Hypoxic BMSC-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT
Metastasis is the main cause of lung cancer mortality. Bone marrow-derived mesenchymal stem cells (BMSCs) are a component of the cancer microenvironment and contribute to cancer progression. Intratumoral hypoxia affects both cancer and stromal cells. Exosomes are recognized as mediators of intercellular communication. Here, we aim to further elucidate the communication between BMSC-derived exosomes and cancer cells in the hypoxic niche.
Exosomal miRNA profiling was performed using a microRNA array. Lung cancer cells and an in vivo mouse syngeneic tumor model were used to evaluate the effects of select exosomal microRNAs. Hypoxic BMSC-derived plasma exosomal miRNAs were assessed for their capacity to discriminate between cancer patients and non-cancerous controls and between cancer patients with or without metastasis.
We demonstrate that exosomes derived from hypoxic BMSCs are taken by neighboring cancer cells and promote cancer cell invasion and EMT. Exosome-mediated transfer of select microRNAs, including miR-193a-3p, miR-210-3p and miR-5100, from BMSCs to epithelial cancer cells activates STAT3 signaling and increases the expression of mesenchymal related molecules. The diagnostic accuracy of individual microRNA showed that plasma exosomal miR-193a-3p can discriminate cancer patients from non-cancerous controls. A panel of these three plasma exosomal microRNAs showed a better diagnostic accuracy to discriminate lung cancer patients with or without metastasis than individual exosomal microRNA.
Exosome-mediated transfer of miR-193a-3p, miR-210-3p and miR-5100, could promote invasion of lung cancer cells by activating STAT3 signalling-induced EMT. These exosomal miRNAs may be promising noninvasive biomarkers for cancer progression.
KeywordsBMSCs, exosomal miRNA Metastasis EMT STAT3
Bone marrow-derived mesenchymal stem cells
Dulbecco’s Modified Eagle’s medium
Epithelial mesenchymal transition
Fetal bovine serum
Human bone marrow-derived mesenchymal stem cells
Hypoxia inducible factor
Heterogeneous nuclear ribonucleoproteins
Lewis lung cancinoma cells
Minimum essential medium
Non-small cell lung cancer
Nanoparticle Tracking Analysis
Oropharyngeal squamous cell carcinoma
Phosphate Buffered Saline
Protein inhibitor of activated stat3
RNA binding proteins
Renal cell carcinoma
Receiver operating characteristic
Suppressor of cytokine signaling
Signal transducers and activators of transcription 3
Transmission electron microscopy
Lung cancer is the leading cause of cancer-related mortality. Exosomes are extracellular membrane vesicles that contain proteins and RNAs. The molecular composition of exosomes is stable and tissue-specific, and can reflect the conditional state of their tissue of origin . mRNAs and microRNAs (miRNAs) have been identified in exosomes . miRNAs are 19-22 nt non-coding RNAs. Circulating miRNAs are protected from degradation by sequestration in exosomes . Increased levels of specific miRNAs assembled on the exosomes have been associated with cancer progression [4, 5, 6]. Exosomes are the major mediators of cell–cell communication because they can be captured by neighboring cells . Exosome-mediated transfer of particular miRNAs from stromal cells to epithelial cancer cells contributes to cancer progression . Exosomes are excreted by many cells and can be detected in bodily fluids including blood, urine and bronchoalveolar secretions, which are considered to ideal source of biomarkers [9, 10, 11]. There is significant interest in roles of exosomal miRNAs in the development of cancer, especially in the non-invasive detection of cancer progression.
Tumor cells and microenvironmental cells are subjected to a range of stressors such as hypoxia, metabolic stress, starvation, oxidative stress . Hypoxia is a feature of solid tumours .The rapid growth of cancer cells exceeds oxygen delivery, producing local hypoxia. Most solid tumours have regions permanently or transiently subjected to hypoxia .Hypoxia is characterized by an oxygen tension below physiological normoxia. Hypoxia enhances the release of exosomes containing CD63 and CD9 . Stress can alter the content of exosomes .
Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent stromal cells that are recruited to tumours and contribute to cancer progression. Although bone marrow is considered a tissue with limited oxygen supply , the oxygen tension in the centre of solid tumours tissue even lower-close to 0.025 kPa (0.1–0.2%). The microenvironment encountered by BMSCs differs between bone marrow and the cancer microenvironment. In this study, we aimed to elucidate the cell-cell communication in the hypoxic cancer microenvironment mediated by BMSC-derived exosomal miRNAs.
Patients and specimens
The clinical characteristics of patients
Chemotherapy or radiation
Reagent, cell culture and hypoxia treatment
STAT3 inhibitor stattics was purchased from Selleck Chemicals (S7024, Houston, TX, USA). GW4869, an inhibitor of exosomes biogenesis/release, was purchased form Sigma (D1692, St. Louis, MO, USA) .miR-193-3p mimics, miR-210-3p mimics, miR-5100 mimics and corresponding inhibitors were designed and synthesized by RIBOBIO (Gongzhou, China).NSCLC cell lines including H358, A549, H460 were cultured in RPMI-1640 medium(SH30809.01B, Hyclone) and murine lewis lung cancinoma(LLC) cells were cultured in DMEM high glucose medium(SH30022.01B, Hyclone) supplemented with penicillin G (100 U/mL) and streptomycin (100 mg/mL) (SV30010, Hyclone) and 10% fetal bovine serum (04–001-1ACS, BI). Cells were obtained from Cell Bank of Cancer Research Institute, Central South University and authenticated by STR profiling. Cells were routinely sub-cultured using 0.25% (w/v) trypsin-EDTA solution(SV30042.01, Hyclone). Murine bone marrow-derived MSCs (mBMSCs) from C57BL/6 mice were purchased from Cyagen (Guangzhou, China). mBMSCs were cultured in MEM medium (SH30024.01, Hyclone) supplemented with penicillin G (100 U/mL) and streptomycin (100 mg/mL) (SV30010, Hyclone)and 10% fetal calf serum (04–001-1 ACS, BI).Human MSCs were obtained from bone marrow aspirates of non-hematological malignant tumor patients and were isolated as described previously [17, 18]. Human bone marrow-derived mesenchymal stem cells (hBMSCs) are cultured in Medium For Mesenchymal Stem Cells (HUXMA-03011-440,cyagen). Cells were grown at 37 °C in a humidified atmosphere with 5% CO2. For hypoxia induction, cells were incubated for 3 days in 0.2% oxygen concentration, 37 °C temperature, 5% CO2 concentration and 90% humidity in Hypoxic Workstation (Don Whitley,UK). The hypoxia treated medium was collected for the following experiments. Cells were lysed for extraction of protein and RNA in the workstation to avoid reoxygenation.
Exosome isolation and quantification
Before exosome isolation, BMSCs were cultured in medium supplemented with exosome-depleted FBS to avoid interference from bovine exosomes [19, 20]. FBS was depleted of exosomes by ultracentrifugation at 1 × 105 g at 4 °C for 16 h (Beckman Coulter Avanti J-30I, USA), then the supernatant of FBS was collected and filtered with a 0.22um filter (millipore,USA). mBMSCs and hBMSCs were cultured in medium with 10% exosome-free FBS. After 72 h, cell culture medium was collected, and exosomes were isolated from the supernatant by differential centrifugation of 300 g for 10 min, 2000 g for 15 min, and 12,000 g for 30 min to remove floating cells and cellular debris. The supernatant was then passed through a 0.22-μm filter(millipore, USA). The resultant supernatant was forced through the membrane by centrifugal filtration at 4 × 103 g for 1 h at 4 °C with the ultrafiltration device (UFC900396,Millipore,USA). Finally, the EXO Quick-TC™ Exosome Isolation Reagent(EXOTC50A-1,System Biosciences,USA) was added in the concentrated solution of supernatant at a ratio of 1:5. Subsequent exosome extraction was conducted according to the kit instructions (SBI, System Biosciences) [21, 22].
