Introduction
Gastric cancer (GC) is one of the most common malignancies with high morbidity and mortality worldwide and ranks fourth in terms of cancer related deaths [
1]. The risk factors for GC include helicobacter pylori infection, age, obesity, excessive intake of salt and nitrates and diets with low fruit and vegetables [
2]. Moreover, mutations, differential gene expression, chromosomal aberrations, epigenetic alterations and abnormal intracellular signaling pathways could also induce the development and metastasis of GC [
3]. Although continual improvements on clinical diagnosis methods and therapeutic strategies have been made in the past decades, most GC patients are still diagnosed at the intermediate or terminal stage because of the lack of early specific symptoms [
2,
4]. Therefore, it is extremely urgent to explore the molecular mechanism of GC progression and find precise biomarkers which could be engineered as therapeutic targets.
Circular RNAs (circRNAs) are an emerging category of non-coding RNA with tissue-specific expression patterns in eukaryotic cells [
5]. Compared with traditional linear RNAs, they have high stability due to the covalently closed structure without a 5′cap or a 3′poly A tail [
5,
6]. Recently, increasing evidence indicates that circRNAs could participate in cancer development due to their indispensable roles in gene regulation [
7,
8], including GC [
9], intrahepatic cholangiocarcinoma (ICC) [
10], colorectal carcinoma (CRC) [
11], breast cancer [
12], glioblastoma [
13], and others. Numerous studies have explored the molecular mechanisms of circRNAs in cancer progression, showing that circRNAs could function as sponges of microRNAs (miRNAs) [
14,
15], interact with RNA-binding proteins [
16], mediate gene transcription [
17], and encode proteins [
18]. Especially, the regulatory network between circRNAs and miRNAs, which is known to function as competing endogenous RNA (ceRNA) [
19,
20], has been widely reported in many cancers.
Exosomes, extracellular membrane-derived vesicles with an approximate diameter of 30–200 nm, can be released into the extracellular environment when multivesicular bodies (MVB) fuse with the plasma membrane [
21,
22]. In the past decades, many studies have reported that exosomes play a critical role in diverse physiological processes through the intercellular exchange of biomolecules such as proteins, lipids, mRNAs, miRNAs and other non-coding RNAs [
23‐
27]. Meanwhile, because of several unique characteristics such as immune compatibility, low toxicity, nano-scale size, and stability in blood, exosomes can also be engineered to act as a nanocarrier for the delivery of target drugs and nucleic acids. [
24]. Notably, exosome-based RNA delivery has shown great promise in the field of cancer therapy. For example, MSCs-Exo can efficiently deliver inhibitors to reduce miR-142-3p levels in vitro and in vivo, which consequently results in a significant inhibitory effect on breast tumor development [
25]. Systematic administration of an anti-cancer drug 5-FU and miR-21 inhibitor oligonucleotide (miR-21i) loaded exosomes inhibits colon cancer cell proliferation and reverses drug resistance in 5-FU-resistant colon cancer cells [
26].
In our previous study, we have identified a new circRNA, circDIDO1, which is down-regulated in GC tissues. It inhibits GC progression via encoding a DIDO1-529aa protein as PARP1 inhibitor and promoting the RBX1-mediated ubiquitination and degradation of PRDX2 [
28]. Considering that the potent tumor suppressor role of circDIDO1, we want to explore the other mechanism responsible for its role and its therapeutic potential. In this study, we reported that circDIDO1 could act as a sponge of miR-1307-3p to regulate SOCS2 expression. Meanwhile, we demonstrated that systematic administration of circDIDO1 by exosome-mediated gene delivery achieved promising anti-cancer effects in vivo and in vitro.
Material and methods
Patients and tissue samples
A total of 17 paired tumor tissues and adjacent nontumor tissues (5 cm away from the tumor edge) were collected from the Department of General Surgery, the Affiliated People’s Hospital of Jiangsu University. All the tumor tissues were frozen in liquid nitrogen and stored at −80 °C for further use. Patients in this study had no previous radiotherapy or chemotherapy experience.
Cell culture and transfection
Human GC cell lines (MGC-803 and HGC-27) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and Saiku Biological Technology (Guangzhou, China).
