Exosomal Transfer of MiR-500a-3p Confers Cisplatin Resistance and Stemness via Targeting FBXW7 in Gastric Cancer CURRENT

Background Chemoresistance has become a major obstacle for gastric cancer (GC) therapy in clinical practice. MiRNAs have been reported to play critical roles in the development of chemoresistance in various tumors, including GC. However, the role of MiR-500a-3p within exosomes in cisplatin-resistant GC cells remains largely unknown. Materials and methods Cell proliferation and exosome delivery assays were performed using CCK-8 and transwell assays, respectively. CD63, CD81, β-tubulin, FBXW7, GAPDH, CD133, CD44 and SOX2 were detected by western blot and immunofluorescence assays. The expression levels of miR-500a-3p and FBXW7 mRNA were measured by real-time qRT-PCR. The interaction between miR-500a-3p and FBXW7 was predicted by bioinformatics software and confirmed by the dual-luciferase reporter. The mechanism of exosomal miR-500a-3p for cisplatin resistance was investigated in vitro and in vivo experiments. Results MiR-500a-3p level was upregulated in cisplatin-resistant GC cells and its downregulation enhanced cisplatin sensitivity. Moreover, extracellular miR-500a-3p could be incorporated into exosomes and transmitted to sensitive cells, thus disseminating cisplatin resistance. Additionally, exosomal miR-500a-3p promoted cisplatin resistance via targeting FBXW7 in vitro and in vivo . Clinically, higher expression of miR-500a-3p in the plasma exosomes of GC patients is correlated with DDP resistance and thereby results in poor progression-free prognosis. Conclusion Our finding highlights the potential of exosomal miR-500a-3p as an alternative modality for the prediction and treatment of GC with chemoresistance, providing a novel avenue for the treatment of GC.


Background
Gastric cancer (GC) is a serious global public health problem that rank the sixth most common malignancy and the third leading cause of cancer-related deaths worldwide [1]. Due to most GC patients diagnosed in advanced or metastatic stages [2], chemotherapy becomes the main strategy to improve prognosis and mitigate adverse symptoms, such as gastric bleeding or obstruction [3]. In this regard, cisplatin(DDP) is still one of the most used drugs in first-line chemotherapy against advanced GC [4]. However, chemoresistance-whether intrinsic or acquired-remains an inevitable obstacle in most GC patients and represents the most important cause of recurrence and mortality in GC [5].
While in cancer research, a plethora of recent evidence indicates that exosomes participate in tumor microenvironment remodeling, angiogenesis, invasion, metastasis and chemoresistance through initiating or suppressing various signaling pathways in the recipient cells [15]. RNA cargo that protected by exosomes from digestion has garnered much attention from researchers, especially microRNAs(miRNAs). MiRNAs are a class of 18-22 nucleotides small single-stranded non-coding RNA molecules that promote mRNA cleavage and subsequent degradation by binding to the complementary 3′ untranslated region (UTR) of the mRNA and thereby regulate protein regulation [16]. Emerging evidence has demonstrated that tumor cell-secreted exosomal miRNAs play a crucial role in regulating tumor growth, metastasis, angiogenesis and chemoresistance [17][18][19][20].
However, the mechanisms of exosomeal miRNAs in DDP resistant GC are still waiting for exposure.
In this study, the effect of exosome-transmitted miRNAs on DDP resistance in GC cells was investigated. Moreover, we identify exosomal miR-500a-3p promote DDP resistance and CSCs An ExoQuick precipitation kit (System Biosciences, LLC, Palo Alto, CA) was used to extract and purify exosomes in accordance with the manufacturer's instruction. Briefly, the culture medium or plasma was harvested and centrifuged at 3,000 g for 15 min for removing cell debris. The obtained supernatant was then mixed with ExoQuick precipitation solution, followed by incubation at 4 °C for 30 min and centrifugation at 1,500 g for 30 min. After carefully removing the supernatant with a pipette, the exosome pellets participated in the bottom were centrifuged for another 5 min at 1,500 g to remove the extra liquid. Finally, the exosome pellets were resuspended in 100 µl phosphatebuffered saline (PBS).