Exosomes were isolated from plasma of cancer patients and healthy control according to the manufacturer’s instructions (SBI, System Biosciences) . Briefly, blood was collected into EDTA - K2 anticoagulant tube and mixed immediately to avoid clotting. After centrifuged at 3000 g for 15 min at 4 °C, the supernatant was collected. A total of 250 uL of plasma was mixed with ExoQuick exosome precipitation solution and exosome isolation was performed according to the manufacturer’s instructions (SBI, System Biosciences) . Exosomal protein was measured by the BCA™ Protein Assay Kit (Pierce, USA) . CD63 and HSP70 (ExoAb Antibody Kit,EXOAB-KIT-1, System Biosciences,USA), β-actin (20536-1-AP, Proteintech, Chicago, USA) were used as positive control and inner control for exosomes. The exosomes were used for following experiments immediately or stored at − 80 degrees.
Transmission electron microscopy (TEM)
The exosomes were observed by TEM . Exosomes were isolated and fixed in 1% glutaraldehyde for 10 min, washed with deionized water. Approximately 10 μL of exosome suspension was placed on formvar carbon-coated 300-mesh copper electron microscopy grids(Agar Scientific Ltd., Stansted,UK), and incubated for 5 min at room temperature. Then exosomes were negatively stained with 2% uranyl oxalate for 1 min at room temperature. The grids were washed with three times of PBS and air dried for 5 min. Images were obtained by TEM (JEM-2100, Jeol, Japan).
Nanoparticle tracking analysis
The size distribution and concentration of exosomes were determined by Nanoparticle Tracking Analysis (NTA) according to the manufacturer’s instruction, which utilizes the properties of both light scattering and Brownian motion (Zetasizer Nano ZS90 instrument, Malvern, UK). Exosomes were resuspended in 1 ml PBS and mixed, then the diluted exosomes were injected into the Zetasizer Nano ZS90 instrument (Malvern, UK).Particles were tracked and size of particles was measured based on Brownian motion and the diffusion coefficient. Filtered PBS was used as controls. All samples were measured with parameters of 44.5 mm and 0.64 V voltage using NP100 membranes. Samples were calibrated by CPC100 standard particles diluted 1000-fold under identical settings . Five videos of typically 60 s’ duration were taken. Data was analyzed by Zetasizer software (Malvern Instruments) which was optimized to identify and track each particle on a frame-by-frame basis.
RNA exaction from cells and exosomes
Isolation of cellular or extracellular RNA was performed using TRIzol reagent (TR118, MRC,USA). Prior to isolation of extracellular RNA (exosomes), 25 fmol of C. elegans cel-mir-39 standard RNA (Ribobio, Guangzhou, China) was added to each sample as a spike-in control [25, 26, 27]. Before isopropanol precipitation, Dr.GenTLE Precipitation Carrier (TAKARA#9094, RR820A, Takara, Japan) was added as a co-precipitant to enhance the yield of extracellular RNA.
Exosomes were isolated from 5 × 106 normoxic or hypoxic mBMSCs and hBMSCs, Cells were planted into 6-well plates one day before treatment. When the cells grew at about 70% of confluent, 200μg of exosomes were directly added into cells. PBS was added as control. Forty-eight hrs after treatment, cells were collected for the following experiments.
Blockade of exosome generation by GW4869
GW4869 (Sigma, St. Louis, MO, USA) was used as an inhibitor of exosomes biogenesis/release. GW4869 was added into the medium with 10% exosome-free FBS before BMSCs were put in hypoxic chamber. 3 days after hypoxic treatment,the conditioned medium of MSCs were collected for exosome isolation as mentioned above.
MiRNA microarray of exosomes
Plasma exosomes from mice that received co-injection of BMSCs and LLC cells or injection of LLC cell alone and exosomes from hypoxia-treated mBMSCs or normoxia-treated mBMSCs were collected for microarray analysis. Agilent Mouse miRNA microarray (v19.0; Agilent Technologies Inc., Santa Clara, CA, USA) was used in the analysis. MiRNAs were labeled and hybridized with miRNA Complete Labeling and Hybridization kit (Agilent Technologies) according to the manufacturer’s protocol. The original data files were processed by Feature Extraction software. Signals were normalized using Gene Spring GX software 11.0 (Agilent Technologies).ANOVA was used to compare the different miRNA expressions. The microarray data have been submitted to the Gene Expression Omnibus and the data could be accessed by the accession numbers GSE119887 and GSE119790.
