# Exosomal miRNA Expression Profiling and the Roles of Exosomal miR-4741, miR-32, miR-3149, and miR-6727 on Gastric Cancer Progression

Authors: Guojun Tang; Jianping Wang; Wuzhen Dong; Kangfu Dai; Jinlin Du
Journal: BioMed Research International
 (2022)

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## Abstract

Objective. Accumulated evidence highlights the biological implications of exosomes in gastric cancer. Herein, we conducted the exosomal miRNA expression profiling and identified potential diagnostic markers for gastric cancer. Methods. Plasma exosomes were isolated and identified from three gastric cancer patients and three healthy participants. Microarrays of exosomal miRNAs were then analyzed. Differentially expressed exosomal miRNAs were screened with fold−change≥2.0 and p≤0.05. Among them, miR-4741, miR-32, miR-3149, and miR-6727 expressions were verified in tissues and plasma of patients and healthy subjects. ROC curves were conducted for evaluating the diagnostic performance. The roles of miR-32, miR-3149, miR-6727, and miR-4741 on gastric cancer progression were observed by cellular experiments. Results. Isolated exosomes were well characterized by Western blot and transmission electron microscopy as well as nanoparticle-tracking analyses. According to the microarrays, 142 exosomal miRNAs were upregulated, and 34 were downregulated in gastric cancer than healthy subjects. miR-4741 upregulation and miR-32, miR-3149, and miR-6727 downregulations were found in tissues and plasma of gastric cancer patients. The AUCs of miR-4741, miR-32, miR-3149, and miR-6727 were separately 0.8554, 0.9456, 0.7683, and 0.8923. Upregulated miR-32, miR-3149, and miR-6727 as well as downregulated miR-4741 lowered proliferative, migratory, and invasive capacities as well as elevated apoptotic levels of gastric cancer cells. Conclusion. Our study successfully isolated and verified exosomes from plasma of gastric cancer as well as proposed four exosomal miRNAs that could act as promising diagnostic markers and suppress gastric cancer progression.


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## Body

## 1. Introduction

Gastric cancer represents the fifth most diagnosed malignancy as well as the third dominating cause of cancer deaths globally [1]. The five-year survival rates range from 20 to 40% [2]. As estimated, the incidence of gastric cancer tends to be younger [3]. Although new diagnostic strategies have been continuously improved in gastric cancer, the existing clinical biomarkers display limited sensitivity and specificity in diagnosing gastric cancer [4]. More effective biomarkers and targets eagerly needed to be discovered to better diagnose and cure gastric cancer.Accumulated evidence has demonstrated that exosomes may emerge as promising diagnostic biomarkers of gastric cancer [5]. Exosomes, nanosized extracellular vesicles (30-150 nm in size), are released by most cells, which are key ways for intercellular interactions through delivering nucleic acid molecules like protein, DNA, mRNA, and noncoding RNA (microRNA (miRNA), etc.) [6]. Due to the lipid bilayer structure, exosomes may stably be in different fluids (such as serum, urine as well as saliva) [7]. Exosomes may mediate carcinogenesis, tumor growth, and metastases as well as drug resistance for gastric cancer [8]. For instance, tumor-derived exosomes facilitate migratory capacity of gastric cancer cells through inducing neutrophil N2 polarization [9]. Exosomal circSHKBP1 accelerates gastric cancer progress through mediating miR-582-3p/HUR/VEGF axis as well as inhibiting HSP90 degradation [10]. Hypoxic tumor-derived exosomes induce progress and metastases of gastric cancer via miR-301a-3p/PHD3/HIF-1α axis [11]. Increasing evidence has highlighted the critical roles of exosomes that carry miRNAs in gastric carcinoma [12]. Exosomal miR-522 inhibits ferroptosis as well as induces acquired resistance to chemotherapy in gastric cancer [12]. Furthermore, exosomes that carry anti-miR-214 ameliorate resistance to cisplatin in gastric cancer [13]. Exosomal miR-139 in tumor-related fibroblasts suppresses gastric cancer development through repression of MMP11 expression [14]. Exosomal miR-21-5p induces peritoneal metastases through mesothelial-to-mesenchymal transition in gastric cancer [15]. Thus, exosomal miRNAs possess the potential for diagnosing as well as curing gastric cancer.This study systemically and comprehensively analyzed exosomal miRNAs in plasma specimens of gastric cancer subjects. Especially, exosomal miR-4741, miR-32, miR-3149, and miR-6727 displayed high accuracy and sensitivity in diagnosing gastric cancer. Also, they participated in mediating proliferative, migratory, and invasive capacities as well as apoptotic levels for gastric cancer cells. Thus, exosomal miR-4741, miR-32, miR-3149, and miR-6727 might mediate gastric cancer progression.


## 2. Materials and Methods

### 2.1. Patients and Samples

Totally, 120 patients who underwent radical gastric cancer surgery were collected at the Jinhua Central Hospital of Zhejiang Province (China) from April 2017 to April 2019. The age ranged from 40 to 65 years old, with an average age of48.80±9.52. These patients included 60 cases of early gastric cancer and 60 cases of middle-advanced gastric cancer. Adjacent control tissues were obtained from gastric cancer subjects. Meanwhile, 57 healthy participants were enrolled in our study. Blood specimens were harvested from these gastric carcinoma subjects and controls. These clinical specimens were immediately stored at -80°C. This experiment was carried out in line with the Declaration of Helsinki, and our research was approved by the Ethics Committee of Jinhua Central Hospital of Zhejiang Province (KY-2017026). All subjects signed written informed consent.


### 2.2. Isolation and Identification of Plasma Exosomes

Exosomes were extracted from plasma specimens of gastric cancer and healthy subjects using ExoQuickTM Kit (System Biosciences, USA). The exosomal specimens were fixed utilizing 5% glutaraldehyde and placed in a carbon-coated copper grid. The grid was covered with a phosphotungstic acid solution (2%, pH 7.0) for 30 s. Transmission electron microscopy (TEM, Tecnai G2 Spirit Bio TWIN, FEI, USA) was used to observe exosomes. Exosomes were diluted via PBS buffer. Utilizing nanoparticle tracking analyses (NTA; ZetaView PMX 110, Particle Metrix, Meerbusch, Germany) and ZetaView 8.04.02 software, the particle size and concentration of exosomes were detected. The ZetaView system was calibrated with 110 nm polystyrene particles at 28.7°C and pH 7.0. Western blot was used to detect the expression levels of exosomal markers CD63, CD9, TSG101, and GM130. Total protein was isolated from the cells utilizing RIPA reagent (Beyotime, Shanghai, China) containing 10μg/mL phosphatase inhibitor as well as 100 μg/mL PMSF (Beyotime). Above proteins were separated via SDS-PAGE and then transferred to PVDF membranes. Following blocking for 1 h, these membranes were incubated by primary antibody including anti-CD63 (ab134045, Abcam, UK), CD9 (ab223052, Abcam), anti-TSG101 (ab30871, Abcam), and GM130 (ab187514, Abcam) at 4°C overnight. It was washed 3 times for 5 min and then incubated in the corresponding secondary antibody (ab7090, Abcam) for 2 h. Then, Western blots were detected by enhanced chemiluminescence (ECL).


### 2.3. Microarray Analysis

The miRNAs were extracted from plasma-derived exosomes with the mirVana miRNA isolation kit (Thermo Scientific, USA). The total RNA of the sample was quantified by NanoDrop ND-2000 (Thermo Scientific, USA), and the RNA integrity was checked by Agilent Bioanalyzer 2100 (Agilent Technologies). After passing the RNA quality inspection, sample labeling, chip hybridization, and elution were presented following the standard process of the chip. Firstly, total RNA was dephosphorylated, denatured, and further labeled with Cyanine-3-CTP (Cy3). The labeled RNA was purified and hybridized with the chip. After elution, the original image was obtained by scanning utilizing Agilent Scanner G2505C (Agilent Technologies). The Feature Extraction software (version 10.7.1.1, Agilent Technologies) was used to process the original image for extracting the original data. Then, Genespring software (version 14.8, Agilent Technologies) was applied to quantile standard and subsequently process. The normalized data was filtered, and at least one set of 100% probes marked as detected in each set of samples utilized for comparisons was left for further analyses. Thep value and fold-change value of the t test were utilized for screening differentially expressed miRNAs. The screening criteria were fold−change≥2.0 and p≤0.05. Then, three databases (TargetScan (http://www.targetscan.org/mamm_31/) [16], MicroRNA.org (http://www.microrna.org) [17], and PITA (https://genie.weizmann.ac.il/pubs/mir07/index.html) [18]) were used to jointly predict the target mRNAs of differentially expressed miRNAs. Next, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out on the target mRNAs for determining the biological functions or pathways mainly influenced by these differentially expressed miRNAs. At last, unsupervised hierarchical clustering analyses were performed based on differentially expressed miRNAs. Furthermore, this study displayed the expression patterns of differentially expressed miRNAs among different samples by heat maps.


