# Delivery of miR-224-5p by Exosomes from Cancer-Associated Fibroblasts Potentiates Progression of Clear Cell Renal Cell Carcinoma

Authors: Yifei Liu; Wenqiang Fu; Xiaoning Cao; Shuopeng Li; Tianyu Xiong; Xiaolei Zhang; Xiaotang Wu; Ling Cheng; Yanbing Wei; Bin Gao
Journal: Computational and Mathematical Methods in Medicine
 (2021)

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## Abstract

Objectives. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma. Cancer-associated fibroblasts (CAFs) as the primary components of cancer stroma can affect tumor progression by secreting exosomes, while exosomes are carriers for proteins, nucleic acids, and other agents that responsible for delivery of biological information. Given this, exosomes derived from CAFs are emerging as promising biomarkers in clinical cancer diagnosis. Nevertheless, their role in clear cell renal cell carcinoma (ccRCC) remains poorly understood. Methods. Here, we separated fibroblasts from ccRCC tissue, extracted exosomes, observed their morphology, and detected the expression of exosome marker proteins including Hsp70, CD9, and CD63. In the meantime, we labeled exosomes and performed coculture experiment to verify the delivery of miR-224-5p from CAFs to 769-P cells with exosomes as a carrier, so as to clarify the effect of CAF-derived exosomes on ccRCC cell malignant behaviors, as well as to discuss how miR-224-5p involves in above regulation. Results. Transmission electron microscopy was firstly applied, and it was noted that the exosomes we isolated were in normal range. Besides, Western blot also confirmed the presence of exosome marker proteins Hsp70, CD9, and CD63. Furthermore, coculture experiments were performed and the CAF-derived exosomes were observed to be able to facilitate the malignant behaviors of ccRCC cells, and the exosomal miR-224-5p could be internalized by ccRCC cells to participate in regulation of cell proliferation, migration, invasion, and apoptosis. Conclusion. To sum up, miR-224-5p can enter ccRCC cells via CAF-derived exosomes, in turn, promoting the malignant behaviors of ccRCC cells, which indicates that miR-224-5p has the potential severing as a therapeutic target for ccRCC.


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## Body

## 1. Introduction

Renal cell carcinoma (RCC) originates from renal epithelium, accounting for approximately 90% of cancers in kidney, of which clear cell renal cell carcinoma (ccRCC) is the most common subtype responsible for the majority of renal cancer deaths [1]. Generally, ccRCC is often diagnosed at an advanced metastatic stage, leading to dramatic decrease in patient’s survival rate [2]. Therefore, early diagnosis of ccRCC and monitoring of disease progression are the critical steps to improve the survival of patients [3].The “tumor” is composed of cancer and stromal cells. Cancer cells are generally believed to be malignant cells that do not undergo differentiation, while stromal cells are nonmalignant cells surrounding the cancer cells. Stromal cells consist of fibroblasts, vascular endothelial cells, and immune cells [4]. Fibroblasts are the basis of cancer stroma, while cancer-associated fibroblasts (CAFs) are the activated subpopulation of fibroblasts [5, 6]. CAFs are a type of perpetually activated fibroblasts that are believed to have a strong capability in tumor regulation and get involved in all stages of cancer progression [7, 8]. As reported, CAFs can regulate cell proliferation, migration, metabolism, and immune response by secreting various growth and cellular factors and proteases [9, 10]. Moreover, CAFs can deliver molecules into other cells via secreting exosomes to change the tumor environment [11].Exosomes are important extracellular vesicles that are cup-shaped, with a diameter of 40-150 nm [12]. Endosomes are firstly generated by endocytosis of cell membrane, and then, numerous vesicles take shape in endosomes. Thereafter, the endosomes fuse with the plasma membrane, which enables the intralumenal vesicles or called exosomes to be released into the extracellular medium [13]. In cancer, tumor cells aberrantly secret massive exosomes to transfer paracrine signals or to contribute to tumor-environment interactions at a distance [11]. Exosomes play a key role in cell-cell communication by transferring certain inclusions to target cells and tissue [14]. Proteins, DNA, and RNA are the main components of the inclusions, of which miRNAs may promote tumor growth, metastasis, angiogenesis, and drug resistance through interfering with tumor immunity and microenvironment [15]. Therefore, exosomal miRNAs are applied as promising biomarkers [16]. In addition to the fact that tumor cells can secrete exosomal miRNAs, CAF-derived exosomal miRNAs also play important roles in tumor growth [17]. Previous studies indicated that miR-224-5p is a kind of miRNA that are commonly aberrantly highly expressed in malignancies. For instance, miR-224-5p in papillary thyroid carcinoma targets EGR2 to promote cancer cell migration and invasion [18]; in breast cancer, miR-224-5p can suppress cancer cell autophagy [19], while in gastric cancer, miR-224-5p acts as an oncogene to facilitate cancer cell malignant behaviors [20]. Nevertheless, the role of miR-224-5p delivered by CAF-derived exosomes in mediating cancer biological behaviors have not been touched. Besides, studies regarding miR-224-5p regulating ccRCC malignant progression are still poor.Here, CAFs in ccRCC patients were identified to have the capacity to secrete exosomes which can make an effect on cancer cell phenotypes like proliferation. Besides, the exosomes were noted to have significantly highly expressed miR-224-5p, while miR-224-5p could be delivered from the exosomes to ccRCC cells to play a part in cancer cell phenotypes. Overall, the findings provided by our analysis help identify a potential biological target for ccRCC treatment.


