WTAP/IGF2BP3-mediated GBE1 expression accelerates the proliferation and enhances stemness in pancreatic cancer cells via upregulating c-Myc

Abstract

Background: Pancreatic cancer (PC) is one of the most malignant cancers with highly aggressiveness and poor prognosis. N6-methyladenosine (m6A) have been indicated to be involved in PC development. Glucan Branching Enzyme 1 (GBE1) is mainly involved in cell glycogen metabolism. However, the function of GBE1 and Whether GBE1 occurs m6A modification in PC progression remains to be illustrated.

Methods: The clinical prognosis of GBE1 was analyzed through online platform. The expression of GBE1 was obtained from online platform and then verified in normal and PC cell lines. Lentivirus was used to generated GBE1 stable-overexpression or knockdown PC cells. Cell Counting Kit (CCK-8), colony formation assay, sphere formation assay and flow cytometry assay were conducted to analyze cell proliferation and stemness ability in vitro. Subcutaneous and orthotopic mouse models were used to verify the function of GBE1 in vivo. RNA immunoprecipitation (RIP) assay, RNA stability experiment and western blots were conducted to explore the molecular regulation of GBE1 in PC.

Results: GBE1 was significantly upregulated in PC and associated with poor prognosis of PC patients. Functionally, GBE1 overexpression facilitated PC cell proliferation and stemness-like properties, while knockdown of GBE1 attenuated the malignancy of PC cells. Importantly, we found the m6A modification of GBE1 RNA, and WTAP and IGF2BP3 was revealed as the m6A regulators to increase GBE1 mRNA stability and expression. Furthermore, c-Myc was discovered as a downstream gene of GBE1 and functional rescue experiments showed that overexpression of c-Myc could rescue GBE1 knockdown-induced PC cell growth inhibition.

Conclusions: Our study uncovered the oncogenic role of GBE1/c-Myc axis in PC progression and revealed WTAP/IGF2BP3-mediated m6A modification of GBE1, which highlight the potential application of GBE1 in the targeted therapy of PC.

Keywords: GBE1; IGF2BP3; Pancreatic cancer; Proliferation; WTAP; m6A modification.

Fig. 1

GBE1 was highly expressed in PC and predicted poor prognosis of PC patients. A GBE1 expression in PC and normal tissues was analyzed by GEPIA platform. We used Log₂(TPM + 1) for log-scale in Y axis. 0.05 was used as p-value cutoff. B XIANTAO platform was used to explore the GBE1 expression in PC and normal tissues. p < 0.001. C Overall survival (OS) of PC patients was analyzed on XIANTAO platform. D Disease specific survival (DSS) of PC patients was analyzed on XIANTAO platform. E Progression free interval (PFI) of PC patients was analyzed on XIANTAO platform. F Relapse free survival (RFS) of PC patients was analyzed on Kaplan–Meier plotter platform. G RNA expression of GBE1 in PC cells and normal HPDE cells (one-way ANOVA test). H Protein expression of GBE1 in PC cells and normal HPDE cells. ***p < 0.0001

Fig. 2

GBE1 overexpression promoted the growth and stemness-like properties of PC cells. A RT-qPCR analysis of GBE1 overexpression efficiency in PC cells (t test). B Western blot analysis of GBE1 overexpression efficiency in PC cells. C GBE1 increased PC cell proliferation revealed by CCK8 assay (t test). D GBE1 enhanced colony formation ability of PC cells, and colony statistical analysis was shown on the right (t test). E GBE1 promoted sphere formation ability of PC cells. GBE1 promoted the percentage of CD24⁺ (F), CD44⁺ (G) and CD133⁺ (H) population of PC cells revealed by flow cytometry. The statistical results of flow cytometry were showed on the right (t test). I GBE1 overexpression promoted tumor growth of PC cells in vivo as compared to negative control (NC) cells. NC or GBE1 BXPC3 cells (1 × 10⁶ cells/150ul, N = 5) were subcutaneously injected into the back of nude mice. Twenty days later, tumors were removed from mice. Tumor volume (J) and mice weight (K) of both NC and GBE1 groups were measured and analyzed every 5 days (t test). *p < 0.05, **p < 0.01, ***p < 0.001. NS No significant