### 2.4. Real-Time Quantitative Polymerase-Chain Reaction (RT-qPCR)

The primers of miR-3149 (5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATACACAC-3′ (forward), 5′-ACACTCCAGCTGGGTTTGTATGGATATGTGT-3′ (reverse)), miR-32p (5′-GCGGCGTATTGCACATTACT-3′ (forward), 5′-TCGTATCCAGTGCAGGGTC-3′ (reverse)), and U6 (5′-CTC GCTTCGGCAGCACA-3′ (forward), 5′-AACGCTTCACGAATTTGCGT-3′ (reverse)) were synthesized by Guangzhou Ruibo Company (China). After resuspending the serum exosomes, total RNA of plasma exosomes was extracted utilizing Trizol reagent. The concentration of extracted total RNA was then determined. Reverse transcription was carried out using RNA reverse transcription kit, conditions: 37°C for 15 min, 85°C for 5 s, 4°C hold. After reverse transcription, RT-qPCR was presented via SYBR Green real-time PCR kit (Sigma, USA), conditions: predenaturation at 95°C for 30 s, denaturation at 95°C for 5 s, renaturation at 60°C for 34 s. The expression of exosomal miRNAs was calculated with 2−ΔΔCt.


### 2.5. Cell Culture and Transfection

MGC-803 gastric cancer cells were obtained from the Cell Resource Center, Shanghai Academy of Biological Sciences (China). These cells were grown with RPMI 1640 medium plus 10% fetal bovine serum and put in a constant temperature incubator at 37°C and 5% CO2. Then, cells were digested with 0.25% trypsin digestion. MGC-803 cells grown in log phase were selected and inoculated in a 6-well plate. When the cell confluence was about 75%, Lipofectamine 2000 (Invitrogen, USA) was used for transfection, including miR-32 mimic, miR-3149 mimic, miR-4741 inhibitor (Shanghai GenePharma Co., Ltd., China), and their negative control (NC). 24 h after transfection, RT-qPCR was used to detect the expression of miRNAs.


### 2.6. Cell Counting Kit-8 (CCK-8)

MGC-803 cells in the logarithmic phase were taken to prepare a cell suspension (5×107/L) and inoculated onto a 96-well plate (100 μL per well). Each group of experiments set up 5 parallel multiple wells, each group was repeated 3 times and routinely cultured in an incubator. The cells were collected at 24, 48, and 72 h following transfections. 10 μL of CCK-8 solution (Dojindo, Japan) was added to each well and incubated for 2 h. Absorbance values were detected with a microplate reader at 490 nm.


### 2.7. Flow Cytometry

After trypsinization, the cells were collected in a 5 mL centrifuge tube, with 3 replicate holes in each group. After centrifugation, the supernatant was discarded. Then, the cell pellet was washed once with PBS and resuspended after centrifugation. The cell suspension was taken and strained with PE-Cy7 (Sigma, USA) for 15 min at room temperature in the dark. Flow cytometry was used to detect the apoptosis of MGC-803 cells.


### 2.8. Wound Healing

After transfection, MGC-803 cells were cultured in 6-well plates. When the cells grown to 70%-80% confluence, a 20μL pipette tip was utilized to make a scratch. Subsequently, the separated cells were washed 3 times with PBS, and the cells were cultured in fresh RPMI-1640 medium. The scratch healing was imaged under an optical microscope at the set time points (0 h and 24 h). The width of the scratch was measured by ImageJ software.


### 2.9. Transwell Invasion Assay

Matrigel was prelaid on the upper chamber of the Transwell chamber (Corning, USA). After 24 h of transfection, the cells in each group were trypsinized and resuspended in serum-free medium to adjust the cell density to2×104/L. 200 μL of the cell suspension was taken and added to the upper layer of the Transwell chamber. Then, 500 μL of 10% serum medium was added to the lower layer of the Transwell chamber and placed it in a 37°C, 5% CO2 incubator. After culturing for 24 h, the cells were washed with PBS. Using a wet cotton swab, the uninvaded cells in the upper chamber were gently wiped off. The cells were fixed utilizing 4% paraformaldehyde lasting 20 min. Following washing 3 times utilizing PBS, cells were stained utilizing 0.1% crystal violet lasting 15 min. Following washing utilizing PBS, 5 fields of view under the microscope were randomly selected, and stained cells were counted.


### 2.10. Statistical Analysis

All experiments were carried out in triplicate. Data are expressed as themeanvalue±standarddeviation. Statistical analyses were achieved with Student’s t-test and one-way or two-way analysis of variance in GraphPad Prism software, with p<0.05 considered statistical significance. Receiver-operator characteristic (ROC) curves were plotted to assess the diagnostic efficacy of indicated factors. From the Gene Expression Omnibus database, miRNA expression profiling of plasma samples from 5 gastric cancer patients and 3 healthy controls was retrieved from the GSE86822 dataset (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86822) on the GPL17107 platform. The expression of miR-4741, miR-32, miR-3149, and miR-6727 was externally verified in plasma of gastric cancer patients and healthy controls.


## 2.1. Patients and Samples

Totally, 120 patients who underwent radical gastric cancer surgery were collected at the Jinhua Central Hospital of Zhejiang Province (China) from April 2017 to April 2019. The age ranged from 40 to 65 years old, with an average age of48.80±9.52. These patients included 60 cases of early gastric cancer and 60 cases of middle-advanced gastric cancer. Adjacent control tissues were obtained from gastric cancer subjects. Meanwhile, 57 healthy participants were enrolled in our study. Blood specimens were harvested from these gastric carcinoma subjects and controls. These clinical specimens were immediately stored at -80°C. This experiment was carried out in line with the Declaration of Helsinki, and our research was approved by the Ethics Committee of Jinhua Central Hospital of Zhejiang Province (KY-2017026). All subjects signed written informed consent.


## 2.2. Isolation and Identification of Plasma Exosomes

Exosomes were extracted from plasma specimens of gastric cancer and healthy subjects using ExoQuickTM Kit (System Biosciences, USA). The exosomal specimens were fixed utilizing 5% glutaraldehyde and placed in a carbon-coated copper grid. The grid was covered with a phosphotungstic acid solution (2%, pH 7.0) for 30 s. Transmission electron microscopy (TEM, Tecnai G2 Spirit Bio TWIN, FEI, USA) was used to observe exosomes. Exosomes were diluted via PBS buffer. Utilizing nanoparticle tracking analyses (NTA; ZetaView PMX 110, Particle Metrix, Meerbusch, Germany) and ZetaView 8.04.02 software, the particle size and concentration of exosomes were detected. The ZetaView system was calibrated with 110 nm polystyrene particles at 28.7°C and pH 7.0. Western blot was used to detect the expression levels of exosomal markers CD63, CD9, TSG101, and GM130. Total protein was isolated from the cells utilizing RIPA reagent (Beyotime, Shanghai, China) containing 10μg/mL phosphatase inhibitor as well as 100 μg/mL PMSF (Beyotime). Above proteins were separated via SDS-PAGE and then transferred to PVDF membranes. Following blocking for 1 h, these membranes were incubated by primary antibody including anti-CD63 (ab134045, Abcam, UK), CD9 (ab223052, Abcam), anti-TSG101 (ab30871, Abcam), and GM130 (ab187514, Abcam) at 4°C overnight. It was washed 3 times for 5 min and then incubated in the corresponding secondary antibody (ab7090, Abcam) for 2 h. Then, Western blots were detected by enhanced chemiluminescence (ECL).


