Exosome‐transmitted miR‐769‐5p confers cisplatin resistance and progression in gastric cancer by targeting CASP9 and promoting the ubiquitination degradation of p53

Abstract Background Cisplatin resistance is the main cause of poor clinical prognosis in patients with gastric cancer (GC). Yet, the exact mechanism underlying cisplatin resistance remains unclear. Recent studies have suggested that exocrine miRNAs found in the tumor microenvironment participate in tumor metastasis and drug resistance. Methods Exosomes isolated from BGC823 and BGC823/DDP culture medium were characterized by transmission electron microscopy and differential ultracentrifugation, and miRNA expression profiles of BGC823 and BGC823/DDP cells derived exosomes were analyzed using miRNA microarray. In vivo and in vitro assays were used to identify roles of exosomal miR‐769‐5p and clarify the mechanism of exosomal miR‐769‐5p regulated the crosstalk between sensitive and resistant GC cells. Results In this study, we found that cisplatin‐resistant GC cells communicated with the tumor microenvironment by secreting microvesicles. MiR‐769‐5p was upregulated in GC tissues and enriched in the serum exosomes of cisplatin‐resistant patients. The biologically active miR‐769‐5p could be integrated into exosomes and delivered to sensitive cells, spreading cisplatin resistance. Underlying cellular and molecular mechanism was miR‐769‐5p targeting CASP9, thus inhibiting the downstream caspase pathway and promoting the degradation of the apoptosis‐related protein p53 through the ubiquitin‐proteasome pathway. Targeting miR‐769‐5p with its antagonist to treat cisplatin‐resistant GC cells can restore the cisplatin response, confirming that exosomal miR‐769‐5p can act as a key regulator of cisplatin resistance in GC. Conclusions These findings indicate that exosome‐transmitted miR‐769‐5p confers cisplatin resistance and progression in gastric cancer by targeting CASP9 and promoting the ubiquitination degradation of p53. These findings reveal exosomal miR‐769‐5p derived from drug‐resistant cells can be used as a potential therapeutic predictor of anti‐tumor chemotherapy to enhance the effect of anti‐cancer chemotherapy, which provides a new treatment option for GC.

20, Netherland), and their particle morphology and size were analyzed. The concentration and number of exosomes were detected by nanoparticle tracking analysis (NTA). Exosome protein markers were identified by Western blot assay and flow cytometry analysis (FACS Calibur, BD Biosciences, USA) .
Human serum exosomes were obtained with ExoQuick Exosome Precipitation Solution (SBI, CA, USA) following the user manual. Briefly, serum was collected and centrifuged at 3000 × g for 15 minutes. Then add the 63 ul ExoQuick Exosome Precipitation Solution to 250 ul supernatant and refrigerate the mixture 30 minutes serum at 4°C. After centrifugation at 1500 × g for 30 minutes, resuspend exosome pellet in 100 µl using sterile 1× PBS.

PKH26 Staining for Exosomes
The isolated exosomes were labeled with PKH26 Red Fluorescent Cell Linker Kits (Sigma). Exosomes were first resuspended in 100 μL Diluent C. A dye solution (4 × 10−6 M) was prepared by adding 0.4 μL PKH26 ethanolic dye solution to 100 μL Diluent C. The 100 μL exosome suspension was then mixed with the 100 μL dye solution by pipetting. After incubating the cell and dye suspension for 5 min with periodic mixing, the staining was stopped by adding 200 μL serum and incubating for 1 min. The stained exosomes were finally washed twice with 1× PBS, and they were resuspended in a fresh sterile conical polypropylene tube.

Lentiviral, plasmid, and microRNA mimics/inhibitors package and cell transfection
The lentivirus encoding miR-769-5p overexpression or knockdown and negative control (769, NC, anti-769, anti-NC) were designed and produced by GENECHEM (Shanghai, China). The lentivirus were added to BGC823 BGC823/DDP, SGC7901 and SGC7901/DDP cells respectively and stable cell lines were obtained by selection with puromycin (Sigma-Aldrich, MO, USA). The infection efficiency was confirmed by fluorescence microscopy and real-time quantitative RT-PCR (qRT-PCR).