Characterization of exosomes 5
The morphology of exosome was observed by transmission electron microscopy. Briefly, Exosomes in PBS were fixed using 1% glutaraldehyde and incubated at 4 °C. Then, 10 µl of the suspension was placed onto formvar/carboncoated copper grids, followed by dyeing with 3% aqueous phospphotungstic aid for 30 seconds. Subsequently, exosomes were observed with a transmission electron microscopy (TEM; Tecnai 12; Philips). Size distribution of exosomes was analyzed by NanoSight LM10 system which was equipped with a fast video capture and particle-tracking software (NanoSight, Amesbury, UK). Western blot analysis was performed to detect exosome markers CD63 and CD81.
Exosomes and miR-500a-3p internalization assays Transfected or exosomes-treated GC cells were fixed in 4% paraformaldehyde for 10 min, blocked with PBS buffer containing 5% bovine serum albumin. Then those cells incubated with antibodies at 4 °C overnight, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated secondary antibody and the nuclear counterstain diaminophenylindole (DAPI). After rinsing, the cells were analyzed using immunofluorescence microscopy.

Sphere formation assay
Transfected or exosomes-treated GC cells were plated in each well of ultra-low-attachment 24-well plates (Corning Life Sciences, Corning, NY, USA) at low density (500 cells per each well) with 0.8% methyl cellulose (Sigma, USA) supplemented with 20 µl/ml B27 supplement (Life Technologies), 20 ng/ml basic fibroblast growth factor (bFGF, Gibco), 10 ng/ml EGF (Gibco), LIF (Gibco), 1% Lglutamine (Gibco) and 1% penicillin-streptomycin sulfate (Thermo Fisher Scientific) for 2 weeks. The number of sphere in each well ≥ 50 µm in diameter were counted under a microscope. Sphere formation rate for each well was the ratio of colony number to total cell number per well.

Western blot assay
Proteins were extracted with a lysis buffer and then quantified by a bicinchoninic acid protein assay.
Equivalent amounts of cell lysates were separated using SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Roche Applied Sciences). Membranes were immunoblotted overnight at 4 °C with antibodies, followed by the appropriate second antibodies(supplement table 1).
The bands were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).
Image density of the immunoblotting was determined by Gel densitometry (Bio-Rad).
RNA extraction, and real-time qRT-PCR Total RNA for cultured cells and exosomes were extracted with using Trizol Reagent (Takara Bio, Inc., Shiga, Japan). The mRNA expressions were detected by the PrimeScript RT Reagent Kit and SYBR Premix Ex Taq (Takara Bio, Inc., Shiga, Japan). GAPDH was used as control. All the primers designed for qPCR were listed in supplemental table 1. All-in-One microRNA qRT-PCR Detection Kits (GeneCopoeia, Inc., Maryland, USA) were used to detect miRNA expression and U6 used as a control.
Every experiment was repeated 3 times according to the manufacturer's protocol. Final data were 7 analyzed with the 2-ΔΔCt method.

Statistics
All in vitro experiments were repeated at least in triplicate. The data was represented as either a scatter plots or bar graphs with means ± standard error deviation of the mean (SEM). The statistical analysis was performed using SPSS software (version 13.0, NY, USA). Statistical significance between two groups was determined using a two-tailed Student's t-test. To compare multiple groups, one-way analysis of variance (ANOVA) followed by a Bonferroni-Dunn test was performed. The GC patients were divided into high expression group and low expression group according to the median ofmiR-500a-3p expression and Kaplan-Meier survival analysis was implemented to compare GC patient progression-free surviva by log-rank test. The receiver operating characteristic (ROC) curve was applied to determine the area under the curve (AUC) values for exosomal miR-500a-3p in plasma by the GraphPad Prism Software (GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered statistically significant.