## 2.3. Microarray Analysis

The miRNAs were extracted from plasma-derived exosomes with the mirVana miRNA isolation kit (Thermo Scientific, USA). The total RNA of the sample was quantified by NanoDrop ND-2000 (Thermo Scientific, USA), and the RNA integrity was checked by Agilent Bioanalyzer 2100 (Agilent Technologies). After passing the RNA quality inspection, sample labeling, chip hybridization, and elution were presented following the standard process of the chip. Firstly, total RNA was dephosphorylated, denatured, and further labeled with Cyanine-3-CTP (Cy3). The labeled RNA was purified and hybridized with the chip. After elution, the original image was obtained by scanning utilizing Agilent Scanner G2505C (Agilent Technologies). The Feature Extraction software (version 10.7.1.1, Agilent Technologies) was used to process the original image for extracting the original data. Then, Genespring software (version 14.8, Agilent Technologies) was applied to quantile standard and subsequently process. The normalized data was filtered, and at least one set of 100% probes marked as detected in each set of samples utilized for comparisons was left for further analyses. Thep value and fold-change value of the t test were utilized for screening differentially expressed miRNAs. The screening criteria were fold−change≥2.0 and p≤0.05. Then, three databases (TargetScan (http://www.targetscan.org/mamm_31/) [16], MicroRNA.org (http://www.microrna.org) [17], and PITA (https://genie.weizmann.ac.il/pubs/mir07/index.html) [18]) were used to jointly predict the target mRNAs of differentially expressed miRNAs. Next, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out on the target mRNAs for determining the biological functions or pathways mainly influenced by these differentially expressed miRNAs. At last, unsupervised hierarchical clustering analyses were performed based on differentially expressed miRNAs. Furthermore, this study displayed the expression patterns of differentially expressed miRNAs among different samples by heat maps.


## 2.4. Real-Time Quantitative Polymerase-Chain Reaction (RT-qPCR)

The primers of miR-3149 (5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATACACAC-3′ (forward), 5′-ACACTCCAGCTGGGTTTGTATGGATATGTGT-3′ (reverse)), miR-32p (5′-GCGGCGTATTGCACATTACT-3′ (forward), 5′-TCGTATCCAGTGCAGGGTC-3′ (reverse)), and U6 (5′-CTC GCTTCGGCAGCACA-3′ (forward), 5′-AACGCTTCACGAATTTGCGT-3′ (reverse)) were synthesized by Guangzhou Ruibo Company (China). After resuspending the serum exosomes, total RNA of plasma exosomes was extracted utilizing Trizol reagent. The concentration of extracted total RNA was then determined. Reverse transcription was carried out using RNA reverse transcription kit, conditions: 37°C for 15 min, 85°C for 5 s, 4°C hold. After reverse transcription, RT-qPCR was presented via SYBR Green real-time PCR kit (Sigma, USA), conditions: predenaturation at 95°C for 30 s, denaturation at 95°C for 5 s, renaturation at 60°C for 34 s. The expression of exosomal miRNAs was calculated with 2−ΔΔCt.


## 2.5. Cell Culture and Transfection

MGC-803 gastric cancer cells were obtained from the Cell Resource Center, Shanghai Academy of Biological Sciences (China). These cells were grown with RPMI 1640 medium plus 10% fetal bovine serum and put in a constant temperature incubator at 37°C and 5% CO2. Then, cells were digested with 0.25% trypsin digestion. MGC-803 cells grown in log phase were selected and inoculated in a 6-well plate. When the cell confluence was about 75%, Lipofectamine 2000 (Invitrogen, USA) was used for transfection, including miR-32 mimic, miR-3149 mimic, miR-4741 inhibitor (Shanghai GenePharma Co., Ltd., China), and their negative control (NC). 24 h after transfection, RT-qPCR was used to detect the expression of miRNAs.


## 2.6. Cell Counting Kit-8 (CCK-8)

MGC-803 cells in the logarithmic phase were taken to prepare a cell suspension (5×107/L) and inoculated onto a 96-well plate (100 μL per well). Each group of experiments set up 5 parallel multiple wells, each group was repeated 3 times and routinely cultured in an incubator. The cells were collected at 24, 48, and 72 h following transfections. 10 μL of CCK-8 solution (Dojindo, Japan) was added to each well and incubated for 2 h. Absorbance values were detected with a microplate reader at 490 nm.


## 2.7. Flow Cytometry

After trypsinization, the cells were collected in a 5 mL centrifuge tube, with 3 replicate holes in each group. After centrifugation, the supernatant was discarded. Then, the cell pellet was washed once with PBS and resuspended after centrifugation. The cell suspension was taken and strained with PE-Cy7 (Sigma, USA) for 15 min at room temperature in the dark. Flow cytometry was used to detect the apoptosis of MGC-803 cells.


## 2.8. Wound Healing

After transfection, MGC-803 cells were cultured in 6-well plates. When the cells grown to 70%-80% confluence, a 20μL pipette tip was utilized to make a scratch. Subsequently, the separated cells were washed 3 times with PBS, and the cells were cultured in fresh RPMI-1640 medium. The scratch healing was imaged under an optical microscope at the set time points (0 h and 24 h). The width of the scratch was measured by ImageJ software.


## 2.9. Transwell Invasion Assay

Matrigel was prelaid on the upper chamber of the Transwell chamber (Corning, USA). After 24 h of transfection, the cells in each group were trypsinized and resuspended in serum-free medium to adjust the cell density to2×104/L. 200 μL of the cell suspension was taken and added to the upper layer of the Transwell chamber. Then, 500 μL of 10% serum medium was added to the lower layer of the Transwell chamber and placed it in a 37°C, 5% CO2 incubator. After culturing for 24 h, the cells were washed with PBS. Using a wet cotton swab, the uninvaded cells in the upper chamber were gently wiped off. The cells were fixed utilizing 4% paraformaldehyde lasting 20 min. Following washing 3 times utilizing PBS, cells were stained utilizing 0.1% crystal violet lasting 15 min. Following washing utilizing PBS, 5 fields of view under the microscope were randomly selected, and stained cells were counted.


## 2.10. Statistical Analysis

All experiments were carried out in triplicate. Data are expressed as themeanvalue±standarddeviation. Statistical analyses were achieved with Student’s t-test and one-way or two-way analysis of variance in GraphPad Prism software, with p<0.05 considered statistical significance. Receiver-operator characteristic (ROC) curves were plotted to assess the diagnostic efficacy of indicated factors. From the Gene Expression Omnibus database, miRNA expression profiling of plasma samples from 5 gastric cancer patients and 3 healthy controls was retrieved from the GSE86822 dataset (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86822) on the GPL17107 platform. The expression of miR-4741, miR-32, miR-3149, and miR-6727 was externally verified in plasma of gastric cancer patients and healthy controls.


## 3. Results

### 3.1. Isolation and Identification of Plasma Exosomes

To identify exosomes isolated from plasma specimens of gastric cancer and control subjects, this study first employed TEM to conduct preliminary identification of the morphology of exosomes. The results were shown in Figure1(a). The exosomes extracted from the patient’s plasma showed a typical electron microscope shape: saucer-shaped or a hemispherical concave on one side. The diameter of most exosomes was around 50-100 nm. Western blot results showed that the exosomes in the patient’s plasma all expressed CD63, CD9, and TSG101 proteins, but almost no GM130 protein (Figure 1(b)). This study further used NTA to determine the diameter and density of exosomes. As shown in our results, the diameter of plasma exosomes from healthy and gastric cancer subjects ranged from 50 to 200 nm, mainly around 75 nm (Figure 1(c)).Figure 1
Isolation and identification of plasma exosomes from gastric cancer patients. (a) Transmission electron microscopy for observing the morphological characteristics of isolated exosomes.Bar=100nm. (b) Western blot for detecting the expression of exosome markers CD63, CD9, TSG1010, and GM130. (c) Nanoparticle tracking analysis for detecting the particle size and concentration of exosomes.
(a)(b)(c)


### 3.2. Microarray Expression Profiles of Exosomal miRNAs for Gastric Cancer

To characterize the exosomal miRNA expression profiles of gastric cancer as well as control subjects, this study carried out microarray analyses. Figure2(a) showed the dispersion of microarray data of each specimen. A scatter plot was drawn on the standardized data in a two-dimensional coordinate to evaluate the central tendency of the overall distribution of the two groups of data. The scatter plot array of the data pairwise comparison was drawn into a matrix diagram, as shown in Figure 2(b). The PCA analysis was performed on all samples, and the distribution of samples was investigated to verify the rationality of the experimental design and the uniformity of biological replicate samples, which was displayed in a two-dimensional graph (Figure 2(c)).Figure 2
Microarray expression profiles of exosomal miRNAs in gastric cancer. (a) Box plot of the dispersion of microarray data. (b) Scatter plots of the overall distribution of the data from gastric cancer and control specimens. (c) PCA for the sample distributions based on the expression profiles.
(a)(b)(c)