All the cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (Bioind, Israel). The HEK-293 T cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and was cultured in high glucose-DMEM medium containing 10% FBS. Cells were incubated at 37 °C with 5% CO2 atmosphere. CircDIDO1 and SOCS2 plasmids, NC and miR-1307-3p mimics were transfected into GC cells through Lipofectamine 2000 (Invitrogen, USA). The experiment was conducted according to the manufacturer’s instructions.
Total RNA was extracted from tissues or cultured cells using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. The purity and concentration of total RNA were evaluated using NanoDrop One spectrophotometer (Thermo, USA). RNA was reversely transcribed to cDNA by using the HiScript 1st Strand cDNA Synthesis Kit (Thermo, USA). Subsequent detection of RNA levels was performed with UltraSYBR Mixture kit (Vazyme, China) through Real-time PCR Detection System (CFX96, Bio-Rad, USA). For miRNA expression assay, miRNA primers were synthesized by QIAGEN and total RNA was reversely transcribed using the miScript II Reverse Transcription Kit (QIAGEN, Germany). qRT-PCR amplification for miRNA was performed by using miScript SYBR Green PCR Kit (QIAGEN, Germany). U6 were used as the internal control genes. The relative RNA expression level was calculated using the 2−△△Ct method.
Isolation and characterization of exosomes
Exosomes were isolated from cell supernatants following our previous protocol [
29]. The protein concentration of the exosomes was determined by a BCA protein assay kit (Gibco, China). Size distribution of exosomes was identified through Nanoparticle tracking analysis (NTA) (Nanosight LM10, Particle Metrix, Germany). The morphology of isolated exosomes was observed by using transmission electron microscopy (Philips, Netherlands). The exosomal markers CD9, CD63, TSG101 and the negative control Calnexin were determined by western blot.
Preparation of RGD-engineered exosomes
293 T cells were transfected with vector and circDIDO1 for 24 h and then cultured in DMEM medium containing 10% exosome-free FBS. After 48 h, cell culture supernatant was collected and exosomes were purified as previously described [
29]. Exosomes from the vector (Exo-vector) and DIDO1 overexpression (Exo-circDIDO1) groups were incubated with 50 µg of DSPE-PEG-RGD (Ruixi Biotechnology, China) at 37 ℃ for 30 min to form RGD-Exo-vector and RGD-Exo-circDIDO1, respectively [
30].
RGD-engineered exosomes labeling and internalization
Diluted exosomes (1 mL) were fluorescently labeled using 5 μL of the membrane dye DiL (red, Invitrogen, USA) for 30 min at 37 °C. A total of 1 × 104 cells was cultured on cover glass in 12-well plates. When 60–70% confluence was reached, DiL-labeled exosomes were added and cells were incubated for 12 h. Then cells were washed with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. Finally, Hoechst 33342 (Sigma, USA) was used for nuclear staining and then intracellular uptake of DiL-labeled exosomes was obtained using a confocal microscope (Beckman Coulter, USA).
The transfected cells were seeded in 24-well plates (1 × 104/well) and counted every 24 h for 5 days. For colony formation assay, a total of 1 × 103 cells were seeded into 6-well plates and the medium was replaced every 2 days. After 8 days of incubation, the colonies were fixed with 4% paraformaldehyde and stained with crystal violet. The number of colonies was observed and counted with a microscope.
Transwell migration and matrigel invasion assays
The cells were harvested and resuspended in serum-free RPMI-1640 medium. Cells (2 × 104 for migration assay and 1 × 104 for invasion assay) in 200 μL of serum-free medium were plated into the upper chambers of a 24-well chamber with an 8.0 μm pore (Corning, USA). For invasion assay, the transwell upper chambers were pre-coated with diluted cold Matrigel (BD Biosciences, USA). The cells were incubated with 600 μL of medium containing 10% FBS in the lower chambers for 24 h (for the migration assay) or 48 h (for the invasion assay). After incubation, cells on the upper surface of the polycarbonate membrane were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 20 min. The cells were imaged and counted under a microscope.