Result DDP resistant GC cells exhibited higher tumorigenesis and CSCs properties
To explore the underlying regulatory mechanism of GC DDP resistance, DDP-resistant cell lines, Accumulating evidences demonstrate that CSCs play important roles in chemoresistance of many human tumors. In our established DDP resistant GC cells lines, higher proportion of CSCs markers CD133+, CD44 + and SOX + were observed in MGC803/DDP and MKN45/DDP cells (Fig. 1G).
Consistently, MGC803/DDP and MKN45/DDP cells could formed larger spheres compared to those sensitive cells (Fig. 1H). These data suggested that DDP resistant GC cells have been successfully constructed and those DDP resistant GC cells exhibited higher tumorigenesis and CSCs properties.
MGC803/DDP-derived exosomes conferred DDP resistance and promote CSCs properties in recipient MGC803 cells Recent studies revealed that exosomes secreted by cancer cells were implicated in chemotherapy resistance [19,20]. We speculated that exosomes from DDP resistant GC cells might exert their effects on recipient cells. In order to verify this conjecture, we isolated exosomes from the conditioned medium(CM) of MGC803 and MGC803/DDP cells. Transmission electron microscopy(TEM) revealed a cup-shaped vesicles with bilayered membranes and the Nanosight particle tracking analysis (NTA) further identified that the predominant size of the vesicles was 100 nm( Fig. 2A), that are typical exosomes. Moreover, MGC803/DDP cells secreted significantly more exosomes than MGC803 cells (Fig. 1B). By western blot analysis, the exosomal markers CD63, CD81 were detected in the exosomes, whereas β-tubulin was enriched in the whole cell lysates (Fig. 2C). After that, PHK-67 labled MGC803 and MGC803/DDP exosomes(green fluorescence) were cocultured with MGC803 CM.
As expected, green fluorescence signals were observed in exosomes treated MGC803 cells while no signal in PBS treated cells (Fig. 2D). The uptake efficiency of exosomes by MGC803 cells escalated in a time-dependent manner and more than 80% cells were positive for PKH67 fluorescence at  Fig. 3A and B, miR-500a-3p expression were significantly higher both in MGC803/DDP and their secreted exosomes by real time qRT-PCR. Thus, we assumed that miR-500a-3p from MGC803/DDP exosomes may confer DDP resistance to recipient MGC803 cells via exosome transfer. In the coincubation experiments, MGC803 intracellular miR-500a-3p levels were dramatically upregulated upon incubation with exosomes from MGC803/DDP with miR-500a-3p higher expression but not with exosomes from MGC803/DDP with miR-500a-3p knockdown by anti-miR-500a-3p transfection (Fig. 3C). While incubation with exosomes from MGC803 with miR-500a-3p overexpression by mimic-miR-500a-3p increased MGC803 intracellular miR-500a-3p level (Fig. 4D). Functionally, MGC803 cells became insensitive to DDP when incubated with exosomes with higher miR-500a-3p, whereas miR-500a-3p downregulation in exosomes abolished this effect (Fig. 3C, D). Moreover, the elevation of miR-500a-3p in recipient cells exhibited a time-dependent manner after incubation with MGC803/DDP exosomes (Fig. 3E). However, the level of pre-miR-500a-3p (precursor of miR-500a-3p) was not changed when incubating with MGC803/DDP exosomes (Fig. 3F), suggesting the miR-500a-3p elevation in recipient cells was not the result of miRNA endogenous synthesis but more likely a direct transfer by exosomes. Subsequently, we found miR-500a-3p expression in MGC803/DDP CM was little changed upon RNase A treatment but significantly decreased when treated with RNase A + Triton X-100 simultaneously (Fig. 3G), indicating that extracellular miR-500a-3p was mainly encased within the membrane instead of directly released. To visualize miR-500a-3p transfer, MGC803 and MGC803/DDP cells transiently transfected with PHK67-tagged miR-500a-3p were cocultured with MGC803 cells for 30 hours in a transwell system, as depicted in Fig. 3H. As a result, the green fluorescently labeled miR-500a-3p was observed in the lower chamber cells through confocal microscopy (Fig. 3H), further suggesting that miR-500a-3p could be transferred by exosomes. In abdominal tumorigenesis model, MGC803/DDP exosomes promoted tumor growth and dissemination under DDP therapy but downregulating miR-500a-3p in MGC803/DDP exosomes could abolished its tumor promoting effect (Fig. 3I-K). These findings revealed that functional exosomal miR-500a-3p from DDP resistant GC cells could be transferred to recipient ones, which subsequently became resistant to DDP in vivo and in vitro.

