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. 2019 Dec 2;18(1):174.
doi: 10.1186/s12943-019-1105-0.

LncRNA LINRIS Stabilizes IGF2BP2 and Promotes the Aerobic Glycolysis in Colorectal Cancer

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Free PMC article

LncRNA LINRIS Stabilizes IGF2BP2 and Promotes the Aerobic Glycolysis in Colorectal Cancer

Yun Wang et al. Mol Cancer. .
Free PMC article

Abstract

Background: Long noncoding RNAs (lncRNAs) play nonnegligible roles in the epigenetic regulation of cancer cells. This study aimed to identify a specific lncRNA that promotes the colorectal cancer (CRC) progression and could be a potential therapeutic target.

Methods: We screened highly expressed lncRNAs in human CRC samples compared with their matched adjacent normal tissues. The proteins that interact with LINRIS (Long Intergenic Noncoding RNA for IGF2BP2 Stability) were confirmed by RNA pull-down and RNA immunoprecipitation (RIP) assays. The proliferation and metabolic alteration of CRC cells with LINRIS inhibited were tested in vitro and in vivo.

Results: LINRIS was upregulated in CRC tissues from patients with poor overall survival (OS), and LINRIS inhibition led to the impaired CRC cell line growth. Moreover, knockdown of LINRIS resulted in a decreased level of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), a newly found N6-methyladenosine (m6A) 'reader'. LINRIS blocked K139 ubiquitination of IGF2BP2, maintaining its stability. This process prevented the degradation of IGF2BP2 through the autophagy-lysosome pathway (ALP). Therefore, knockdown of LINRIS attenuated the downstream effects of IGF2BP2, especially MYC-mediated glycolysis in CRC cells. In addition, the transcription of LINRIS could be inhibited by GATA3 in CRC cells. In vivo experiments showed that the inhibition of LINRIS suppressed the proliferation of tumors in orthotopic models and in patient-derived xenograft (PDX) models.

Conclusion: LINRIS is an independent prognostic biomarker for CRC. The LINRIS-IGF2BP2-MYC axis promotes the progression of CRC and is a promising therapeutic target.