C57BL/6 mice were subcutaneously injected with LLC-RFP with or without BMSCs. When the size of tumours reached 150–200 mm3, the red fluorescent protein positive LLC cells were collected from the tumour sites by flow cytometry cell sorting and subjected to RNA sequencing analysis. The total RNA was isolated from the cell using TRIzol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The extracted RNA was then quantified and assessed for integrity using the NanoDrop (Thermo, USA). The sample quality control, library preparation and sequencing were performed by BGI, China. Briefly, library preparation was performed using oligo-dT beads for enrichment with mRNA containing poly-A tails. RNA was then fragmented and reversely transcribed to double-stranded cDNA (dscDNA) using random hexamer primers. These cDNA fragments then have the addition of a single ‘A’ base and subsequent ligation of the adapter. Then quantified the PCR products by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. Each library was then sequenced at a depth of 10G clean reads on the BGISEQ500 platform (BGI, China). The dscDNA was then end-repaired and ligated to the bubble adapter with protruding T of 3′ end. During PCR amplification step, the fragments were separated into single strands, amplified, and cyclized to form ‘DNA nanoballs’. The DNA nanoballs were loaded into the patterned nanoarrays and pair-end reads of 100 bp were read through on the BGISEQ-500 platform for the following data analysis study. The RNA sequencing data have been submitted to the Gene Expression Omnibus and the data could be accessed by the accession numbers GSE120349.
Cellular internalization of exosomes
Exosomes isolated from culture medium were labelled with the green-fluorescing, lipophilic dye PKH67 according to the manufacturer’s recommendations (Sigma, St. Louis, MO, USA) . Briefly, exosomes were resuspended in 1 mL Diluent C mixed with 4 μL PKH67 and then incubated for 4 mins at room temperature. An equal volume of exosome-free FBS was added to stop the reaction before repelleting. Exosomes were washed twice with exosome-free FBS/ RPMI-1640 to remove excess PKH67. Labelled exosomes were resuspended in 1 × PBS and used immediately or stored at − 20 °C.
Fluorescently labelled exosomes prepared above were resuspended and added to LLC-RFP cells that were at 80% confluence, and incubated for 5 h. Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were then washed with PBS for three times and stained with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI, Vector, Shield, Burlingame, CA) for 5 mins. After staining, cells were washed twice with PBS to remove excess DAPI. LLC-RFP cells that uptook the labelled exosomes were observed under a fluorescence microscope (BX53, Olympus, Japan).
Cell migration and invasion assays
Transwell migration assay was performed using a transwell insert that contains polycarbonate filters with 8-μm pores (cat. no. 3422; Corning).Transfected cells (5 × 104) were suspended in 200 μl of serum-free medium and added to the transwell membrane in the upper chamber. Exosomes that were suspended in complete medium were added to the lower chamber. Cells were incubated at 37 °C for 24 h to allow cell migration through the membrane. Migrated cells were fixed in 4% paraformaldehyde and stained with crystal violet. Migrated cell images were observed and imaged under microscope (CKX41, Olympus, Japan). Cell migration was quantitated by counting in 10 random fields on the lower membrane surface.
Invasion capacity of cells was measured by Matrigel matrix gel invasion assay using a Transwell system (BD Biosciences, CA, USA). The surface of the filter (8-μm pore size) of the upper chamber was coated with 1 mg/ml Matrigel matrix. Exosomes suspended in complete medium were added to the bottom wells. Twenty four hours after incubation, the non-invasive cells were removed with a cotton swab. Migrated cells were fixed in 4% paraformaldehyde and stained with crystal violet. Cell invasion was observed and imaged under microscope (CKX41, Olympus, Japan). Cell invasion was quantitated by counting in 10 random fields on the lower membrane surface.