FBXW7 reversed the DDP resistance of exosomal miR-500a-3p by inhibiting CSCs properties
To determine the potential mechanisms underlying the role of FBXW7 in abrogating the DDP resistance by miR-500a-3p, we investigated the CSCs properties in GC. In sphere formation assay, MGC803/DDP exosomes induced more number and size of sphere formation were abrogated by FBXW7 overexpression (Fig. 5A and B). Additionally, upregulation of cell stemness markers CD133, CD44 and SOX2 by MGC803/DDP exosomes could be inhibited by reintroduction of FBXW7 (Fig. 5C-F).
These above data demonstrated that exosomal miR-500a-3p/ FBXW7 axis enhances DDP resistance in MGC803 cells by CSCs properties activation.
Plasma exosomal miR-500a-3p is related to DDP resistance in III stage GC patients Clinically, we investigated the plasma exosomal miR-500a-3p level in III stage GC patients who would receive DDP-based chemotherapy. As presented in Fig. 6A, plasma exosomal miR-500a-3p levels were significantly higher in DDP resistant patients than in that of sensitive patients. Moreover, Kaplan-Meier analysis revealed that high exosomal miR-500a-3p levels in III stage GC patient plasma were correlated with reduced overall survival (Fig. 6B). Importantly, receiver operating characteristic (ROC) curve analysis demonstrated that the ability to discriminate between the resistant and sensitive group with the plasma exosomal miR-500a-3p level was acceptably accurate(AUC = 0.843, Fig. 6C).
Above all, the plasma exosomal miR-500a-3p might be applied as the noninvasive biomarker for DDP resistance in GC.

Discussion
In spite of DDP-based chemotherapy is particularly effective in a large number of cancers, the emerge of DDP resistance has become a main obstacle for the treatment of cancer patients [21,22], especially in GC [23,24]. The platinum compound DDP can bind covalently to DNA, forming adducts that inhibit DNA replication subsequently causing transcription inhibition, cell-cycle arrest, DNA repair deficiency and apoptosis [25]. Unfortunately, the overall 5-year survival rate for GC patients who received DDP based chemotherapy after surgery remains dismal, while for late stage cases, DDP has shown little benefits due to dissatisfactory treatment efficiency, resulting in tumor progression and reduced prognosis [5]. Therefore, investigating the molecular mechanisms underlying DDP resistance may be of great significance for improving GC patient outcome [4,26,27]. In current study, the effects and mechanism of exosomal miR-500a-3p in DDP resistance were explored in GC cell. Our data suggested that miR-500a-3p abundance was elevated in DDP resistant GC cells and their secreted exosomes.
Moreover, we found that exosomal miR-500a-3p could contribute to DDP resistance in recipient GC cells by downregulating FBXW7 expression via enhancing stemness cells properties.  [19]. MiR-32-5p is delivered from HCC multidrug-resistant cells to sensitive cells via exosomes and activates the PI3K/Akt pathway to induce multidrug resistance [20]. Our results proved that exosomes from DDP resistant GC cells enhances recipient cells resistance to DDP by miR-500a-3p/FBXW7 pathway in vitro and in vivo.
MiR-500a-3p has been reported to be implicated in the chemotherapeutic resistance, invasion and migration via GSK-3β and LY6K in different types of cancers [40][41][42]. However, the biological roles of miR-500a-3p and the underlying molecular mechanisms responsible for GC have not been reported. In this study, we found that miR-500a-3p was elevated in exosomes from DDP resistant GC cells and clinical upregulation of miR-500a-3p in exosomes from III stage GC patients' plasma correlated with DDP-based chemoresistance and GC progression, which might be used as a noninvasive predictor of chemotherapy in GC Patients. Furthermore, FBXW7 was identified as the target of miR-500a-3p in GC.

Conclusion
In sammary, we provide evidence that DDP resistance GG cells can secret miR-500a-3p enriched exosomes to promote stemness and DDP resistance by targeting FBXW7 in GC cells (Fig. 6D).
Moreover, exosomal miR-500a-3p is up-regulated in the plasma of GC patients with DDP resistance, which thereby results in poor progression-free prognosis. We envision that blocking the function of exosomal miR-500a-3p could be potentially used as an alternative modality for the prediction and treatment of GC with chemoresistance.

Availability of Data and Materials
The data used to support findings of the study are available from the corresponding author upon request.

Ethics Approval and Consent to Participate
The study was approved by the medical ethics committee of Affiliated Xuzhou Hospital of Dongnan University Medical College.

Consent for Publication
We have received consent from individual patients who have participated in this study. The consent forms will be provided upon request.
Author contributors H.L. and C.Z. performed primer design and experiments. L.Z. and P.L. contributed to the animal experiments. H.L. contributed to RT-PCR and qRT-PCR. P.S. analyzed the data. L.Z. and P.L. wrote the paper. All authors read and approved the final manuscript.

Conflicts of interest
The authors declare they have no competing interests.      Plasma exosomal miR-500a-3p is related to DDP resistance in III stage GC patients. A Plasma exosomal miR-500a-3p level was detected in III stage GC patients responding or not