### 3.3. Differentially Expressed Exosomal miRNAs for Gastric Cancer

Based on the microarrays, we screened differentially expressed exosomal miRNAs withfold−change≥2.0 and p≤0.05. As a result, 142 exosomal miRNAs were upregulated (Table 1), and 34 were downregulated (Table 2) in gastric cancer compared to healthy subjects (Figures 3(a) and 3(b)). By three databases microRNAorg, PITA, and TargetScan, 6823 target mRNAs were predicted for these differentially expressed exosomal miRNAs (Figure 3(c); Supplementary Table 1).Table 1
Upregulated exosomal miRNAs in gastric cancer.
miRNAsp valueFold-changemiRNAsp valueFold-changehsa-miR-6727-5p1.50E-0498.41242hsa-miR-3614-5p0.0013215.420335hsa-miR-47411.35E-0584.10587hsa-miR-46882.91E-075.338095hsa-miR-5585-3p3.49E-0461.08826hsa-miR-39453.66E-065.113267hsa-miR-45302.23E-0658.76396hsa-miR-6021.17E-055.065191hsa-miR-142-5p6.11E-0555.08285hsa-miR-2276-3p2.26E-064.952288hsa-miR-494-3p4.83E-0548.00325hsa-miR-186-5p0.0031374.919776hsa-miR-23a-3p0.04723544.9474hsa-miR-6890-3p2.43E-054.879964hsa-miR-30e-5p1.70E-0444.5984hsa-miR-6757-3p1.98E-054.753316hsa-miR-130a-3p0.04934640.1765hsa-miR-80732.16E-064.750058hsa-miR-6791-5p1.16E-0535.17456hsa-miR-1914-3p1.06E-054.634837hsa-miR-31382.06E-0633.31427hsa-miR-193b-5p6.42E-074.594794hsa-miR-7180.00466529.61524hsa-miR-6857-3p6.12E-054.579425hsa-miR-61260.01508423.6956hsa-miR-223-3p0.038844.501325hsa-miR-27b-3p0.02486523.45536hsa-miR-3613-3p4.37E-054.491396hsa-miR-211-3p0.00288123.01478hsa-miR-5751.11E-054.489378hsa-miR-30c-5p0.02093122.89729hsa-miR-6780b-5p6.35E-064.470476hsa-miR-331-3p0.01967822.28292hsa-let-7g-5p0.0479034.458645hsa-miR-134-5p0.01388521.13077hsa-miR-47063.28E-074.456074hsa-miR-4632-5p0.00146220.79573hsa-miR-6511b-5p2.98E-064.425767hsa-miR-144-5p0.01077619.00057hsa-miR-652-5p2.63E-054.342584hsa-miR-23b-3p0.02604118.41381hsa-miR-345-3p5.19E-064.331377hsa-miR-7108-5p0.00227417.76675hsa-miR-4666b6.64E-054.322666hsa-miR-199a-5p0.03559717.26349hsa-miR-96-5p2.90E-044.248471hsa-miR-29b-3p0.01216517.26232hsa-miR-4419a4.90E-044.242825hsa-miR-374a-5p0.02898217.12239hsa-miR-5634.27E-044.204622hsa-miR-7846-3p0.01228916.88478hsa-miR-60863.10E-044.193719hsa-miR-42539.23E-0416.36829hsa-miR-6734-5p4.61E-064.14653hsa-miR-151b0.02194314.60052hsa-miR-1233-5p6.23E-074.100964hsa-miR-146a-5p0.03406814.0491hsa-miR-183-5p3.44E-064.098074hsa-miR-6786-5p0.00290414.02458hsa-miR-135a-3p4.10E-044.085765hsa-miR-5001-5p0.00130913.43416hsa-miR-31475.22E-044.067993hsa-miR-43100.00846613.00651hsa-miR-31311.35E-064.047892hsa-miR-590-5p0.01687912.24443hsa-miR-98-3p1.93E-044.016676hsa-miR-3667-5p0.00218812.07358hsa-miR-210-3p0.0016783.958811hsa-miR-3940-5p0.02009911.56078hsa-miR-18b-5p5.58E-043.953054hsa-miR-4840.0103111.19741hsa-miR-3194-5p6.85E-073.900124hsa-miR-181a-5p0.04515911.07253hsa-miR-664a-3p7.63E-053.88288hsa-miR-44760.04527410.85468hsa-miR-1185-2-3p0.0013833.862222hsa-miR-6819-5p0.0376889.898777hsa-miR-4758-5p7.82E-053.842414hsa-miR-140-5p0.0284129.550995hsa-let-7d-3p6.58E-043.826686hsa-miR-18a-5p0.0464469.54463hsa-miR-4695-5p2.08E-043.821445hsa-miR-5195-3p0.0028599.140968hsa-miR-361-5p2.36E-053.804533hsa-miR-3180-5p0.003219.092558hsa-miR-378a-3p7.26E-043.781667hsa-miR-4745-5p0.0390119.050331hsa-miR-4646-5p6.62E-043.761816hsa-miR-6833-5p0.0219318.956721hsa-miR-486-3p6.37E-043.681377hsa-miR-660-5p0.0202878.722665hsa-miR-195-5p1.45E-043.608673hsa-miR-4433a-3p0.0037258.720988hsa-miR-6776-3p5.99E-043.600524hsa-miR-80637.43E-068.708197hsa-miR-17-3p6.12E-043.592142hsa-miR-130b-3p0.0469438.396238hsa-miR-150-5p0.0297623.567105hsa-miR-47390.0396978.289042hsa-miR-6722-3p6.77E-043.542223hsa-miR-60684.80E-047.934393hsa-miR-378i4.34E-053.502339hsa-miR-193a-5p4.43E-047.75249hsa-miR-3940-3p9.02E-043.498083hsa-miR-1224-5p0.0043647.742238hsa-miR-4485-3p0.0026773.442792hsa-miR-39112.79E-047.694816hsa-miR-6341.67E-043.441633hsa-miR-7845-5p6.02E-047.552491hsa-miR-6787-3p0.003723.441111hsa-miR-30a-5p0.0290127.426994hsa-miR-514b-5p2.73E-043.394248hsa-miR-46985.20E-047.31334hsa-miR-28610.0378563.355749hsa-miR-14710.0098177.098284hsa-miR-1981.07E-043.355455hsa-miR-149-5p9.09E-046.732034hsa-miR-7-5p2.00E-043.32485hsa-miR-4713-3p6.83E-046.298571hsa-miR-44285.76E-053.290763hsa-miR-6776-5p4.04E-046.297086hsa-miR-6880-5p5.72E-063.155543hsa-miR-39178.92E-046.22132hsa-miR-46510.0142242.89093hsa-miR-3156-5p9.85E-046.071382hsa-miR-874-3p0.0087732.882781hsa-miR-60738.90E-046.025009hsa-miR-320c0.0108282.859505hsa-miR-80643.26E-045.989857hsa-miR-129-1-3p0.0092932.727352hsa-miR-5010-3p5.73E-045.836541hsa-miR-129-2-3p0.0062082.7257hsa-miR-301a-3p0.021775.759432hsa-miR-4685-5p0.0033592.484358hsa-miR-23c7.66E-045.753131hsa-miR-71500.0202382.12366hsa-miR-6778-5p5.87E-045.742155hsa-miR-6769b-5p0.0497032.113569hsa-miR-44960.0010295.662955hsa-miR-44430.0163192.110317hsa-miR-4728-5p9.17E-045.617614hsa-miR-80690.0230952.060308Table 2
Downregulated exosomal miRNAs in gastric cancer.
miRNAsp valueFold-changehsa-miR-12020.004258-2.41459hsa-miR-1249-3p6.59E-04-7.00656hsa-miR-1268b0.017473-2.80673hsa-miR-188-5p0.028927-3.4416hsa-miR-3124-5p1.31E-04-5.7789hsa-miR-31490.007595-74.7889hsa-miR-3151-3p0.034851-2.57356hsa-miR-3162-5p1.71E-04-2.32991hsa-miR-32-3p0.005302-95.2611hsa-miR-42980.016032-2.16797hsa-miR-43230.001012-6.27546hsa-miR-4433a-5p0.048353-2.48552hsa-miR-44550.047934-10.1695hsa-miR-4664-3p0.046873-2.36319hsa-miR-4665-3p0.030008-2.55911hsa-miR-4701-3p0.011413-32.2hsa-miR-4738-3p0.001106-3.71895hsa-miR-4749-3p0.048681-2.24699hsa-miR-5571-5p0.003511-5.16292hsa-miR-574-5p0.035445-9.07774hsa-miR-5950.006773-50.1615hsa-miR-60760.002865-2.05885hsa-miR-60870.021551-2.55464hsa-miR-61270.001076-2.17353hsa-miR-61652.60E-05-2.55632hsa-miR-6360.010713-6.53533hsa-miR-6508-5p0.049217-2.09058hsa-miR-6737-3p0.02317-2.74204hsa-miR-6785-3p0.049271-3.27601hsa-miR-6824-3p0.042667-4.52891hsa-miR-6870-3p0.016952-5.32729hsa-miR-6879-5p1.86E-04-2.13504hsa-miR-6880-3p0.020752-4.65907hsa-miR-9330.033641-4.54467Figure 3
Differentially expressed exosomal miRNAs and their target mRNAs in gastric cancer. (a) Volcano map of differentially expressed exosomal miRNAs in gastric cancer and healthy specimens. Red: upregulation; blue: downregulation. (b) Hierarchical clustering analysis of differentially expressed exosomal miRNAs in gastric cancer and healthy specimens. (c) Venn diagram of the shared target mRNAs of differentially expressed exosomal miRNAs by three databases: microRNAorg, PITA, and TargetScan.
(a)(b)(c)


