CircRHBDD1 promotes immune escape via IGF2BP2/PD-L1 signaling and acts as a nanotherapeutic target in gastric cancer

Abstract

Background: Circular RNAs (circRNAs) have been implicated in the development and progression of gastric cancer (GC). However, it remains unclear whether dysregulated circRNA affects immune escape and the efficacy of immunotherapy in GC. Our aim is to investigate the molecular mechanism of circRNA affecting GC immunotherapy and identify effective molecular therapeutic targets.

Methods: The differential expression profile of circRNAs was established through circRNA sequencing, comparing three paired GC tissues with their adjacent non-cancerous gastric tissues. The expression level of circRHBDD1 in GC tissues was then assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The biological characteristics of circRHBDD1 were verified through a series of experiments, including agarose gel electrophoresis assays, RNase R treatment, and actinomycin D experiments. The prognostic value of circRHBDD1 in GC was evaluated by conducting both univariate and multivariate survival analyses. Furthermore, loss- and gain-of-function approaches were utilized to investigate the impact of circRHBDD1 on GC immune escape. RNA-sequencing, immunoprecipitation, flow cytometry, and methylated RNA immunoprecipitation (meRIP) analysis were performed to elucidate the underlying molecular mechanisms.

Results: We discovered that circRHBDD1 exhibited remarkably high expression levels in GC tissues and cell lines. Notably, the high expression of circRHBDD1 was significantly correlated with poor overall survival and disease-free survival among GC patients. Both in vitro and in vivo experiments revealed that circRHBDD1 upregulated the expression of PD-L1 and impeded the infiltration of CD8⁺ T cells. Further, we found that circRHBDD1 binds to IGF2BP2, disrupting the interaction between E3 ligase TRIM25 and IGF2BP2, and ultimately inhibiting IGF2BP2 ubiquitination and degradation. Intriguingly, IGF2BP2 enhances PD-L1 mRNA stability through m⁶A modification. Additionally, we developed Poly (lactide-co-glycolic acid) (PLGA)-Polyethylene glycol (PEG)-based nanoparticles loaded with circRHBDD1 siRNA. In vivo experiments validated that the combination of PLGA-PEG(si-circRHBDD1) and anti-PD-1 offers a safe and efficacious nano-drug regimen for cancer immunotherapy.

Conclusion: Our results demonstrated that circRHBDD1 promoted GC immune escape by upregulating the expression of PD-L1 and reprogramming T cell-mediated immune response. Inhibition of circRHBDD1 expression could potentially enhance the response of GC patients to immunotherapy, thus improving treatment outcomes. Additionally, the development of a nanodrug delivery system provides a feasible approach for future clinical applications.

Keywords: CircRNAs; Gastric cancer; Immune escape; N6-methyladenosine; Nanotherapy; PD-L1; Ubiquitination.

Fig. 1

CircRHBDD1 was a circRNA highly expressed in GC and predicted poor prognosis. A Volcano map of the discrepantly expressive circRNAs in GC. B qRT-PCR was utilized to test CircRHBDD1 expression level in 45 pairs of GC tissues and adjacent tissues. C FISH was utilized to examined circRHBDD1 expression level in GC and adjacent tissues. Scale bar = 50 μm. D CircRHBDD1 expression level in four GC cell strains and normal gastric mucosa cell strains was measured via qRT-PCR. E Sanger sequencing showed the reverse splicing locus of circRHBDD1. F Gelose Gel Electropho to verify the stability of circRHBDD1. G RNase R assay was used to analyze the consistency of circRHBDD1 and RHBDD1 in HGC-27 and MKN-45 cells. H After the HGC-27 and MKN-45 cells were managed with actinomycin D at specific time points, qRT-PCR was utilized to test the mRNA expression levels of circRHBDD1 and RHBDD. I The localization of circRHBDD1 in HGC-27 and MKN-45 cells was tested by FISH. Scale bar = 10 μm. J The localization of circRHBDD1 was examined by nuclear and cytoplasmic fractions assay. K The pertinence of expression levels between circRHBDD1 and OS (p ˂ 0.0001). L The pertinence of expression levels between circRHBDD1 and DFS (p ˂ 0.0001). M Multivariate analysis revealed that the independent risk factors for OS. N Multivariate analysis showed that the independent risk factors affecting DFS. Data are presented with the means ± SD of three independent experiments. **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

