Engineered exosome-mediated delivery of circDIDO1 inhibits gastric cancer progression via regulation of MiR-1307-3p/SOCS2 Axis

Abstract

Background: Our previous study has identified a novel circRNA (circDIDO1) that is down-regulated in gastric cancer (GC) and significantly inhibits GC progression. The purpose of this study is to identify the molecular mechanism for circDIDO1 and to evaluate the therapeutic effect of circDIDO1 in GC.

Methods: By combining bioinformatic analysis with RNA sequencing data, we predicted the potential target of circDIDO1 and further validated the regulatory mechanisms for its tumor suppressor function in GC. RIP assay, luciferase reporter assay and in vitro cell function assays were performed to analyze circDIDO1-regulated downstream target genes. For the therapeutic study, circDIDO1-loaded, RGD-modified exosomes (RGD-Exo-circDIDO1) were constructed and its anti-tumor efficacy and biological safety were evaluated in vitro and in vivo.

Results: CircDIDO1 inhibited GC progression by regulating the expression of the signal transducer inhibitor SOSC2 through sponging miR-1307-3p. Overexpression of circDIDO1 or SOSC2 antagonized the oncogenic role of miR-1307-3p. RGD-Exo-circDIDO1 could efficiently deliver circDIDO1 to increase SOCS2 expression in GC cells. Compared with PBS and RGD-Exo-vector treatment, RGD-Exo-circDIDO1 treatment significantly inhibited the proliferation, migration and invasion of GC cells while promoted cell apoptosis. The therapeutic efficacy of RGD-Exo-circDIDO1 was further confirmed in a mouse xenograft tumor model. In addition, major tissues including the heart, liver, spleen, lungs and kidneys showed no obvious histopathological abnormalities or lesions in the RGD-Exo-circDIDO1 treated group.

Conclusion: Our findings revealed that circDIDO1 suppressed the progression of GC via modulating the miR-1307-3p/SOSC2 axis. Systemic administration of RGD modified, circDIDO1 loaded exosomes repressed the tumorigenicity and aggressiveness of GC both in vitro and in vivo, suggesting that RGD-Exo-circDIDO1 could be used as a feasible nanomedicine for GC therapy.

Keywords: Cancer therapy; CirDIDO1; Exosomes; Gastric cancer; MiR-1307-3p; SOCS2.

Fig. 1

MiR-1307-3p plays oncogenic roles in GC progression. A Luciferase reporter assays for screening of miRNAs that potentially regulate circDIDO1. B Prediction of binding sites in circDIDO1 for miR-1307-3p. C HGC-27 cells were co-transfected with miR-1307-3p and wild-type (WT) or mutant (MUT) luciferase reporter vector for circDIDO1. The luciferase activity was determined as indicated. D RNA immunoprecipitation (RIP) assay for the binding of circDIDO1 to Ago2 protein. E Analysis of miR-1307-3p expression in tumor tissues and adjacent normal tissues from GC patients by using TCGA data. F qRT-PCR analyses of circDIDO1 expression in control and miR-1307-3p overexpressing GC cells. G–J Cell growth curve, colony formation, transwell migration, and matrigel invasion assays for control and miR-1307-3p overexpressing GC cells. Data are shown as mean ± SD (n = 3, **P < 0.01, *P < 0.05). Scale bars = 100 μm

Fig. 2

CircDIDO1 acts as a ceRNA for miR-1307-3p. A–D Cell growth curve, colony formation, transwell migration, and matrigel invasion assays for control, miR-1307-3p, and miR-1307-3p+circDIDO1 transfected GC cells. Data are shown as mean ± SD (n = 3, **P < 0.01, *P < 0.05). Scale bars = 100 μm