### 3.4. Biological Functions of Target mRNAs of Differentially Expressed Exosomal miRNAs

Functional annotations were utilized for characterizing biological implications of target mRNAs of differentially expressed exosomal miRNAs. In Figure4(a), above mRNAs were distinctly linked to Wnt pathways, cell adhesion, chromatin modification, protein autophosphorylation, protein dephosphorylation, and protein phosphorylation processes. Also, they were markedly related to different cellular components like cytoplasm, nucleoplasm, postsynaptic density, and nucleus (Figure 4(b)). As shown in Figure 4(c), molecular functions such as protein binding, protein kinase binding, and ubiquitin protein ligase binding were significantly enriched by above mRNAs. In Figure 4(d), pathways in cancer, focal adhesion, regulation of actin cytoskeleton, signaling pathways regulating pluripotency of stem cells, PI3K-Akt pathways, EGFR tyrosine kinase inhibitor resistance, insulin, Hippo, CMAP, FoxO pathway, and AMPK pathways were markedly correlated to these mRNAs.Figure 4
Functional annotation analyses of target mRNAs of differentially expressed exosomal miRNAs. (a) Biological processes; (b) cellular components; (c) molecular functions; (d) KEGG pathways.
(a)(b)(c)(d)


### 3.5. Exosomal miR-4741, miR-32, miR-3149, and miR-6727 as Potential Markers Diagnosing Gastric Cancer

This study enrolled 60 early gastric cancer patients, 60 middle-advanced patients, and 57 healthy participants. Firstly, no statistical significance in age was found among them (Figure5(a)). The serum levels of cancer biomarkers ProGRP, CEA, and CA199 were detected in each subject. In Figure 5(b), there was no significance in serum ProGRP levels between early and middle-advanced gastric cancer subjects. Moreover, serum CEA and CA199 levels were not significant among healthy, early, and middle-advanced gastric cancer subjects (Figures 5(c) and 5(d)). Among all differentially expressed exosomal miRNAs, we validated the expressions of miR-32, miR-3149, and miR-4741 as well as miR-6727 in healthy and gastric cancer subjects through RT-qPCR. As a result, miR-32, miR-3149, and miR-6727 were all markedly downregulated while miR-4741 was markedly upregulated in gastric cancer than control tissues (Figure 5(e)). Furthermore, the expressions of miR-4741, miR-32, and miR-3149 as well as miR-6727 were detected in plasma specimens of healthy, early, and middle-advanced gastric cancer subjects. Compared to healthy controls, exosomal miR-4741 exhibited increased expression in early and middle-advanced patients (Figure 5(f)), and lower exosomal miR-4741 expression was observed in middle-advanced than early patients. Meanwhile, exosomal miR-32, miR-3149, and miR-6727 displayed decreased expression in early and middle-advanced patients (Figures 5(g)–5(i)). Also, lower expression of miR-3149 was found in middle-advanced than early patients. ROC curves were drawn for assessing the diagnostic performance of exosomal miR-4741, miR-32, miR-3149, and miR-6727 in gastric cancer. The AUCs of miR-4741, miR-32, miR-3149, and miR-6727 were separately 0.8554, 0.9456, 0.7683, and 0.8923 (Figures 5(j)–5(m)). The above data indicated that exosomal miR-4741, miR-32, and miR-6727 exhibited higher sensitivity and accuracy in diagnosing gastric cancer. The expression of miR-4741, miR-32, miR-3149, and miR-6727 was further validated in plasma from gastric cancer patients and healthy controls in the GSE86822 dataset. Consistently, circulating miR-4741 exhibited higher expression while circulating miR-32, miR-3149, and miR-6727 had lower expression in gastric cancer patients compared with healthy controls (Figures 5(n)–5(q)).Figure 5
Exosomal miR-4741, miR-32, miR-3149, and miR-6727 as promising markers for diagnosing gastric cancer. (a) Comparisons of age distribution among healthy, early gastric cancer and middle-advanced gastric cancer. (b) Differences in serum ProGRP levels in early and middle-advanced gastric cancer. (c) Comparisons of serum CEA levels among healthy, early gastric cancer and middle-advanced gastric cancer. (d) Differences in serum CA199 levels among healthy, early gastric cancer and middle-advanced gastric cancer. (e) Comparisons of the expression of exosomal miR-3149, miR-32, miR-4741, and miR-6727 in healthy subjects and gastric cancer patients. (f–i) Comparisons of the expressions of exosomal (f) miR-4741, (g) miR-32, (h) miR-3149 as well as (i) miR-6727 among healthy, early gastric cancer and middle-advanced gastric cancer. (j–m) ROC curves of evaluating the efficacy of exosomal (j) miR-4741, (k) miR-32, (l) miR-3149, and (m) miR-6727 expression for diagnosing gastric cancer. (n–q) External validation of exosomal (n) miR-4741, (o) miR-32, (p) miR-3149, and (q) miR-6727 expression in gastric cancer patients and healthy subjects in the GSE86822 dataset. Ns: not significant;∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)(o)(p)(q)


### 3.6. MiR-32, miR-3149, and miR-6727 Upregulations Weaken Proliferative Ability and Enhance Apoptotic Levels of Gastric Cancer Cells

For investigating the effects of miR-32, miR-3149, and miR-6727 in gastric cancer progression, miR-32 mimic, miR-3149 mimic, and miR-6727 mimic were transfected into MGC-803 cells to enhance their expressions (Figures6(a)–6(c)). CCK-8 assay was carried out for observing the proliferative capacities of gastric cancer cells. Our data demonstrated that cell viability was markedly weakened by miR-32 mimic, miR-3149 mimic, and miR-6727 mimic in MGC-803 cells in comparison to their controls (Figures 6(d)–6(f)). Apoptotic levels were examined by flow cytometry. Our data suggested that miR-32 mimic, miR-3149 mimic, and miR-6727 mimic markedly elevated the apoptotic levels of MGC-803 cells than their controls (Figures 6(g)–6(l)).Figure 6
MiR-32, miR-3149, and miR-6727 upregulations weaken proliferative capacity and elevate apoptotic levels in gastric cancer cells. (a–c) RT-qPCR for examining the expressions of miR-32, miR-3149 as well as miR-6727 in MGC-803 cells under transfections with miR-32 mimic, miR-3149 mimic, and miR-6727 mimic and their controls. (d–f) CCK-8 for the cell viability of MGC-803 cells under transfections with miR-32 mimic, miR-3149 mimic, and miR-6727 mimic and their controls. (g–l) Flow cytometry for the apoptotic levels of MGC-803 cells under transfections with (g, h) miR-32 mimic, (i, j) miR-3149 mimic, and (k, l) miR-6727 mimic and their controls.∗∗p<0.01; ∗∗∗p<0.001; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)MiR-32, miR-3149, and miR-6727 upregulations weaken migratory and invasive capacities of gastric cancer cells.The migratory capacities of MGC-803 cells were evaluated through wound healing assays. After transfections with miR-32 mimic, miR-3149 mimic, and miR-6727 mimic, migratory distances were distinctly diminished in MGC-803 cells than their controls (Figures7(a)–7(d)). Moreover, invasive levels of MGC-803 cells were tested utilizing Transwell assays. As a result, we found that invasive abilities were markedly decreased by miR-32 mimic, miR-3149 mimic, and miR-6727 mimic in MGC-803 cells compared to their controls (Figures 7(e)–7(h)).Figure 7
MiR-32, miR-3149, and miR-6727 upregulations weaken migratory and invasive capacities of gastric cancer cells. (a–d) Wound healing for migration distances of MGC-803 cells under transfections with (b) miR-32 mimic, (c) miR-3149 mimic, and (d) miR-6727 mimic and their controls. (e–h) Transwell assay for invasive levels of MGC-803 cells under transfections with (f) miR-32 mimic, (g) miR-3149 mimic, and (h) miR-6727 mimic and their controls.∗∗∗p<0.001; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.7. Inhibition of miR-4741 Weakens Proliferation, Migration, and Invasion and Enhances Apoptosis of Gastric Cancer Cells