Western blot analysis
The protein lysate used for western blotting was extracted using mRIPA buffer (Biotime, Hangzhou, China) containing protease inhibitors (Roche, Basel, Switzerland). Proteins were quantified using the BCA™ Protein Assay Kit (Pierce, USA).A western blot system was set up using a Bio-Rad Bis-Tris Gel system, according to the manufacturer’s instructions (Bio-Rad, CA, USA). The cell protein lysates were separated on 10% SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidenedifluoride membranes (Millipore, Danvers, MA, USA). The primary antibody solution was prepared in 5% blocking buffer. Primary antibodies against STAT3, p-STAT3 (Cell Signaling Technology, USA) were incubated with the membrane at 4 °C overnight, followed by a brief wash and incubation with secondary antibody for 1 h at room temperature. An anti-β-actin antibody control was purchased from Proteintech (Chicago,USA) and was used as a loading control. Finally, a 40:1 solution of peroxide and luminol was added to cover the blot surface for five minutes at room temperature. The chemiluminescent signals were captured, and the intensity of the bands was quantified using a Bio-Rad ChemiDoc XRS system (Bio-Rad, CA, USA).
Quantitative real-time PCR
Forward primer (5′- to 3′)
Reverse primer (5′- to 3′)
The primers of RNU6 and cel-miR-39 were purchased from Guangzhou RiboBio Co.,Ltd. (Guangzhou, China). Relative miRNA and mRNA expression levels were calculated by the 2-ΔΔCt method. All samples were tested thrice.
miR-193a-3p and miR-210-3p or miR-5100 inhibitors and mimics (2′-O-methyl modification) were synthesized by RiboBio Company. Lung cancer cells were seeded into six-well plates 24 h before transfection. When cells reached 50% confluency, miRNA mimics (50 nM) or miRNA inhibitor (100 nM) was transfected with Lipofectamine 3000 according to the manufacturer’s instructions(Life Technologies). Briefly, 3 μl of lipofectamine 3000 reagent was diluted in 100 μl of opti-MEM medium. 5 μl of mimics/10 μl of inhibitors were diluted in 100 μl of opti-MEM medium. Upon mixing above diluted reagents, the mixture was incubated at room temperature for 20 mins and added to the cells. Exosomes were added immediately after inhibitor transfection mixture addition. 48 h after transfection, cells were harvested for the following experiments.
Six-week-old female C57BL/6 mice were used to examine allograft tumor growth. All animal experiments were conducted following protocols approved by Central South University, China. A total of 1 × 106 of murine Lewis Lung Cancer LLC-luciferase cells with or without murine BMSCs were injected subcutaneously into mice. Animals were randomly divided into four groups. When the mean tumor volume reached 100mm3, hypoxic mBMSC-secreted exosomes or normoxic mBMSC-secreted exosomes were intratumorally injected into mice at a dose of 200μg per mouse every 2 days for 10 times. Lung metastasis were measured by H&E staining on lung paraffin sections, or detected by ex vivo luciferase based noninvasive bioluminescence imaging using IVIS Lumina II (PerkinElmer). Artagain black paper (Strathmore, USA) was used to cover the primary tumours to avoid signal saturation.
All statistical analyses were performed using the SPSS 18.0 software, and the graphs were generated using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). The comparisons of means among groups were analyzed by one way ANOVA, and the Dunn Multiple Comparison Test was further used to determine significant difference between groups. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were established to determine the sensitivity and specificity of the miRNAs for the prediction of tumorigenesis and metastasis in the patients. Logistic regression was used to develop a combined miRNA panel to predict the probability of developing tumours as previously described . Spearman’s correlation analysis was conducted to evaluate whether the expression levels of the 2 miRNAs were correlated with one another and with other common clinical biomarkers. A value of P < 0.05 was considered statistically significant.
