CircGPR137B/miR-4739/FTO feedback loop suppresses tumorigenesis and metastasis of hepatocellular carcinoma

Abstract

Background: Emerging evidence indicates that circular RNAs (circRNAs) and m⁶A RNA methylation participate in the pathogenesis and metastasis of multiple malignancies including hepatocellular carcinoma (HCC). However, it remains undocumented how circRNAs form a feedback loop with the m⁶A modification contributing to HCC.

Methods: A novel hsa_circ_0017114 (circGPR137B) was identified from three pairs of primary HCC and adjacent normal tissues by circRNA expression profiling. The association of circGPR137B and miR-4739 with clinicopathological parameters and prognosis in patients with HCC was analyzed by RT-qPCR, fluorescence in situ hybridization and TCGA cohorts. The role of circGPR137B in HCC was estimated in vitro and in vivo. RT-qPCR, western blot, m⁶A dot blot, RIP, MeRIP and dual-luciferase reporter assays were used to validate the reciprocal regulation of the feedback loop among circGPR137B, miR-4739 and m⁶A demethylase FTO. Meanwhile, the expression, function and prognosis of FTO in HCC were investigated by RT-qPCR, western blot, TCGA and rescue experiments.

Results: We identified a new dramatically downregulated circGPR137B in HCC tissues, and found that downregulation of circGPR137B or upregulation of miR-4739 was associated with poor prognosis in patients with HCC. Ectopic expression of circGPR137B strikingly repressed the proliferation, colony formation and invasion, whereas knockdown of circGPR137B harbored the opposite effects. Moreover, restored expression of circGPR137B inhibited tumor growth and lung metastasis in vivo. Further investigations showed that circGPR137B, co-localized with miR-4739 in the cytoplasm, acted as a sponge for miR-4739 to upregulate its target FTO, which mediated m⁶A demethylation of circGPR137B and promoted its expression. Thus, a feedback loop comprising circGPR137B/miR-4739/FTO axis was formed. FTO suppressed cell growth and indicated favorable survival in patients with HCC.

Conclusion: Our results demonstrate that circGPR137B inhibits HCC tumorigenesis and metastasis through the circGPR137B/miR-4739/FTO feedback loop. This positive feedback mechanism executed by functional coupling between a circRNA sponge and an m⁶A modification event suggests a model for epigenetics.

Keywords: CircGPR137B; Demethylation; FTO; Hepatocellular carcinoma; m6A; miR-4739.

Fig. 1

Downregulation of circGPR137B is associated with poor survival in patients with HCC. A CircRNA profiling analysis of the differentially-expressed circRNAs between HCC and adjacent normal tissues. B Heatmap analysis of 96 upregulated circRNAs and 51 downregulated circRNAs in HCC according to the restrictive conditions (P < 0.05 and FC > 1.5). C Volcano plot of a new substantially downregulated hsa_circ_0017114 (circGPR137B) in HCC tissues. D CircRNA profiling analysis of the expression of circGPR137B in 3 paired HCC tissues. E RT-qPCR analysis of the expression of circGPR137B in 5 paired HCC tissues. F, G FISH validation of the expression of circGPR137B in 87 paired HCC tissues. Blue color: DAPI; Green color: circGPR137B. H, I Kaplan–Meier analysis of the association of circGPR137B high or low expression with overall survival in HCC and early stage ones

Fig. 2

Characteristics of circGPR137B in HCC cells. A Genomic loci of the GPR137B and circGPR137B. B RT-qPCR analysis of the enrichment levels of GPR137B and circGPR137B after treatment with RNase R in HCC cell lines. C RT-qPCR analysis of the transcription half-life of GPR137B and circGPR137B after treatment with actinomycin D in HCC cell lines. D, E RT-qPCR and FISH analysis of the localization of circGPR137B and GPR137B in HCC cells and tissues. Blue color: DAPI; Green color: circGPR137B. Data are the means ± SEM of three experiments. ***P < 0.001

Fig. 3

CircGPR137B inhibits in vitro cell growth. A RT-qPCR analysis of the expression levels of circGPR137B in different HCC cell lines. B RT-qPCR analysis of the overexpression efficiency of circGPR137B lentiviruses in HepG2 and Hep3B cell lines and the knockdown efficiency of sh-circGPR137B lentiviruses in SK-hep-1 cells. C MTT. Colony formation (D) and Transwell analysis (E) of the effects of circGPR137B overexpression or knockdown on cell proliferation, colony formation and cell invasion in HCC cell lines. Data are the means ± SEM of three experiments. *P < 0.05; **P < 0.01, ***P < 0.001

Fig. 4

CircGPR137B has a negative correlation with miR-4739 in HCC. A, B Identification of the binding sites between circGPR137B and 5 miRNAs. C. Luciferase activity of circGPR137B 3’UTR was evaluated in HCC cells exposed to the treatment of 5 miRNA mimics. D, E FISH analysis of the expression levels of miR-4739 in 87 pairs of HCC tissue samples. F Pearson correlation analysis of the correlation of circGPR137B with miR-4739 in HCC. G TCGA analysis of the expression levels of miR-4739 in 220 HCC tissue samples. H Kaplan–Meier analysis of the association of miR-4739 high or low expression with overall survival in HCC