Then, MGC-803 cells were transfected with miR-4741 inhibitor to lower its expression (Figure8(a)). miR-4741 inhibitor lowered the cell viability (Figure 8(b)) and enhanced apoptotic level (Figures 8(c) and 8(d)) as well as suppressed migratory and invasive capacities (Figures 8(e)–8(h)) of MGC-803 cells.Figure 8
Inhibition of miR-4741 weakens proliferation, migration, and invasion and enhances apoptosis of gastric cancer cells. (a) RT-qPCR of miR-4741 expression in MGC-803 cells under transfections with miR-4741 inhibitor or their controls. (b) CCK-8 of the cell viability of MGC-803 cells under transfections with miR-4741 inhibitor or their controls. (c, d) Flow cytometry for the apoptotic levels of MGC-803 cells under transfections with miR-4741 inhibitor or their controls. (e, f) Wound healing for migration distances of MGC-803 cells transfected with miR-4741 inhibitor or their controls. (g, h) Transwell assay for invasive levels of MGC-803 cells transfected with miR-4741 inhibitor or their controls.∗∗∗p<0.001; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.1. Isolation and Identification of Plasma Exosomes

To identify exosomes isolated from plasma specimens of gastric cancer and control subjects, this study first employed TEM to conduct preliminary identification of the morphology of exosomes. The results were shown in Figure1(a). The exosomes extracted from the patient’s plasma showed a typical electron microscope shape: saucer-shaped or a hemispherical concave on one side. The diameter of most exosomes was around 50-100 nm. Western blot results showed that the exosomes in the patient’s plasma all expressed CD63, CD9, and TSG101 proteins, but almost no GM130 protein (Figure 1(b)). This study further used NTA to determine the diameter and density of exosomes. As shown in our results, the diameter of plasma exosomes from healthy and gastric cancer subjects ranged from 50 to 200 nm, mainly around 75 nm (Figure 1(c)).Figure 1
Isolation and identification of plasma exosomes from gastric cancer patients. (a) Transmission electron microscopy for observing the morphological characteristics of isolated exosomes.Bar=100nm. (b) Western blot for detecting the expression of exosome markers CD63, CD9, TSG1010, and GM130. (c) Nanoparticle tracking analysis for detecting the particle size and concentration of exosomes.
(a)(b)(c)


## 3.2. Microarray Expression Profiles of Exosomal miRNAs for Gastric Cancer

To characterize the exosomal miRNA expression profiles of gastric cancer as well as control subjects, this study carried out microarray analyses. Figure2(a) showed the dispersion of microarray data of each specimen. A scatter plot was drawn on the standardized data in a two-dimensional coordinate to evaluate the central tendency of the overall distribution of the two groups of data. The scatter plot array of the data pairwise comparison was drawn into a matrix diagram, as shown in Figure 2(b). The PCA analysis was performed on all samples, and the distribution of samples was investigated to verify the rationality of the experimental design and the uniformity of biological replicate samples, which was displayed in a two-dimensional graph (Figure 2(c)).Figure 2
Microarray expression profiles of exosomal miRNAs in gastric cancer. (a) Box plot of the dispersion of microarray data. (b) Scatter plots of the overall distribution of the data from gastric cancer and control specimens. (c) PCA for the sample distributions based on the expression profiles.
(a)(b)(c)


## 3.3. Differentially Expressed Exosomal miRNAs for Gastric Cancer

Based on the microarrays, we screened differentially expressed exosomal miRNAs withfold−change≥2.0 and p≤0.05. As a result, 142 exosomal miRNAs were upregulated (Table 1), and 34 were downregulated (Table 2) in gastric cancer compared to healthy subjects (Figures 3(a) and 3(b)). By three databases microRNAorg, PITA, and TargetScan, 6823 target mRNAs were predicted for these differentially expressed exosomal miRNAs (Figure 3(c); Supplementary Table 1).Table 1
Upregulated exosomal miRNAs in gastric cancer.
miRNAsp valueFold-changemiRNAsp valueFold-changehsa-miR-6727-5p1.50E-0498.41242hsa-miR-3614-5p0.0013215.420335hsa-miR-47411.35E-0584.10587hsa-miR-46882.91E-075.338095hsa-miR-5585-3p3.49E-0461.08826hsa-miR-39453.66E-065.113267hsa-miR-45302.23E-0658.76396hsa-miR-6021.17E-055.065191hsa-miR-142-5p6.11E-0555.08285hsa-miR-2276-3p2.26E-064.952288hsa-miR-494-3p4.83E-0548.00325hsa-miR-186-5p0.0031374.919776hsa-miR-23a-3p0.04723544.9474hsa-miR-6890-3p2.43E-054.879964hsa-miR-30e-5p1.70E-0444.5984hsa-miR-6757-3p1.98E-054.753316hsa-miR-130a-3p0.04934640.1765hsa-miR-80732.16E-064.750058hsa-miR-6791-5p1.16E-0535.17456hsa-miR-1914-3p1.06E-054.634837hsa-miR-31382.06E-0633.31427hsa-miR-193b-5p6.42E-074.594794hsa-miR-7180.00466529.61524hsa-miR-6857-3p6.12E-054.579425hsa-miR-61260.01508423.6956hsa-miR-223-3p0.038844.501325hsa-miR-27b-3p0.02486523.45536hsa-miR-3613-3p4.37E-054.491396hsa-miR-211-3p0.00288123.01478hsa-miR-5751.11E-054.489378hsa-miR-30c-5p0.02093122.89729hsa-miR-6780b-5p6.35E-064.470476hsa-miR-331-3p0.01967822.28292hsa-let-7g-5p0.0479034.458645hsa-miR-134-5p0.01388521.13077hsa-miR-47063.28E-074.456074hsa-miR-4632-5p0.00146220.79573hsa-miR-6511b-5p2.98E-064.425767hsa-miR-144-5p0.01077619.00057hsa-miR-652-5p2.63E-054.342584hsa-miR-23b-3p0.02604118.41381hsa-miR-345-3p5.19E-064.331377hsa-miR-7108-5p0.00227417.76675hsa-miR-4666b6.64E-054.322666hsa-miR-199a-5p0.03559717.26349hsa-miR-96-5p2.90E-044.248471hsa-miR-29b-3p0.01216517.26232hsa-miR-4419a4.90E-044.242825hsa-miR-374a-5p0.02898217.12239hsa-miR-5634.27E-044.204622hsa-miR-7846-3p0.01228916.88478hsa-miR-60863.10E-044.193719hsa-miR-42539.23E-0416.36829hsa-miR-6734-5p4.61E-064.14653hsa-miR-151b0.02194314.60052hsa-miR-1233-5p6.23E-074.100964hsa-miR-146a-5p0.03406814.0491hsa-miR-183-5p3.44E-064.098074hsa-miR-6786-5p0.00290414.02458hsa-miR-135a-3p4.10E-044.085765hsa-miR-5001-5p0.00130913.43416hsa-miR-31475.22E-044.067993hsa-miR-43100.00846613.00651hsa-miR-31311.35E-064.047892hsa-miR-590-5p0.01687912.24443hsa-miR-98-3p1.93E-044.016676hsa-miR-3667-5p0.00218812.07358hsa-miR-210-3p0.0016783.958811hsa-miR-3940-5p0.02009911.56078hsa-miR-18b-5p5.58E-043.953054hsa-miR-4840.0103111.19741hsa-miR-3194-5p6.85E-073.900124hsa-miR-181a-5p0.04515911.07253hsa-miR-664a-3p7.63E-053.88288hsa-miR-44760.04527410.85468hsa-miR-1185-2-3p0.0013833.862222hsa-miR-6819-5p0.0376889.898777hsa-miR-4758-5p7.82E-053.842414hsa-miR-140-5p0.0284129.550995hsa-let-7d-3p6.58E-043.826686hsa-miR-18a-5p0.0464469.54463hsa-miR-4695-5p2.08E-043.821445hsa-miR-5195-3p0.0028599.140968hsa-miR-361-5p2.36E-053.804533hsa-miR-3180-5p0.003219.092558hsa-miR-378a-3p7.26E-043.781667hsa-miR-4745-5p0.0390119.050331hsa-miR-4646-5p6.62E-043.761816hsa-miR-6833-5p0.0219318.956721hsa-miR-486-3p6.37E-043.681377hsa-miR-660-5p0.0202878.722665hsa-miR-195-5p1.45E-043.608673hsa-miR-4433a-3p0.0037258.720988hsa-miR-6776-3p5.99E-043.600524hsa-miR-80637.43E-068.708197hsa-miR-17-3p6.12E-043.592142hsa-miR-130b-3p0.0469438.396238hsa-miR-150-5p0.0297623.567105hsa-miR-47390.0396978.289042hsa-miR-6722-3p6.77E-043.542223hsa-miR-60684.80E-047.934393hsa-miR-378i4.34E-053.502339hsa-miR-193a-5p4.43E-047.75249hsa-miR-3940-3p9.02E-043.498083hsa-miR-1224-5p0.0043647.742238hsa-miR-4485-3p0.0026773.442792hsa-miR-39112.79E-047.694816hsa-miR-6341.67E-043.441633hsa-miR-7845-5p6.02E-047.552491hsa-miR-6787-3p0.003723.441111hsa-miR-30a-5p0.0290127.426994hsa-miR-514b-5p2.73E-043.394248hsa-miR-46985.20E-047.31334hsa-miR-28610.0378563.355749hsa-miR-14710.0098177.098284hsa-miR-1981.07E-043.355455hsa-miR-149-5p9.09E-046.732034hsa-miR-7-5p2.00E-043.32485hsa-miR-4713-3p6.83E-046.298571hsa-miR-44285.76E-053.290763hsa-miR-6776-5p4.04E-046.297086hsa-miR-6880-5p5.72E-063.155543hsa-miR-39178.92E-046.22132hsa-miR-46510.0142242.89093hsa-miR-3156-5p9.85E-046.071382hsa-miR-874-3p0.0087732.882781hsa-miR-60738.90E-046.025009hsa-miR-320c0.0108282.859505hsa-miR-80643.26E-045.989857hsa-miR-129-1-3p0.0092932.727352hsa-miR-5010-3p5.73E-045.836541hsa-miR-129-2-3p0.0062082.7257hsa-miR-301a-3p0.021775.759432hsa-miR-4685-5p0.0033592.484358hsa-miR-23c7.66E-045.753131hsa-miR-71500.0202382.12366hsa-miR-6778-5p5.87E-045.742155hsa-miR-6769b-5p0.0497032.113569hsa-miR-44960.0010295.662955hsa-miR-44430.0163192.110317hsa-miR-4728-5p9.17E-045.617614hsa-miR-80690.0230952.060308Table 2
Downregulated exosomal miRNAs in gastric cancer.
miRNAsp valueFold-changehsa-miR-12020.004258-2.41459hsa-miR-1249-3p6.59E-04-7.00656hsa-miR-1268b0.017473-2.80673hsa-miR-188-5p0.028927-3.4416hsa-miR-3124-5p1.31E-04-5.7789hsa-miR-31490.007595-74.7889hsa-miR-3151-3p0.034851-2.57356hsa-miR-3162-5p1.71E-04-2.32991hsa-miR-32-3p0.005302-95.2611hsa-miR-42980.016032-2.16797hsa-miR-43230.001012-6.27546hsa-miR-4433a-5p0.048353-2.48552hsa-miR-44550.047934-10.1695hsa-miR-4664-3p0.046873-2.36319hsa-miR-4665-3p0.030008-2.55911hsa-miR-4701-3p0.011413-32.2hsa-miR-4738-3p0.001106-3.71895hsa-miR-4749-3p0.048681-2.24699hsa-miR-5571-5p0.003511-5.16292hsa-miR-574-5p0.035445-9.07774hsa-miR-5950.006773-50.1615hsa-miR-60760.002865-2.05885hsa-miR-60870.021551-2.55464hsa-miR-61270.001076-2.17353hsa-miR-61652.60E-05-2.55632hsa-miR-6360.010713-6.53533hsa-miR-6508-5p0.049217-2.09058hsa-miR-6737-3p0.02317-2.74204hsa-miR-6785-3p0.049271-3.27601hsa-miR-6824-3p0.042667-4.52891hsa-miR-6870-3p0.016952-5.32729hsa-miR-6879-5p1.86E-04-2.13504hsa-miR-6880-3p0.020752-4.65907hsa-miR-9330.033641-4.54467Figure 3
Differentially expressed exosomal miRNAs and their target mRNAs in gastric cancer. (a) Volcano map of differentially expressed exosomal miRNAs in gastric cancer and healthy specimens. Red: upregulation; blue: downregulation. (b) Hierarchical clustering analysis of differentially expressed exosomal miRNAs in gastric cancer and healthy specimens. (c) Venn diagram of the shared target mRNAs of differentially expressed exosomal miRNAs by three databases: microRNAorg, PITA, and TargetScan.
(a)(b)(c)