Keywords: Autophagy; CRC; IGF2BP2; LINRIS; MYC.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Identification of LINRIS as an oncogenic lncRNA in CRC. a Heatmap for differentially expressed lncRNAs in CRC tissues compared with that in normal tissues. b qPCR detection shows that LINRIS was highly expressed in CRC tissues (T, n = 118) compared with the expression in normal colon tissues (N). *P < 0.05, **P < 0.01. c Kaplan-Meier analysis of the OS curves for CRC patients with LINRIS-low (n = 58) or LINRIS-high (n = 60) expression (log-rank test). d The overall LINRIS expression in multiple human cancers from TCGA. e LINRIS levels in different CRC cell lines compared with the level in the normal colon cell line CCD841. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01. f qPCR detection shows that LINRIS was highly expressed in ESCC (n = 42), GC tissues (n = 35) and PDAC (n = 27) tissues compared with the expression in normal esophageal, gastric and pancreatic tissues respectively. *P < 0.05, **P < 0.01. g BrdU assays of the indicated cells with LINRIS knockdown by shRNAs compared with the control. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01. h and i Images (h) and quantification (i) of the 3D culture of the indicated cells with or without knocking down LINRIS after 1 week. Scale bar, 100 μm. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01. j FISH assays identifying the subcellular location of LINRIS in HCT116 cells. Scale bar, 100 μm. k RNAScope® ISH detection of LINRIS expression (arrows) in CRC cells. Scare bar: 20 μm. l Location of LINRIS in the cytoplasmic and nuclear extractions from CRC cells with qPCR
Fig. 2
Fig. 2
LINRIS was associated with IGF2BP2 in CRC. a IGF2BP2 was pulled down by biotin-labeled sense LINRIS (S) but not LINRIS anti-sense (AS) RNA in the indicated cells. b RIP assays were applied using anti-IGF2BP2 antibodies with extractions from HCT116 cells. Relative enrichment (mean ± SD) represents the RNA levels associated with the indicated protein relative to an input control from three independent experiments after immunoprecipitation with the anti-IGF2BP2 antibody compared with that with the IgG antibody. MYC mRNA was uesd as the positive control and GAPDH mRNA was used as the negative control. c Expression vectors for FLAG-tagged MCP and MS2-tagged LINRIS were transfected into CRC cells to establish the FLAG-MCP-MS2 system. And IGF2BP2 was then pulled down using the anti-FLAG® M2 affinity gel followed by the Western blot analysis. d Western blot detection of IGF2BP2 binding to LINRIS after FLAG-MCP-MS2 pull-down assays. e In vitro-synthesized full-length (FL), N-terminal (NT) and C-terminal (CT) fragments of LINRIS were incubated with protein lysates from HCT116 cells. RNA pull-down and Western blotting assays were then performed. The data shown represent three independent experiments. f Western blot analysis shows the levels of IGF2BP2 in 11 CRC cell lines with GAPDH as the loading control. g IGF2BP2 expression was positively correlated with LINRIS expression in CRC cells. The r values and P values are from Pearson’s correlation analysis. h Western blot analysis shows the expression of IGF2BP2 with or without knockdown of LINRIS in the indicated cells. i CRC cells transfected with shRNAs specific for LINRIS or a scrambled control. Cell lysates were immunoprecipitated with either an antibody against IGF2BP2 or an IgG control and then analyzed by immunoblotting with a ubiquitin (Ub)-specific antibody
Fig. 3
Fig. 3
LINRIS is involved in the autophagic degradation of IGF2BP2. a Schematic structures of IGF2BP2 proteins and three truncated mutants (T1: 1–193; T2: 157–427; T3: 341–913) of IGF2BP variants used in this study. Orange boxes are RRM domains, and blue boxes are KH domains. b RIP assays were performed using anti-FLAG antibodies and HCT116 cells transfected with vectors expressing the FLAG-tagged FL and the truncated mutants (T1-T3) of IGF2BP2. The data shown represent three independent experiments. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01. c IP assays were performed using anti-FLAG antibodies and HCT116 cells transfected with vectors expressing the FLAG-tagged WT or IGF2BP2 mutants (K77R and K139R). d Western blot analysis following RNA pull-down assays shows that the K139R mutation of IGF2BP2 blocked its binding to LINRIS in CRC cells. GAPDH was used as the loading control. e CRC cells with or without shRNAs specific for LINRIS were treated with 20 μg/ml CHX or a vehicle for the indicated periods of time. IGF2BP2 levels were analyzed by western blotting. f CRC cells with LINRIS knockdown and control cells were treated with or without MG132 (5 μM) for 12 h. Cell lysates were analyzed by Western blotting with GAPDH as the loading control. g Illustration of the protein degradation process via two main pathways. h CRC cells with LINRIS knockdown were treated with or without Baf A1 (100 nM, 24 h) and NH4Cl (10 mM, 4 h). Cell lysates were analyzed by Western blotting with GAPDH as the loading control. i Western blotting shows the levels of IGF2BP2 in the indicated cells after treatment with or without EBSS for 3–6 h or Rap (100 nM) for 24 h. GAPDH was used as the loading control. j Confocal microscopy of HCT116 cells treated with or without EBSS for 3 h. Scale bar, 50 μm
Fig. 4
Fig. 4