RNA immunoprecipitation
RNA immunoprecipitation experiments were performed by the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA), following the manufacturer’s instructions. Briefly, the transfected cells were pelleted and resuspended in lysis buffer containing a protease inhibitor cocktail and RNase inhibitor. The lysates were immunoprecipitated with magnetic beads conjugated with anti-Ago2 antibody or anti-mouse IgG at 4 °C overnight. Then, the beads were washed by RIP wash buffer and incubated with proteinase K to remove proteins. Finally, the immunoprecipitated RNA was purified and used for qRT-PCR analysis.
Luciferase reporter assay
The wild-type (WT) or mutant (MUT) luciferase reporter vectors were constructed by Hanheng Biological Technology (Shanghai, China). GC cells were co-transfected with WT or MUT reporter vectors and miRNA mimics by using Lipofectamine 2000 (Invitrogen, USA). After 48 h incubation, cells were harvested, and the luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega, USA).
Cell apoptosis assay
Cell apoptosis was analyzed by using the Annexin V-Alexa Fluor 647/propidium iodide (PI) apoptosis detection kit (Fcmacs, China). After treatment with RGD-engineered exosomes for 24 h, the cells were detached and dissociated with collagenase. Cell pellets were resuspended in binding buffer and stained with Annexin V-Alexa Fluor 647 and PI for 15 min at room temperature. Cell apoptosis ratio was analyzed by CytoFLEX flow-cytometry (Beckman, USA).
In vivo animal studies
For the therapeutic study, 15 nude mice were inoculated subcutaneously with MGC-803 cells. After 2 weeks, the mice were randomly divided into 3 groups. PBS, RGD-Exo-vector and RGD-Exo-circDIDO1 were intravenously injected to mice via the tail vein every 4 days for 28 days (30 μg/μL in protein concentration; 200 μL per mouse). The protocol was approved by the Animal Use and Care Committee of Jiangsu University.
Statistical analysis
GraphPad Prism 7.0 (GraphPad Software, USA) were used for statistical analysis. All data were presented as mean ± standard deviation (SD). The significant difference between different groups was analyzed by Student’s t-test and one-way ANOVA test according to actual conditions. P < 0.05 was considered as statistically significant.
Discussion
Increasing evidence suggests that circRNAs widely participate in GC development. For instance, circ-RanGAP1 regulates VEGFA expression by targeting miR-877-3p to facilitate GC invasion and metastasis [
9]. CircLMO7 acts as a miR-30a-3p sponge to inhibit GC progression by regulating the WNT2/β-Catenin pathway [
31]. In our previous study, circDIDO1 was identified as a novel circRNA which was down-regulated in GC [
28], but its molecular mechanisms and potential application value in GC were not fully revealed. Our present research verified that circDIDO1 could function as a sponge of miR-1307-3p to induce the expression of SOCS2, thereby suppressing GC progression. Furthermore, we found that exosomes can be used as nanocarriers to deliver circDIDO1 to GC and show an efficient anti-tumor effect (Fig.
8).
Our study has revealed the importance of circDIDO1 in tumor development. Many studies suggest that circRNAs could function as miRNA sponges to modulate GC development [
9,
31]. Since the bioinformatic predictions showed that circDIDO1 has the potential to bind to Ago2 protein, we speculated that circDIDO1 might act as a ceRNA to compete for miRNA binding, thereby negatively regulating miRNA activity. Subsequently, the results confirmed that circDIDO1 inhibits GC progression by sponging miR-1307-3p. A few previous studies have shown that miR-1307-3p plays an important role in cancer progression. It has been proved that miR-1307-3p is upregulated in breast cancer tissues and significantly contributes to breast cancer development by targeting SMYD4 [
32]. Another study indicates that circPPP1CB inhibits human bladder cancer growth, metastasis, and EMT process by modulating the miR-1307-3p/SMG1 axis [
33]. However, the biological role of miR-1307-3p in GC has not been reported yet. Our findings suggest that up-regulation of miR-1307-3p in GC promotes tumor progression and circDIDO1 could reverse the effects of miR-1307-3p, which is consistent with other studies on miR-1307-3p [
32,
33].