Fig. 3

SOCS2 is a target of miR-1307-3p. A ENCORI Pan-Cancer Analysis Platform was used to analyze the correlation of miR-1307-3p and SOCS2 in 372 cases of STAD patients. B qRT-PCR analyses of the expression of SOCS2 and circDIDO1 in tumor tissues of 17 GC patients. C Luciferase reporter assay in GC cells co-transfected with wide-type (WT) or mutant (MUT) SOCS2 plasmid together with miR-1307-3p mimic or NC. D The relative level of SOCS2 expression after transfection with circDIDO1 in GC cells. E The relative level of SOCS2 expression in control and miR-1307-3p overexpressing GC cells. F–I Cell growth curve, colony formation, transwell migration, and matrigel invasion assays for control and SOCS2 overexpressing GC cells. Data are shown as mean ± SD (n = 3, **P < 0.01, *P < 0.05). Scale bars = 100 μm

Fig. 4

SOCS2 reverses the oncogenic roles of miR-1307-3p in GC cells. A–D Cell growth curve, colony formation, transwell migration, and matrigel invasion assays for control, miR-1307-3p, and miR-1307-3p+SOCS2 transfected GC cells. Data are shown as mean ± SD (n = 3, **P < 0.01, *P < 0.05). Scale bars = 100 μm

Fig. 5

Characterization and the targeting ability of RGD-Exo-circDIDO1. A The size distribution of RGD-Exo-circDIDO1 was detected by nanosight tracking analysis. B The morphology of exosomes was observed using a transmission electron microscope (scale bar = 40 nm). C The exosomal markers were analyzed by western blotting. D qRT-PCR analyses of circDIDO1 expression in RGD-Exo-circDIDO1. E The internalization of DiI-labeled RGD-Exo-circDIDO1 in GC cells was observed by confocal fluorescence microscope (scale bar = 20 μm). F qRT-PCR analyses of circDIDO1 expression in RGD-Exo-circDIDO1 treated GC cells. Data are shown as mean ± SD (n = 3, **P < 0.01, *P < 0.05)

Fig. 6

RGD-Exo-circDIDO1 treatment inhibits GC cell growth while promotes apoptosis. A–E Flow cytometry analysis of cell apoptosis (A), cell growth curve (B), colony formation (C), transwell migration (D) and matrigel invasion assays (E) were performed for PBS, RGD-Exo, and RGD-Exo-circDIDO1 treated GC cells. Data are shown as mean ± SD (n = 3, **P < 0.01, *P < 0.05). Scale bars = 100 μm

Fig. 7

RGD-Exo-circDIDO1 efficiently inhibits GC growth in mice. A PBS, RGD-Exo, and RGD-Exo-circDIDO1 were intravenously injected into nude mice bearing subcutaneous xenograft tumors. At 28 days after treatment, the mice were sacrificed and the volume and weight of tumors were recorded. Data are shown as mean ± SD (n = 5 for each group, **P < 0.01). B HE, Ki-67 and TUNEL staining of xenograft tumor tissues treated with PBS, RGD-Exo, and RGD-Exo-circDIDO1. Scale bars = 100 μm. C qRT-PCR assays were used to examine miR-1307-3p and SOCS2 expression in GC cells and tissues treated with PBS, RGD-Exo, and RGD-Exo-circDIDO1. Data are shown as mean ± SD (n = 3, **P < 0.01, *P < 0.05). D Western blotting analyses of treated GC cells and tumor tissues. E Histopathological analyses of heart, liver, spleen, lung and kidney sections stained with HE. Scale bars = 60 μm. F Blood biochemistry for liver function markers: ALT, AST and ALP and kidney function markers: BUN and CRE. Data are shown as mean ± SD (n = 5 for each group)

Fig. 8

Schematic diagram shows that RGD-Exo-circDIDO1 inhibits GC progression through miR-1307-3p/SOSC2 axis. CircDIDO1 is delivered to GC cells by RGD-modified engineered exosomes. CircDIDO1 sponges miR-1307-3p to upregulate SOCS2 expression, thus inhibiting GC progression