RNA extraction and quantitative RT-PCR
Total cellular and exosomal RNA was extracted from exosomes, co-cultured cells or GC cells, and frozen xenograft tumor tissues using TRIzol reagent (Invitrogen, CA, USA). Isolated RNA was used for the reverse transcription reaction with HiScript Q RT SuperMix for qPCR (Vazyme, Jiangsu, China). Quantitative RT-PCR was carried out with SYBR Green PCR Master Mix (Vazyme) using an ABI Prism 7900 Sequence detection system (Applied Biosystems, Canada). The relative expression of miR-769 was normalized to U6 levels, and CASP9, RNF20, p53 mRNA expression were normalized to GAPDH by qPCR using Power SYBR Green (Takara, Dalian, China). Data were calculated by the2 (−ΔΔCT) method. The related primers are synthesized by Ribobio (Guangzhou, China) and listed in Additional file 2: Table   S2.
The cells were washed and lysed with the passive lysis buffer from the Dual-Luciferase Reporter Assay System (Promega Corp). About 24 h later, a Dual-Luciferase Reporter Assay kit (Promega, USA) was used to measure the luciferase and renilla activity of these samples according to the manufacturer's instructions. Relative luciferase activity was first normalized with Renilla luciferase activity and then compared with those of the respective control. Wild-type and mutated CASP9 or RNF20 3′ UTRs were synthesized and inserted into the p-MIR-REPORT plasmid by Genechem, Shanghai, China.

Colony formation assay
GC cells (500 cells/well in six-well) were performed to detect the proliferation capacity. After incubation at 37°C, 5% CO2 for two weeks, the plates were washed with PBS, fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, washed three times with water, and analyzed. The assay was repeated three times in duplicate, and the numbers of colony formation counted.

Cell viability assay
Cells (1 × 104/well) were seeded in 96-well plates and treated with cisplatin from 0.2 to 6.4 μg/ml for 24 h. A CCK-8 assay was performed to detect cells viability using a Cell Counting Kit 8 (Dojindo, Japan) and a OD450 nm (Synergy4; BioTek, Winooski, VT, USA). Based on protocols of CCK-8 kits cells were seeded, cultured for 24 h, and further cultured in 100 μL medium with 10 μL CCK-8 reagent. Absorbance at 450 nm was determined using a Multiscan FC plate reader (Thermo Fisher).

Cell Migration Assay
The migratory capacity of GCs was tested by using a Transwell Boyden Chamber (6.5 mm, Costar) with polycarbonate membranes (8-μm pore size) on the bottom of the upper compartment. A total of 2 × 104 cells was suspended in serum-free media.
Meanwhile, the lower chambers were loaded with 0.5 mL RPMI1640 containing 5% FBS, and the plates containing Transwell inserts were incubated. After incubation at 37°C, 5% CO2 for 12 h, the upper chamber was cleaned with a cotton swab, and the lower chamber was washed with PBS. The cells that penetrated through the membrane were fixed with 90% ethanol for 15 min at room temperature, stained with 0.1% crystal violet solution, washed three times with water, and imaged by Inversion Microscope (Zeiss, Germany). The assay was repeated three times in duplicate. We obtained images of migrated cells by using a photomicroscope, and we quantified cell migration by blind counting with five fields per chamber.

Apoptosis assay
The  The slides were visualized for immunofluorescence with a laser scanning microscope (Zeiss, Germany).

Western blot, immunohistochemistry (IHC), and immunoprecipitation (IP) assay
Cell or tissue samples were lysed by RIPA buffer mixed with protease and phosphatase inhibitor cocktails. Serum proteins were extracted with Serum Protein Extraction Kit (Qcheng Bio, China). The proteins were then separated by 10% SDS-PAGE and transferred onto PVDF membranes. Western blot assays were performed according to previously reported data [1] The immune-complexes were detected with ECL Western Blotting Substrate (Thermo Fisher) and visualized with BIO-RAD (BIO-RAD Gel Doc XR+, USA).
Immunohistochemistry and immunoprecipitation were done as previously reported [2].

Statistical analysis
Statistical data were expressed as mean ± SD. One-way analysis of variance was used for three groups and more than three groups. All of the statistical analyses were assessed by software SPSS version 13.0 (SPSS, Chicago, IL, USA)and GraphPad Prism (GraphPad Software, Inc., SanDiego, CA, USA) software, comparisons among groups were done by the independent sample two-sided Student t-test. The ANOVA was performed to evaluate the statistical differences among groups. P-value of 0.05 or less was considered as statistical significance.