Fig. 3

GBE1 deficiency suppressed the PC cell growth and stemness-like properties. A RT-qPCR analysis of GBE1 knockdown efficiency in PC cells. t test. B Western blot analysis of GBE1 knockdown efficiency in PC cells. C GBE1 knockdown suppressed PC cell proliferation revealed by CCK8 assay (t test). D GBE1 knockdown reduced colony formation ability of PC cells, and colony statistical analysis was shown on the right (t test). E GBE1 knockdown suppressed sphere formation ability of PC cells. GBE1 knockdown decreased the percentage of CD24⁺ (F), CD44⁺ (G) and CD133⁺ (H) population of PC cells revealed by flow cytometry. The statistical results of flow cytometry were showed on the right (t test). I GBE1 knockdown suppressed tumor growth of PC cells in vivo when compared to negative control (sh-NC) cells. sh-NC or sh-GBE1 BXPC3 cells (5 × 10⁵ cells/150ul) were subcutaneously injected into the back of nude mice. Twenty days later, tumors were removed from mice. Tumor volume (J) and mice weight (K) of both sh-NC and sh-GBE1 groups were measured and analyzed every 5 days (t test). L GBE1 knockdown suppressed tumor growth of PC cell as determined by orthotopic mouse model. sh-NC or sh-KD BXPC3 cells (3 × 10⁶ cells/50ul, N = 5) were orthotopically injected into the pancreas of nude mice. One month later, tumors were removed from mice. Mice weight (M) of both sh-NC and sh-GBE1 groups were measured and analyzed every 5 days (t test). *p < 0.05, **p < 0.01, ***p < 0.001. NS No significant

Fig. 4

WTAP and IGF2BP3 mediated the m6A modification of GBE1. A m6A enrichment of GBE1 RNA by m6A RIP assay with m6A antibody (t test). mRNA (B) and protein (C) expression of GBE1 in BXPC3 cells after treatment with DAA (one way ANOVA test). D The positive correlation between WTAP and GBE1 mRNA expression as revealed in GEPIA platform. Overexpression of WTAP increased both RNA (E) and protein (F) expression of GBE1 (t test). Knockdown of WTAP by transfection with siRNAs reduced both RNA (G) and protein (H) expression of GBE1 (one way ANOVA test). I IGF2BP3 interacted with GBE1 RNA as revealed by RIP assay with IGF2BP3 antibody (t test). J The positive correlation between WTAP and GBE1 mRNA expression as revealed in GEPIA platform. Overexpression of IGF2BP3 increased both RNA (K) and protein (L) expression of GBE1 (t test). Knockdown of IGF2BP3 by transfection with siRNAs reduced both RNA (M) and protein (N) expression of GBE1 (one way ANOVA test). O Knockdown of IGF2BP3 by siRNA reduced GBE1 RNA stability in BXPC3 cells. BXPC3 cells were transfected with IGF2BP3 siRNAs and negative control siRNA. Twenty-four hours later, Actinomycin D was added to the transfected BXPC3 cells at a final concentration of 2ug/ml and total RNA was harvested at different time points. RT-qPCR analysis of GBE1 expression was performed (one way ANOVA test). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

GBE1 enhanced PC cell proliferation by upregulating c-Myc. RT-qPCR (A) and Western blot (B) results of BXPC3 cells with knockdown of c-Myc after GBE1 overexpression (one way ANOVA test). C Colony formation assay indicated that c-Myc knockdown suppressed GBE1-induced cell proliferation. D Statistical analysis of colony formation results in C (one way ANOVA test). E CCK8 assay indicated that c-Myc knockdown suppressed GBE1-induced cell proliferation (one way ANOVA test). RT-qPCR (F) and Western blot (G) results of BXPC3 cells with overexpression of c-Myc after GBE1 knockdown (one way ANOVA test). H Colony formation assay indicated that c-Myc overexpression rescued GBE1 knockdown-induced cell proliferation suppression. I Statistical analysis of colony formation results in Figure H (one way ANOVA test). J CCK8 assay indicated that c-Myc rescued the cell proliferation inhibition induced by GBE1 knockdown (one way ANOVA test). *p < 0.05, **p < 0.01, ***p < 0.001