Identifying exosomal miRNAs secreted from hypoxic BMSCs that improve the invasion of lung cancer cells
In addition to BMSCs, other cell types such as cancer cells were also affected by hypoxic microenvironment and released exosomes. We then investigate to what extent hypoxia affects the exosome release of BMSCs. The BMSCs were cultured in a hypoxic chamber for 3 days. The exosomes were collected and isolated from the cell culture medium. The exosomes collected from hypoxic BMSC medium were smaller than those from normoxic BMSC medium, with average diameters of 50 nm and 90 nm, respectively (Additional file 1: Figure S1D). The exosomes were observed by transmission electron microscopy (Additional file 1: Figure S1E). The exosomes were positive for exosomal proteins CD63 and HSP70 (Additional file 1: Figure S1F). We then examined BMSC exosomal miRNA expression profile. Total RNAs were extracted and subjected to miRNA microarray. Some exosomal miRNAs including miR-146a-5p, miR-574-3p, miR-328-3p, miR-326-3p, miR-193a-3p, miR-5100 and miR-210-3p were increased, while others such as miR-6404, miR-6995-3p, miR-5112 were decreased by more than 2-fold in hypoxic BMSCs compared to normoxic BMSCs (Fig. 1e and f). We identified overlap of shared exosomal miRNAs in vitro and in vivo that are specific exosomal miRNAs derived from BMSCs (Fig. 1g). The expression of microRNAs was verified by qPCR, showing that exo-miR-210-3p, exo-miR-328-3p, exo-miR-574-5p, exo-miR-25-3p, exo-miR-652-3p, exo-miR-193a-3p and exo-miR-5100 secreted by hypoxic BMSCs were upregulated compared to normoxic BMSCs secreted exosomes (Fig. 1h). We selected exo-miR-193a-3p, exo-miR-210-3p and exo-miR-5100, which showed similar expression pattern in vitro and in vivo for further investigation.
Exosome-mediated transfer of miR-193a, miR-210-3p and miR-5100 from hypoxic BMSCs promotes invasion of cancer cells
Exosomal miRNAs from BMSCs promote metastasis by STAT3 driven EMT
Exosomal miRNA can be biomarkers for lung cancer metastasis
Exosomes are the major mediators of cell–cell communication because they can be captured by neighboring cells . The direct transfer of exosomal miR-193a-3p, miR210-3p and miR-5100 from hypoxic BMSCs to neighboring cancer cells enhances metastatic properties.
BMSCs are multipotent stromal cells that are recruited to tumours and contribute to cancer progression. The effect of hypoxia on BMSCs has been extensively investigated. In this study, we focused on the paracrine effect exosomes secreted by hypoxic BMSCs on the cancer cells. Exosomes are released by most cell types. In order to identify hypoxic BMSC-derived exosomes, we selected overlap of shared exosomal miRNAs in vitro and in vivo. The composition of exosomal microRNAs differs significantly depending on different contexts, indicating selectivity for their loading into exosomes . The proportion of miRNA is higher in exosomes than in their donor cells [2, 31]. RNA binding proteins (RBPs) serve as key players on biosynthesis of exosomes by forming complexes with RNAs and transporting them into exosomes. The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a complex and diverse family of RNA binding proteins. hnRNP-dependent pathways are reported to be related to miRNA secretion into exosomes. NSMase2 is an enzyme for ceramide generation that is involved in exosome secretion. The higher expression of NSMase2, hnRNPA1 and hnRNPH1 in hypoxia may contribute to the higher proportion of miRNA on the exosomes [32, 33, 34]. Potential hypoxia inducible factor (HIF) binding sites were identified in the promoter of hnRNPA1 . On the other hand, the exosomes secreted from hypoxic cells have a larger surface area because of their smaller size, which may carry more proteins or microRNAs. In this study, we found that the 3’ end of miRNA comprises the main exosomal microRNAs, including miR-193a-3p and miR210-3p, verifying that 3’ portion or the 3’ end of the miRNA sequence contains a critical sorting signal. Exosomal miRNA signatures are composed of a distinct set of miRNAs [12, 36, 37]. In this study, after quantile normalization of the data, miRNAs were differentially expressed in hypoxia-treated versus normoxia-treated BMSCs. Exosomes from B cells, dendritic cells, T cells and colorectal cancer cells have been demonstrated to carry miR-193-3p. miR-210-3p is considered to be hypoxia-related and is carried by exosomes secreted from colorectal cancer cells. Exosome-mediated transfer of particular microRNAs from stromal cells to epithelial cancer cells contributes to cancer progression.