The alteration of MYC-mediated glycolysis under the influence of LINRIS. a The mRNA levels of MYC and downstream genes, including GLUT-1, PKM2 and LDHA, when inhibiting LINRIS in HCT116 cells. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01. b Western blot analysis showed the levels of MYC and downstream genes, including GLUT-1, PKM2 and LDHA, when inhibiting LINRIS in CRC cells. c Correlations between the LINRIS levels and the mRNA levels of MYC, GLUT-1, HK2, PKM2 and LDHA in CRC (n = 74). RNA levels were determined by qPCR relative to the levels of GAPDH. The r values and P values are from Pearson’s correlation analysis. d The ECAR was detected in the indicated cells with sh-1, sh-2 or control using an XF Extracellular Flux Analyzer. Glucose, oligomycin and 2-DG were injected sequentially at different time points as indicated. The data shown represent three independent experiments. e Statistical analysis of the effects of LINRIS knockdown on glycolytic activity. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01. f Statistical analysis of 13C-labeled 3-PG/2-PG, 13C-labeled glyceraldehyde 3-phosphate and 13C-labeled pyruvate. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05. g BrdU assay showing that the overexpression (OE) of IGF2BP2 rescued the proliferation inhibition of the indicated cells with the knockdown of LINRIS. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01. h The extracellular acidification rate (ECAR) was detected in HCT116 cells with or without sh-1 and overexpressed IGF2BP2 using an XF Extracellular Flux Analyzer. Glucose, oligomycin and 2-DG were injected sequentially at different time points as indicated. The data shown represent three independent experiments. i Overexpression of IGF2BP2 reversed the suppression of LINRIS knockdown on glycolytic activity in HCT116 cells. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01
Fig. 5
Fig. 5
In vivo experiments elucidated the effect of the inhibition of LINRIS in CRC. a Image of orthotopic tumors with or without LINRIS knockdown resected from nude mice. b The tumor weights of the orthotopic tumors with or without LINRIS knockdown. Error bars, SD of five independent experiments. *P < 0.05 or **P < 0.01 versus the control. c and d The volume growth curves of tumors (c) and the tumor weights (d) of two PDX models are shown. Error bars, SD of four independent experiments. *P < 0.05 or **P < 0.01 versus the control. e Representative images of H&E staining and IHC staining of Ki-67, IGF2BP2 and MYC from the tumor sections. Scale bar, 50 μm. f Quantification of the proliferation index (Ki-67 proportion), IGF2BP2 and MYC levels in the tumor sections. *P < 0.05, **P < 0.01
Fig. 6
Fig. 6
LINRIS could be inhibited by GATA3 in CRC. a Predicted GATA3 DNA-binding sequences are present in the human LINRIS promoter region. b GATA3 expression in several CRC microarray datasets from Oncomine database. c Kaplan-Meier analysis of the OS curves for CRC patients with different GATA3 expression from TCGA database (log-rank test). d LINRIS mRNA levels were upregulated with knockdown of GATA3 in CRC cells. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01. e ChIP-PCR of LINRIS in CRC cells. Error bars, SD of three independent experiments. *P < 0.05 or **P < 0.01. f Relative LINRIS luciferase promoter activity in the indicated cells with GATA3 overexpression (OE-GATA3). Error bars, SD of three independent experiments. *P < 0.05 or **P < 0.01 versus the control. g Relative LINRIS luciferase promoter activity in the indicated cells with GATA3 knockdown. Error bars, SD of three independent experiments. *P < 0.05 or **P < 0.01 versus the control. h Relative LINRIS luciferase promoter activity in the indicated cells with or without the mutation of GATA3 binding sequences. Error bars, SD of three independent experiments. *P < 0.05 or **P < 0.01 versus the control. i Correlations between the LINRIS levels and the mRNA levels of GATA3 in CRC (n = 76). RNA levels were determined by qPCR relative to the levels of GAPDH. The r values and P values are from Pearson’s correlation analysis. j Representative IHC images of GATA3 in tumor tissues from patients with CRC (n = 220) with low or high levels of LINRIS. Scale bar, 50 μm. k Percentages of specimens showing different levels of GATA3 in the low or high LINRIS expression groups (n = 220, Chi-square test, **P < 0.01)
Fig. 7
Fig. 7
Illustration of LINRIS-IGF2BP2-MYC axis in CRC. a Representative IHC images of Ki-67, IGF2BP2, MYC, GLUT-1 and PKM2 and LDHA in tumor tissues from patients with CRC (n = 220) with low or high levels of LINRIS. Scale bar, 50 μm. b Percentages of specimens showing different levels of Ki-67, IGF2BP2, MYC, GLUT-1 and PKM2 and LDHA in the low or high LINRIS expression groups (n = 220, Chi-square test, **P < 0.01). c and d Representative IHC images (c) and statistical analysis (d) of IGF2BP2 expression in CRC and matched normal tissues. The data are shown as the mean ± SD; n = 220, two-tailed Student’s t-test. e Kaplan-Meier analysis of the OS curves for CRC patients with low (n = 118) or high (n = 102) IGF2BP2 expression (log-rank test). f Kaplan-Meier analysis of the OS curves for CRC patients with LINRIS/IGF2BP2-high (both levels of LINRIS and IGF2BP2 were high, n = 76), LINRIS/IGF2BP2-low (both levels of LINRIS and IGF2BP2 were low, n = 76) or intermediate (n = 68) expression (log-rank test). g Proposed working model of this study. LINRIS stabilized IGF2BP2 by binding to its K139 ubiquitination site and subsequently maintained the MYC-induced glycolysis and proliferation of CRC cells. Otherwise, inhibition of LINRIS, such as by GATA3, resulted in more degradation of IGF2BP2 through the ubiquitination-autophagy pathway.

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References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2018;69:7–34. - PubMed
    1. Zheng RS, Sun KX, Zhang SW, Zeng HM, Zou XN, Chen R, Gu XY, Wei WW, He J. Report of cancer epidemiology in China, 2015. Zhonghua Zhong Liu Za Zhi. 2019;41:19–28. - PubMed
    1. Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, Rinn JL. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 2011;25:1915–1927. - PMC - PubMed
    1. Carpenter S, Aiello D, Atianand MK, Ricci EP, Gandhi P, Hall LL, Byron M, Monks B, Henry-Bezy M, Lawrence JB, et al. A long noncoding RNA mediates both activation and repression of immune response genes. Science. 2013;341:789–792. - PMC - PubMed
    1. Lee S, Kopp F, Chang TC, Sataluri A, Chen B, Sivakumar S, Yu H, Xie Y, Mendell JT. Noncoding RNA NORAD regulates genomic stability by sequestering PUMILIO proteins. Cell. 2016;164:69–80. - PMC - PubMed

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