A N7-methylguanosine modified circular RNA, circIPP2A2, promotes malignant behaviors in hepatocellular carcinoma by serving as a scaffold in modulating the Hornerin/PI3K/AKT/GSK3β axis

Abstract

Despite the advancements in treatment strategies, the long-term survival of hepatocellular carcinoma (HCC) is still pessimistic. Therefore, understanding the mechanisms of hepatocellular carcinoma may offer substantial benefits for patients. Our previous research has revealed that Hornerin promoted HCC progression by regulating the AKT signaling pathway. To investigate the upstream regulatory mechanism, the results from RNA Immunoprecipitation and RNA pull-down indicated that the specific region of circIPP2A2 interacted with Hornerin. Additionally, patients with circIPP2A2 upregulation exhibited a poorer survival outcome following surgery compared to the cases with downregulated circIPP2A2. After the structure verification of circIPP2A2, loss-of-function studies using a lentiviral vector revealed that circIPP2A2 downregulation significantly inhibited HCC tumorigenesis and progression both in vitro and in vivo. Mechanistically, the m7G-MeRIP results demonstrated significant enrichment of circIPP2A2. Subsequent studies validated that METTL1 influenced the stability of circIPP2A2 and its binding affinity with Hornerin. Immunoprecipitation and immunofluorescence indicated that circIPP2A2 served as a molecular scaffold to facilitate Hornerin to interact with PI3K. In conclusion, our findings reveal that circIPP2A2, regulated by N7-methylguanosine modification, promotes malignant behaviors in HCC by serving as a molecular scaffold in modulating the Hornerin/PI3K/AKT/GSK3β axis. Targeting circIPP2A2 may be a promising therapeutic strategy for patients with HCC.

Fig. 1. Hornerin had the capability to interact with circIPP2A2.

a catRAPID results showed the top five circRNAs that interacting with Hornerin. Ranking based on Interaction Propensity. b RIP assay using anti-Hornerin antibody in normal liver cells HL-7702 and HCC cells HCCLM3. The binding affinity was assessed using qPCR. c, d Small interfering RNAs targeted at Hornerin were used to downregulate Hornerin expression. qPCR and western blotting assay were used to verify the successful downregulation. e qPCR was used to detect the expression of circIPP2A2 in HL-7702 and HCCLM3. f qPCR was employed for the verification of circIPP2A2 silencing. g, h qPCR and western blotting assay were used to measure the expression of Hornerin in circIPP2A2 downregulated cells. i RNA pull-down was performed using Biotinylation labeled circIPP2A2 back splice sequence. Western blotting assay was used to detect the Hornerin signal in HL-7702 and HCCLM3. j Schematic diagram of circIPP2A2 primers design. k RIP assay was used to unveil the putative sequences with which Hornerin interacted. l RNA pull-down using a specific circIPP2A2 probe was employed to validate the RIP result. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2. The expression and characteristics of circIPP2A2.

a Sixteen pairs of HCC and corresponding normal liver tissues were used to detect the circIPP2A2 expression by qPCR. b qPCR assay was used to detect the circIPP2A2 expression in HCC cell lines (Hep3B, HCCLM3, Huh7, PLC/PRF5, MHCC97H), hepatoblastoma cell line (HepG2), and normal liver cell lines (HL-7702). c qPCR was employed to determine the expression of circIPP2A2 in 78 HCC and 44 normal liver tissues. d The expression of circIPP2A2 in 78 HCC tissues was divided into high expression (N = 37) and low expression (N = 41) group. Kaplan–Meier curves were used to depict the association between circIPP2A2 expression and clinical outcome following operation. e FISH assay was used to determine the subcellular location of circIPP2A2 in HCC. f Sanger sequencing was used to confirm the back-splicing sequence of circIPP2A2. g RNase R digestion assay was conducted to assess the stability of circIPP2A2 in comparison to linear IPP2A2. h Convergent primers were applied to amplify GAPDH and linear IPP2A2, while divergent primers were used for circIPP2A2 with cDNA or gDNA as the templates. i Actinomycin D treatment impeded the synthesis of linear IPP2A2, while it exhibited no impact on circIPP2A2. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3. circIPP2A2 downregulation suppressed HCC malignant behaviors in vitro and in vivo.