## 3.4. Biological Functions of Target mRNAs of Differentially Expressed Exosomal miRNAs

Functional annotations were utilized for characterizing biological implications of target mRNAs of differentially expressed exosomal miRNAs. In Figure4(a), above mRNAs were distinctly linked to Wnt pathways, cell adhesion, chromatin modification, protein autophosphorylation, protein dephosphorylation, and protein phosphorylation processes. Also, they were markedly related to different cellular components like cytoplasm, nucleoplasm, postsynaptic density, and nucleus (Figure 4(b)). As shown in Figure 4(c), molecular functions such as protein binding, protein kinase binding, and ubiquitin protein ligase binding were significantly enriched by above mRNAs. In Figure 4(d), pathways in cancer, focal adhesion, regulation of actin cytoskeleton, signaling pathways regulating pluripotency of stem cells, PI3K-Akt pathways, EGFR tyrosine kinase inhibitor resistance, insulin, Hippo, CMAP, FoxO pathway, and AMPK pathways were markedly correlated to these mRNAs.Figure 4
Functional annotation analyses of target mRNAs of differentially expressed exosomal miRNAs. (a) Biological processes; (b) cellular components; (c) molecular functions; (d) KEGG pathways.
(a)(b)(c)(d)


## 3.5. Exosomal miR-4741, miR-32, miR-3149, and miR-6727 as Potential Markers Diagnosing Gastric Cancer

This study enrolled 60 early gastric cancer patients, 60 middle-advanced patients, and 57 healthy participants. Firstly, no statistical significance in age was found among them (Figure5(a)). The serum levels of cancer biomarkers ProGRP, CEA, and CA199 were detected in each subject. In Figure 5(b), there was no significance in serum ProGRP levels between early and middle-advanced gastric cancer subjects. Moreover, serum CEA and CA199 levels were not significant among healthy, early, and middle-advanced gastric cancer subjects (Figures 5(c) and 5(d)). Among all differentially expressed exosomal miRNAs, we validated the expressions of miR-32, miR-3149, and miR-4741 as well as miR-6727 in healthy and gastric cancer subjects through RT-qPCR. As a result, miR-32, miR-3149, and miR-6727 were all markedly downregulated while miR-4741 was markedly upregulated in gastric cancer than control tissues (Figure 5(e)). Furthermore, the expressions of miR-4741, miR-32, and miR-3149 as well as miR-6727 were detected in plasma specimens of healthy, early, and middle-advanced gastric cancer subjects. Compared to healthy controls, exosomal miR-4741 exhibited increased expression in early and middle-advanced patients (Figure 5(f)), and lower exosomal miR-4741 expression was observed in middle-advanced than early patients. Meanwhile, exosomal miR-32, miR-3149, and miR-6727 displayed decreased expression in early and middle-advanced patients (Figures 5(g)–5(i)). Also, lower expression of miR-3149 was found in middle-advanced than early patients. ROC curves were drawn for assessing the diagnostic performance of exosomal miR-4741, miR-32, miR-3149, and miR-6727 in gastric cancer. The AUCs of miR-4741, miR-32, miR-3149, and miR-6727 were separately 0.8554, 0.9456, 0.7683, and 0.8923 (Figures 5(j)–5(m)). The above data indicated that exosomal miR-4741, miR-32, and miR-6727 exhibited higher sensitivity and accuracy in diagnosing gastric cancer. The expression of miR-4741, miR-32, miR-3149, and miR-6727 was further validated in plasma from gastric cancer patients and healthy controls in the GSE86822 dataset. Consistently, circulating miR-4741 exhibited higher expression while circulating miR-32, miR-3149, and miR-6727 had lower expression in gastric cancer patients compared with healthy controls (Figures 5(n)–5(q)).Figure 5
Exosomal miR-4741, miR-32, miR-3149, and miR-6727 as promising markers for diagnosing gastric cancer. (a) Comparisons of age distribution among healthy, early gastric cancer and middle-advanced gastric cancer. (b) Differences in serum ProGRP levels in early and middle-advanced gastric cancer. (c) Comparisons of serum CEA levels among healthy, early gastric cancer and middle-advanced gastric cancer. (d) Differences in serum CA199 levels among healthy, early gastric cancer and middle-advanced gastric cancer. (e) Comparisons of the expression of exosomal miR-3149, miR-32, miR-4741, and miR-6727 in healthy subjects and gastric cancer patients. (f–i) Comparisons of the expressions of exosomal (f) miR-4741, (g) miR-32, (h) miR-3149 as well as (i) miR-6727 among healthy, early gastric cancer and middle-advanced gastric cancer. (j–m) ROC curves of evaluating the efficacy of exosomal (j) miR-4741, (k) miR-32, (l) miR-3149, and (m) miR-6727 expression for diagnosing gastric cancer. (n–q) External validation of exosomal (n) miR-4741, (o) miR-32, (p) miR-3149, and (q) miR-6727 expression in gastric cancer patients and healthy subjects in the GSE86822 dataset. Ns: not significant;∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)(o)(p)(q)