Fig. 5

CircGPR137B acts as a sponge for miR-4739 in HCC. A Schematic representation of the binding sites between miR-4739 and WT circGPR137B 3’UTR. B RT-qPCR analysis of the effects of circGPR137B overexpression on miR-4739 expression in HepG2 and Hep3B cells. C Comparison of the luciferase activity of circGPR137B 3’UTR after treatment with miR-4739 mimics in HepG2 and Hep3B cells. D. RIP analysis of the enrichment levels of circGPR137B and miR-4739 pulled down from Ago2 or IgG protein in HepG2 and Hep3B cells. E FISH analysis of the co-localization of circGPR137B with miR-4739 in HepG2 cells. Blue color: DAPI, Red color: miR-4739, Green color: circGPR137B. MTT (F) and Transwell analysis (G) of the cell proliferation and invasion after transfection with circGPR137B lentiviruses and (or) miR-4739 mimics in HepG2 and Hep3B cells. Data are the means ± SEM of three experiments. *P < 0.05, **P < 0.01

Fig. 6

FTO is regulated by circGPR137B/miR-4739 axis in HCC. A Schematic representation of the binding sites between miR-4739 and FTO 3’UTR. B Comparison of the luciferase activity of WT or Mut FTO 3’UTR after treatment with miR-4739 mimics in HepG2 and Hep3B cells. C, D RT-qPCR and western blot analysis of the expression of FTO, p21, p27, cleaved caspase-3/− 9, N-cadherin, E-cadherin and vimentin after the transfection with circGPR137B lentiviruses and (or) miR-4739 mimics in HepG2 and Hep3B cells. E, F, G MTT, colony formation and transwell analysis of the cell proliferation, colony number and cell invasion after the transfection with FTO plasmids and (or) miR-4739 mimics in HepG2 and Hep3B cells. Data are the means ± SEM of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

FTO downregulation indicates poor prognosis in HCC. A, B RT-qPCR and western blot analysis of the expression levels of FTO in 10 pairs of HCC samples. C TCGA analysis of the expression levels of FTO in 50 pairs of HCC samples and 371 cases. D Kaplan–Meier analysis of the association of FTO expression with overall survival in HCC

Fig. 8

FTO mediates m⁶A modification of circGPR137B to form a positive feedback loop with circGPR137B. MeRIP (A) and m⁶A dot blot (B) analyses of the effects of FTO on the total m⁶A levels in HepG2 and Hep3B cells. C MeRIP analysis of the effect of FTO on the m⁶A levels of circGPR137B in HepG2 and Hep3B cells. RT-qPCR (D) and western blot (E) analysis of the effect of FTO overexpression or knockdown on the RNA levels of circGPR137B in HCC cells. RT-qPCR (F) and western blot (G) analysis of the effect of circGPR137B knockdown or miR-4739 inhibitor on FTO expression in SK-hep-1 cells. H RIP analysis of the binding between FTO and circGPR137B in HepG2 and Hep3B cells. MTT (I) and transwell (J) analysis of the cell proliferation and invasion after transfection with FTO and si-circGPR137B plasmids in HepG2 and Hep3B cells. Data are the means ± SEM of three experiments. *P < 0.05, **P < 0.01

Fig. 9

CircGPR137B inhibits in vivo HCC tumorigenesis. A Ex vivo imaging of the luciferase signals in tumors formed by stable transfection with circGPR137B or control group into luciferase-labeled HepG2 cells. B, C Schematic representation of the comparison of HepG2 xenograft tumors between circGPR137B and control groups and statistical analysis of the tumor size and weight between circGPR137B and control groups. D HE staining of the tumor tissue cells and IHC analysis of the Ki-67 proliferation index and FTO expression between circGPR137B and control groups. E RT-qPCR analysis of the expression levels of circGPR137B, miR-4739 and FTO between circGPR137B and control groups. F Schematic representation of the comparison of SK-hep-1 xenograft tumors between sh-circGPR137B and sh-NC groups, and statistical analysis of the tumor size and weight between sh-circGPR137B and sh-NC groups. G IHC analysis of the Ki-67 proliferation index and FTO expression levels between sh-circGPR137B and sh-NC groups. H. RT-qPCR analysis of the expression levels of circGPR137B, miR-4739 and FTO between sh-circGPR137B and sh-NC groups

Fig. 10

CircGPR137B inhibits in vivo lung metastasis of HCC. A Ex vivo imaging of the luciferase signals in orthotopic liver tumors with peritoneal infiltration by stable transfection with circGPR137B or control group into luciferase-labeled HepG2 cells. B Schematic representation of the comparison of orthotopic liver tumors, and statistical analysis of the total liver weight between circGPR137B and control groups. C Schematic representation of the comparison of metastatic pulmonary tumors, and statistical analysis of the total lung weight between circGPR137B and control groups. D HE staining of the tumor tissue cells from orthotopic liver tumors and metastatic pulmonary tumors in circGPR137B and control groups and IHC analysis of the Ki-67 proliferation index and FTO expression levels in orthotopic liver tumors and metastatic pulmonary tumors between circGPR137B and control groups. E. RT-qPCR analysis of the expression levels of circGPR137B, miR-4739 and FTO in orthotopic liver tumors between circGPR137B and control groups

Fig. 11

Schematic illustration of circGPR137B suppressing proliferation and metastasis of liver cancer through a positive feedback loop. CircGPR137B induces FTO protein expression by sponging miR-4739 which binds to FTO mRNA in order to inhibit its expression. FTO protein enters into cellular nuclear to mediate m6A demethylation of circGPR137B and promotes its expression. Thus, a positive feedback forms on circGPR137B production, resulting in strongly repressing liver cancer cell proliferation, colony formation, invasion, and lung migration