BMS‐754807 (25 mg kg−1 every 5 days, intraperitoneal injection) or rapamycin (10 mg kg−1 every 5 days, intraperitoneal injection) (n = 5 mice/group). E–G) The effects of IGFRIL knockdown or overexpression on the cell growth, migration, and invasion (E,F), and the activities of the IGF1R‐AKT‐mTOR axis (G) in IGFRIL high and IGFRIL low patient‐derived primary tumor cells (PDCs), respectively. H) The effects of BMS‐754807 (left) or rapamycin (right) treatment on the growth of IGFRIL high or IGFRIL low…