a qPCR was used to verify the successful establishment of circIPP2A2 downregulation HCC cell lines. b CCK-8 assay was performed to assess the proliferation ability after circIPP2A2 downregulation. c Colony formation assay was conducted to evaluate the tumorigenesis change in the context of circIPP2A2 knockdown. d Wound healing assay was employed to detect the migration ability after circIPP2A2 downregulation. e Transwell chambers with or without pre-coated matrigel were used to simulate the invasion and migration in HCC. f, g IVIS image showed the metastasis ability change after circIPP2A2 downregulation in nude mice. Fluorescence intensity represented the lung metastasis ability. Metastatic nodes were confirmed by H&E staining. h Representative images of xenograft tumors in five nude mice. i Changes in tumor volume after circIPP2A2 downregulation. j Tumor growth curve illustrated the impact of circIPP2A2 knockdown on proliferation ability. In vitro data are presented as the mean ± SD of at least three independent experiments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4. circIPP2A2 was regulated by METTL1.

a Anti-METTL1 and anti-WDR4 antibodies were used for RIP assay. qPCR was employed to detect the interaction between circIPP2A2 and METTL1 or WDR4. b RNA pull-down was performed using Biotinylation labeled circIPP2A2 back splice sequence to capture the METTL1 or WDR4 protein. Western blotting was used to detect the indicated protein signal. c METTL1 protein expression after circIPP2A2 downregulation was measured by western blotting. d Confirmation of METTL1 silenced by siRNAs. e The expression of circIPP2A2 and linear IPP2A2 in the context of METTL1 downregulation. f Schematic diagram of prediction m7G site within circIPP2A2. g m7G-MeRIP-qPCR was performed to assess the impact of METTL1 on the enrichment of circIPP2A2. h RNase R digestion assay was used to measure the stability of circIPP2A2 in the context of METTL1 downregulation. i, j RIP assay using anti-Hornerin antibody was conducted to investigate the influence of METTL1 on the interaction between Hornerin and circIPP2A2. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5. Clinical and survival analysis of METTL1 in various tumors.

a, b A pan-cancer survival analysis was performed to determine the expression of METTL1 in various cancers. c The association between METTL1 and clinical outcome was measured using Kaplan–Meier curves in patients with HCC. d Violin plot depicted the correlation between METTL1 expression and tumor postoperative pathological grade in HCC. e qPCR assay was used to measure the expression of METTL1 in 16 pairs of HCC and corresponding normal liver tissues. f, g Western blotting and IHC were performed to assess the protein level of METTL1 in HCC and corresponding normal liver tissues. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6. METTL1 overexpression promoted HCC tumorigenesis and metastasis.

a Western blotting was used to validate the successful overexpression of METTL1 in circIPP2A2 stable knockdown HCC cell lines. b Colony formation assay was employed to assess the tumorigenesis ability induced by METTL1 overexpression in the context of circIPP2A2 knockdown. c CCK-8 was conducted to detect the impact of METTL1 expression on the proliferation ability. d Wound healing assay was performed to measure the migration ability change caused by METTL1 overexpression in circIPP2A2 stable knockdown cells. e Transwell assays with or without pre-coated matrigel were used to determine the alterations in migration and invasion induced by METTL1 overexpression, respectively. ns, not significant; */#/& P < 0.05; **/##/&& P < 0.01; ***/###/&&& P < 0.001; ****/####/&&&& P < 0.0001.

Fig. 7. circIPP2A2 served as a scaffold in modulating the Hornerin/PI3K/AKT/GSK3β axis.

a Western blotting was used to detect the key protein level of the PI3K/AKT/GSK3β signaling pathway in circIPP2A2 stable knockdown and Hornerin overexpression. b The activity of the PI3K/AKT/GSK3β signaling pathway was measured by western blotting after METTL1 downregulation. c IP assay was performed to assess the binding affinity of Hornerin with METTL1, PI3K, AKT, and GSK3β. d RIP assay was employed to measure the interaction between PI3K and the specific sequences of circIPP2A2. e Immunofluorescence using anti-Hornerin (red) and anti-PI3K (green) antibodies were conducted to evaluate the alteration in affinity between Hornerin and PI3K upon silencing of circIPP2A2. f IP assay was used to observe alterations in the interaction between PI3K and Hornerin within circIPP2A2 knockdown HCC cells. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.