The roles of miR-193a-3p, miR-210-3p and miR-5100 in cancer progression have been reported. miR-193a-3p was demonstrated to suppress the proliferation and migration of lung cancer cells and colorectal adenocarcinoma cells by targeting K-ras [38, 39]. miRNA-193a-3p inhibits tumor proliferation migration and chemoresistance in human gastric cancer by regulating the PTEN gene . On the other hand, the level of miR-193a-3p was significantly increased in renal cell carcinoma (RCC) tissues and cell lines. miR-193a-3p increases cell proliferation and migration of RCC cells by targeting the ST3GalIV via PI3K/Akt pathway . Lower expression of miR-193-3p is associated with poor prognosis in colorectal cancer, but better prognosis in oropharyngeal squamous cell carcinoma (OPSCC) [42, 43]. Similar to the controversial roles of miR-193a-3p, miR-210-3p was also suggested to have oncogenic or tumor suppression effects on the development of cancer, depending on the target [44, 45, 46]. A higher level of serum miR-210-3p has been detected in people who live in a high-altitude hypoxic environment . A high level of miR-210-3p in urine was also detected in RCC patients . In this study, we found that cellular expression of specific miRNAs were decreased when these miRNAs were enriched in exosomes, suggesting the differential effects of miRNAs inside and outside of the cells.
Emerging evidence has demonstrated the feasibility of circulating miRNAs as robust, non-invasive biomarkers for the diagnosis of colorectal cancer. Due to their presence in the bodily fluids, exosomes are considered candidate biomarkers. The use of a combination of miRNAs as biomarkers, instead of one unique miRNA, has been considered to be a more powerful tool due to the possible overlap in miRNA targeting . A miRNA may dampen or amplify a signal by participating in a negative or positive feedback loop . In this study, we found a combination of miR-193a-3p, miR-210-3p and miR-5100 promotes the activation of the STAT3 pathway. STAT3 signaling is active when miRNAs target the STAT3 inhibitors SOCS or PIAS3 . MiR-210-3p targets the negative regulator of STAT3 signaling, SOCS1 . Thus, we sought to apply a combination of miRNAs as a biomarker to discriminate between cancer and non-cancerous patients, or between metastatic and non-metastatic patients. A combination of miR-193a-3p, miR-210-3p and miR-5100 can discriminate between the groups with high sensitivity and specificity.
In summary, hypoxic BMSC-derived exosomal miR-193a-3p, miR-210-3p and miR-5100 were identified by microRNA array. Exosome-mediated transfer of particular microRNAs, including miR-193a-3p, miR-210-3p and miR-5100, could promote invasion of cancer cells by activating STAT3 signaling-induced EMT. These exosomal miRNAs may be promising noninvasive biomarkers for cancer progression.
Exosome-mediated transfer of miR-193a-3p, miR-210-3p and miR-5100, could promote invasion of lung cancer cells by activating STAT3 signaling-induced EMT. These exosomal miRNAs may be promising noninvasive biomarkers for cancer progression.
This work was supported by National Natural Science Foundation, China (grant number 81472695, 81773147); Strategic Priority Research Program of Central South University (ZLXD2017004); National Training and Research Base for Talents of principles of carcinogenesis foundation (111 project, 111–2-12).
Availability of data and materials
All the data generated or analyzed during this study are included in this published article and its supplementary files. The RNA-seq data and microarray data have been submitted to the Gene Expression Omnibus and the data could be accessed by the accession numbers GSE120349, GSE119887 and GSE119790.
XNZ and JJX conceived and designed the experiments. XNZ, BQS, FW, LJW, YHW and LLZ performed the experiments. XNZ, JQT and JJX analysed the data and prepared all the figures and wrote the manuscript. GYL, JQT and JJX provided technical support. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The research presented here has been performed in accordance with the Declaration of Helsinki and has been approved by the ethics committee of the Second Xiangya Hospital, Central South University, China. The patients were informed about the sample collection and had signed informed consent forms.
Consent for publication
All authors of this article have directly participated in the planning and drafting and all authors listed have read and approved the final version including details and images. The written informed consent for the publication has been obtained from all the authors. The patients were informed about the publication and had signed informed consent forms.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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