SOCS2, which is a member of the suppressor of cytokine signaling (SOCS) family, can repress the cytokine-induced signaling transduction, thus inhibiting cancer progression [
34,
35]. In addition, SOCS2 has been investigated as the molecular target of diverse miRNAs, including miR-196a/miR-196b [
36], miR-194 [
37] and miR-767-5p [
38]. Herein, we validated that the expression level of SOCS2 was decreased in GC, and similar to circDIDO1, overexpression of SOCS2 suppressed GC cell proliferation, migration and invasion. More importantly, circDIDO1 could positively modulate the expression of SOCS2 in GC cells via acting as a sponge of miR-1307-3p. This ceRNA regulatory network might provide a new therapeutic target for GC treatment.
Targeting circRNAs has been suggested as a new approach for cancer therapy [
39‐
41]. For instance, cell-penetrating inhibitory peptides that block the interaction between oncogenic circRNAs and proteins have been used to inhibit the growth and metastasis of GC cells [
41]. Similarly, synthetic circRNA that mimics its natural counterpart has been produced to suppress GC cell proliferation by acting as miRNA sponge [
42]. Following this strategy, we further explored the possibility of utilizing circDIDO1 as a target for GC therapy. In recent years, exosomes have emerged as a new vehicle for drug delivery [
43]. Exosome-mediated deliveries of siRNA, miRNAs or anti-miRNA oligos, and drugs, have been widely tested in the treatment of various cancers. RGD modification further improves the targeting ability and therapeutic effect of exosome-mediated drug delivery [
44,
45]. Thus, we prepared RGD modified, circDIDO1 loaded exosomes by using active loading and passive modification strategies. Our findings revealed that systemic administration of RGD modified, circDIDO1 loaded exosomes could efficiently repress the tumorigenicity and aggressiveness of GC cells. More importantly, the engineered exosome-based delivery system could significantly up-regulate SOCS2 expression in treated GC cells and tissues. These results indicate that miR-1307-3p/SOCS2 axis plays an essential role in the anti-tumor effect of exosome-mediated delivery of circDIDO1. Finally, the safety of the engineered exosomes in mice after treatment was evaluated, and in vivo studies demonstrated that no significant cytotoxicity or systemic toxicity was induced by the engineered exosomes.
During the last decade, several clinical trials using engineered exosomes to activate immune response or deliver therapeutic nucleic acids have been conducted [
46]. For example, a phase-2 clinical trial verified the activation of NK cells after dendritic cell-derived exosome administration achieved immunotherapeutic effect in advanced non-small cell lung cancer patients without obvious toxicity [
47]. In addition, MSC-derived exosomes loaded with KrasG12D siRNA are now being utilized in a phase-1 clinical trial against pancreatic cancer (NCT 03608631) [
48]. Therefore, considering that RGD-modified, circDIDO1-loaded exosomes we constructed have shown significant anti-tumor effects without observable toxicity in vitro and in vivo, exosome-based circRNA delivery could be developed as a promising therapeutic strategy with high biosafety for GC.
Due to their stability, safety and homing characteristics, exosomes have been widely explored as delivery vehicles for a variety of cargos [
49]. The accumulation of exosomes in the liver after administration provides a venue to load them with antioxidants to reduce endogenous ROS generation [
50,
51]. Eftekhari et al
. have reported an enhanced antioxidative effect after nano-carrier encapsulation to achieve better hepatoprotective effect [
52]. Therefore, the application of exosomes to deliver antioxidants may reduce drug-induced hepatotoxicity caused by ROS generation and subsequent NF-κB activation [
53,
54]. In addition, identifying effective serum markers is important to improve the survival rate of GC patients. Ma et al
. demonstrate that circDIDO1 expression level in serum exosomes could be used as a promising indicator for liver failure [
55]. In addition, serum miR-1307-3p has been suggested as an effective diagnostic marker for early breast cancer [
56]. Considering the critical role of circDIDO1 and miR-1307-3p in GC progression, serum circDIDO1 and miR-1307-3p have great potential as measurable biomarkers for GC screening and early diagnosis. Furthermore, peritoneal metastasis mouse model will be established to further confirm the anti-tumor efficiency of RGD-Exo-circDIDO1 in GC metastasis.
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