## 3.6. MiR-32, miR-3149, and miR-6727 Upregulations Weaken Proliferative Ability and Enhance Apoptotic Levels of Gastric Cancer Cells

For investigating the effects of miR-32, miR-3149, and miR-6727 in gastric cancer progression, miR-32 mimic, miR-3149 mimic, and miR-6727 mimic were transfected into MGC-803 cells to enhance their expressions (Figures6(a)–6(c)). CCK-8 assay was carried out for observing the proliferative capacities of gastric cancer cells. Our data demonstrated that cell viability was markedly weakened by miR-32 mimic, miR-3149 mimic, and miR-6727 mimic in MGC-803 cells in comparison to their controls (Figures 6(d)–6(f)). Apoptotic levels were examined by flow cytometry. Our data suggested that miR-32 mimic, miR-3149 mimic, and miR-6727 mimic markedly elevated the apoptotic levels of MGC-803 cells than their controls (Figures 6(g)–6(l)).Figure 6
MiR-32, miR-3149, and miR-6727 upregulations weaken proliferative capacity and elevate apoptotic levels in gastric cancer cells. (a–c) RT-qPCR for examining the expressions of miR-32, miR-3149 as well as miR-6727 in MGC-803 cells under transfections with miR-32 mimic, miR-3149 mimic, and miR-6727 mimic and their controls. (d–f) CCK-8 for the cell viability of MGC-803 cells under transfections with miR-32 mimic, miR-3149 mimic, and miR-6727 mimic and their controls. (g–l) Flow cytometry for the apoptotic levels of MGC-803 cells under transfections with (g, h) miR-32 mimic, (i, j) miR-3149 mimic, and (k, l) miR-6727 mimic and their controls.∗∗p<0.01; ∗∗∗p<0.001; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)MiR-32, miR-3149, and miR-6727 upregulations weaken migratory and invasive capacities of gastric cancer cells.The migratory capacities of MGC-803 cells were evaluated through wound healing assays. After transfections with miR-32 mimic, miR-3149 mimic, and miR-6727 mimic, migratory distances were distinctly diminished in MGC-803 cells than their controls (Figures7(a)–7(d)). Moreover, invasive levels of MGC-803 cells were tested utilizing Transwell assays. As a result, we found that invasive abilities were markedly decreased by miR-32 mimic, miR-3149 mimic, and miR-6727 mimic in MGC-803 cells compared to their controls (Figures 7(e)–7(h)).Figure 7
MiR-32, miR-3149, and miR-6727 upregulations weaken migratory and invasive capacities of gastric cancer cells. (a–d) Wound healing for migration distances of MGC-803 cells under transfections with (b) miR-32 mimic, (c) miR-3149 mimic, and (d) miR-6727 mimic and their controls. (e–h) Transwell assay for invasive levels of MGC-803 cells under transfections with (f) miR-32 mimic, (g) miR-3149 mimic, and (h) miR-6727 mimic and their controls.∗∗∗p<0.001; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.7. Inhibition of miR-4741 Weakens Proliferation, Migration, and Invasion and Enhances Apoptosis of Gastric Cancer Cells

Then, MGC-803 cells were transfected with miR-4741 inhibitor to lower its expression (Figure8(a)). miR-4741 inhibitor lowered the cell viability (Figure 8(b)) and enhanced apoptotic level (Figures 8(c) and 8(d)) as well as suppressed migratory and invasive capacities (Figures 8(e)–8(h)) of MGC-803 cells.Figure 8
Inhibition of miR-4741 weakens proliferation, migration, and invasion and enhances apoptosis of gastric cancer cells. (a) RT-qPCR of miR-4741 expression in MGC-803 cells under transfections with miR-4741 inhibitor or their controls. (b) CCK-8 of the cell viability of MGC-803 cells under transfections with miR-4741 inhibitor or their controls. (c, d) Flow cytometry for the apoptotic levels of MGC-803 cells under transfections with miR-4741 inhibitor or their controls. (e, f) Wound healing for migration distances of MGC-803 cells transfected with miR-4741 inhibitor or their controls. (g, h) Transwell assay for invasive levels of MGC-803 cells transfected with miR-4741 inhibitor or their controls.∗∗∗p<0.001; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)


## 4. Discussion

Although therapeutic strategies have made distinct progress, prognoses of gastric cancer patients at late stages are unfavorable [19–21]. Due to asymptomatic features at early stages, patients are usually delayed in diagnosis, thereby missing the best time for surgery [22]. Hence, diagnosing gastric cancer as early as possible is of importance for prolonging patients’ survival duration [23]. This study conducted the microarray analyses of exosomal miRNAs in gastric cancer as well as identified exosomal miR-4741, miR-32, miR-3149, and miR-6727 as novel noninvasive markers possessing high sensitivity and specificity for diagnosing gastric cancer.As previously reported, miR-32 displayed upregulation in gastric cancer tissues [24]. miR-32 exerted an inhibitory effect on the proliferative and invasive capacities of gastric cancer cells [25]. Consistently, our data demonstrated the upregulation of miR-32 in gastric cancer tissues as well as plasma specimens. The upregulation weakened proliferation, migration, and invasion as well as enhanced apoptosis in gastric cancer cells. Except for gastric cancer, the implications of miR-32 have been investigated in different cancers. For instance, miR-32 induced tumorigenesis of colorectal cancer through targeting BMP5 [26]. miR-32 mediated radiation sensitivity, migratory, and invasive levels of colorectal cancer cells through TOB1 [27]. miR-32 facilitated esophageal squamous cell cancer metastases through CXXC5 [28]. miR-32 enhanced proliferation and motility of ovarian cancer cells through SMG1 [29]. miR-32 induced cellular proliferative ability of breast cancer cells through suppression of PHLPP2 expression [30]. miR-32 exerted an inhibitory role on proliferative ability and metastases through TWIST1 in non-small-cell lung cancer cells [31]. The AUC of exosomal miR-32 was 0.938, which was indicative of the well diagnostic performance in gastric cancer. A meta-analysis reported the circulating miR-21 as an accurate marker for diagnosing oral cancer [32]. Exosomal miR-32 upregulation may enhance multidrug-resistance of hepatocellular carcinoma through PI3K/Akt pathway [33]. Exosomal miR-32 may be predictive of the effects of chemotherapy and survival for non-small-cell lung cancer subjects [34]. Collectively, exosomal miR-32 possessed the potential for diagnosing and curing gastric cancer. Except for miR-32, miR-3149 and miR-6727 had lower expression as well as miR-4741 had higher expression in gastric cancer tissue and plasma specimens. Also, their upregulations exerted suppressive roles on gastric cancer progression.There are several weaknesses in our study. First, the diagnostic performance of exosomal miR-4741, miR-32, miR-3149, and miR-6727 requires to be verified in a larger cohort. Second, their roles on gastric cancer progression need to be observed in vivo. Third, the downstream targets of miR-4741, miR-32, miR-3149, and miR-6727 will be validated in our future studies.


## 5. Conclusion

Collectively, our research characterized the expression profiling of exosomal miRNAs for gastric cancer as well as proposed four exosomal miR-4741, miRNAs miR-32, miR-3149, and miR-6727 as promising markers for diagnosing gastric cancer. Upregulations of miR-32, miR-3149, and miR-6727 as well as downregulation of miR-4741 distinctly weakened proliferative, migratory, and invasive abilities as well as enhanced apoptotic levels in gastric cancer cells. Our findings were indicative of the potential of exosomal miR-4741, miR-32, miR-3149, and miR-6727 as diagnosed and therapeutic markers against gastric cancer.



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*Source: 1263812-2022-07-04.xml*