CircRHBDD1 upregulated the expression of PD-L1 in GC cells. A KEGG analysis indicated enriched pathways. B, C qRT-PCR and Western blotting were carried out to detected the expression levels of PD-L1 mRNA and protein after circRHBDD1 knockdown in HGC-27 and MKN-45 cells. D Flow cytometry was utilized to examined the surface PD-L1 expression of the cytomembrane after circRHBDD1 knockdown in HGC-27 and MKN-45 cells. E Correlation analysis between circRHBDD1 and the expression level of PD-L1 in GC tissues (n = 30). F Immunofluorescence assays to detect the combination intensity between PD-L1 and PD-1 on HGC-27 and MKN-45 cells conducted by shRNA for circRHBDD1. Scale bar = 50 μm. Data are appeared with the means ± SD of three independent experiments. ***p < 0.001; ****p < 0.0001

Fig. 3

Knockdown of circRHBDD1 accelerated the infiltration and killing ability of CD8⁺ T cells. A GO analysis showed the enriched pathways. B The consequences of T cell-induced tumor cell cytotoxicity assays in circRHBDD1-silenced HGC-27 and MKN-45 cells. C The content and percentum of CD4⁺ T cells and CD8⁺ T cells in CD45⁺ cells were examined by flow cytometry after sh-circRHBDD1-1 and sh-circRHBDD1-2 infection. D The expressions of IFN-γ, TNF-α and granzyme B were measured by flow cytometry after circRHBDD1 knockdown. E The expressions of PD-1, TIM-3 and LAG-3 in CD8⁺ T cells were examined by flow cytometry in MKN-45 cells with circRHBDD1 knockdown. Data are presented with the means ± SD of three independent experiments. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant

Fig. 4

CircRHBDD1 interacted with IGF2BP2 and inhibited its ubiquitination and degradation. A The enrichment status of circRHBDD1 in the AGO2 IP was detected using qRT-PCR through the RIP assay. B Venn diagram showing the intersection of starbase and catRAPID database prediction and mass spectrometry analysis. C IGF2BP2 protein bound to circRHBDD1, as determined by mass spectrometry. D Detection of proteins by Western blotting after RNA pull-down. E RIP detections indicated the combination between IGF2BP2 and circRHBDD1. F FISH of circRHBDD1 and immunofluorescence detection of IGF2BP2 in HGC-27 and MKN-45 cells. Scale bar = 10 μm. G qRT-PCR was utilized to detect the mRNA expression level of IGF2BP2 after knocking down or overexpressing circRHBDD1. H Western blotting was utilized to analyze the expression level of IGF2BP2 protein after knocking down or overexpressing circRHBDD1. I In circRHBDD1-silenced MKN-45 and MKN-28 cells, Western blotting revealed the protein levels of IGF2BP2 under treatment with MG-132 or chloroquine. J After treated with MG-132, western blotting was employed to assess the role of circRHBDD1 on the ubiquitination level of IGF2BP2 in MKN-45 and MKN-28 cells. Data are presented with the means ± SD of three independent experiments. ***p < 0.001; ns not significant

Fig. 5

CircRHBDD1 inhibited the ubiquitination of IGF2BP2 by impeding the interaction of E3 ligase TRIM25 and IGF2BP2. A Prediction of the E3 ligases of IGF2BP2. B E3 ligase TRIM25 affecting the ubiquitination of IGF2BP2 was determined by mass spectrometry. C Immunofluorescence indicated that IGF2BP2 and TRIM25 were co-located in MKN-45 and MKN-28 cells. Scale bar = 50 μm. D Co-IP experiment revealed that IGF2BP2 interacted with TRIM25. E After MG-132 treatment, western blotting was used to detect the impact of TRIM25 on the ubiquitination level of IGF2BP2 in MKN-45 and MKN-28 cells. F Co-IP experiment showed the combination between IGF2BP2 and TRIM25 in circRHBDD1-silencing MKN-45 cells. G The interaction between IGF2BP2 and TRIM25 in circRHBDD1-overexpressing MKN-28 cells was detected by Co-IP experiment

Fig. 6

IGF2BP2 enhanced the stability of PD-L1 mRNA via m⁶A modification in GC. A qRT-PCR was utilized to test the knockdown level of IGF2BP2 in HGC-27 and MKN-45 cells. B Western blotting was utilized to test the knockdown level of IGF2BP2 in HGC-27 and MKN-45 cells and the effects of IGF2BP2 knockdown on the protein expression level of PD-L1. C, D The expression level of PD-L1 was tested by flow cytometry. E qRT-PCR was utilized to determine the effects of knocking down IGF2BP2 on the expression of PD-L1 m⁶A in HGC-27 and MKN-45 cells. F In HGC-27 and MKN-45 cells, IGF2BP2 was knocked down at indicated time points following treatment with actinomycin D. qRT-PCR was utilized to test the expression of PD-L1. G Pertinence assessment between IGF2BP2 and PD-L1 expression in GC tissues (n = 30). H FISH and immunofluorescence images revealing the expression levels of circRHBDD1, IGF2BP2 and PD-L1 in circRHBDD1-sliencing GC cells. Scale bar = 50 μm. Data are presented with the means ± SD of three independent experiments. **p < 0.01; ***p < 0.001

Fig. 7

CircRHBDD1 promoted tumor growth in C57BL/6 mouse models. A Injecting gastric cancer cells with knocked down or overexpressed circRHBDD1 into C57BL/6 mice, and the transplanted tumor was photographed and recorded 21 days later. B The volume of the transplanted tumors. C The weight of the transplanted tumors. D FISH and immunofluorescence assays of circRHBDD1, IGF2BP2 and PD-L1. Scale bar = 50 μm. E Immunofluorescence analysis of CD8⁺ T cells. Scale bar = 50 μm. F Flow cytometry assays of the percentage and number of CD8⁺ T cells. G Presence of IFN-γ, TNF-α and granzyme B. H Presence of PD-1, TIM-3 and LAG-3 on the cover of CD8⁺ T cells. Data are presented with the means ± SD of three independent experiments. **p < 0.01; ***p < 0.001

Fig. 8

Characterization and treatment effect of PLGA-PEG(si-circRHBDD1) NPs in C57BL/6 GC models. A Typical TEM picture of PLGA-PEG(si-circRHBDD1) NPs. Scale bar = 200 nm. B Incubate MKN-28 cells with Coumarin-6 labeled NPs to evaluate the cell uptake of MKN-28 cells by NPs. Scale bar = 50 μm. C Cellular internalization and lysosomal escape of Coumarin-6 NPs observed by CLSM. Scale bar = 10 nm. D, E Dynamic fluorescence imaging in vivo following intravenous administration of dissociative DiR or DiR-NPs. Red arrow indicates the location of heteroplastic tumor. F, G The fluorescence images of tumor and isolated organs of dissociative DiR or DiR-NPs. H Validating the therapeutic effects of PLGA-PEG using a C57BL/6 mouse model. (si-circRHBDD1) combined with anti-PD-1. I Statistical analysis of tumor volume. J Analysis of tumor weight statistics. K HE staining of representative sections from the heart, liver, spleen, lungs, and kidneys. Scale bar = 100 μm. Data are presented with the means ± SD of three independent experiments. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant

Fig. 9

A, B Schematic diagram of circRHBDD1 in promoting immune escape through the IGF2BP2/PD-L1 axis and serving as a nanotherapeutic target in GC