## 2. Materials and Methods

### 2.1. Bioinformatics Analysis

Normal tissue and cancer tissue samples of 12 ccRCC patients in GSE109368 dataset were obtained from GEO database (http://www.ncbi.nlm.nih.gov/geo). Gene differential analysis (∣logFC∣>2, padj<0.05) based on the obtained samples was performed via applying the “edgeR” package, and differentially expressed miRNAs (DEmiRNAs) were obtained. Relevant literature was searched to identify the target miRNA, and the expression of the exosomal miRNA of interest was localized using the EVmiRNA database (http://bioinfo.life.hust.edu.cn/EVmiRNA).


### 2.2. Isolation of Human Primary Fibroblasts

A total of three patients undergoing partial or radical nephrectomy in Tangshan Gongren Hospital from December 2018 to October 2019 were included in this study. Tissue samples from the above patients including tumor and adjacent normal tissues were collected, and the normal part should be at least 3 cm away from the tumor margin. All patients met the criteria as follows: (1) all patients were pathologically diagnosed with ccRCC; (2) none of the patients had lymphatic metastasis or distant metastasis; (3) none of the patients had preoperative radiotherapy or chemotherapy; (4) all patients had signed informed consent prior to the beginning of the study, and this study was approved by the Ethics Committee of Tangshan Gongren Hospital.Freshly resected tissue samples were minced into approximately 1 mm3 cube chunks, and these chunks were then digested with collagenase. Cells were centrifugated at 1000 rpm for 10 min after being filtrated through a 75 μm filter. Next, Dulbecco’s modified Eagle medium (DMEM) which was supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA) as well as appropriate 100 μg/ml streptomycin and 100 U/ml penicillin (Gibco, Thermo Fisher Scientific, Inc.) was used for cell culture, and the culture condition was set to 5% CO2 and 37°C. After about 10 days, a uniform array of fibroblasts formed around cell debris. Fibroblasts that had been passaged more than 10 times were used for subsequent experiments.


### 2.3. ccRCC Cell Culture

ccRCC cell line 769-P (ATCC®CRL-1933) provided by American Type Culture Collection (Manassas, VA, USA) was placed in 10% FBS-supplemented DMEM for culture in an incubator containing 5% CO2 at a temperature of 37°C, with an addition of appropriate 100 μg/ml streptomycin and 100 U/ml penicillin (Gibco, Thermo Fisher Scientific, Inc.).


### 2.4. Exosomes Extraction

The Hieff™ exosome rapid isolation kit (Yeasen, Shanghai, China) was employed to extract exosomes, and the operation was in accordance with the manufacturer’s protocol. Firstly, fibroblasts were washed with phosphate-buffered saline (PBS) at 4°C when they were growing to 80% in confluence and then subjected to centrifugation at 2500 ×g lasting 10 min to isolate cell debris and dead cells in medium. Subsequently, 10 ml treated medium was collected into a new centrifuge tube mixed with 2.5 ml extraction reagent, standing for 2 h. Thereafter, the mixture was centrifuged at 10,000 ×g for 1 h at 4°C. The precipitate was collected for follow-up resuspension in 100μl PBS loading buffer. After that, the solution was transferred to an EP tubular and centrifuged again (12,000 ×g, 2 min, 4°C) to collect the supernatant with exosomes.


### 2.5. Transmission Electron Microscopy

The isolated fraction with exosomes was exposed to HEPES buffer (Invitrogen), after which an aliquot of the suspension was removed onto the carbon-coated grid of the JEOL JEM-2100 transmission electron microscope (100 kV). Thereafter, the grid was transferred into fresh PBS (Hyclone) for wash (3 times, 5 min per), fixed in 3% glutaraldehyde (Sigma-Aldrich) for 10 min, washed by dH2O three times (5 min per), and finally, exposed to 2% uranyl acetate (Sigma-Aldrich). The sample was then observed under the transmission electron microscope after several minutes of drying.


### 2.6. Coculture

Exosomes were mixed with PKH67 dye after resuscitated in loading buffer and incubated for 5 min at room temperature. The samples were then ultracentrifuged at 100,000 ×g for 1 h to remove the free dye with the addition of PBS plus 5% FBS. Stained exosomes were then cocultured with 769-P cell line for 6 h and fixed with 4% paraformaldehyde for 20 min at 25°C. At last, whether PKH67-labled exosomes are present in 769-P cells was determined via a fluorescence microscope.


### 2.7. qRT-PCR

Total RNA was extracted from cells or exosomes using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.). cDNA was synthesized using PrimeScript RT reagent kit (Takara, Japan) according to the manufacturer’s instructions. miRNA cDNA was synthesized with the miRcute Plus miRNA First-strand cDNA synthesis kit (Tianjin, China). qRT-PCR was performed on Applied Biosystems ABI 7500 real-time PCR system (Thermo Fisher Scientific, Inc.) using FastStart Universal SYBR Green Master (Rox) reagent kit (Roch, Basel, Switzerland). Normalization for miRNA expression was done using U6 expression. miR-224-5p forward primer: 5′-CTGGTAGGTAAGTCACTA-3′, reverse primer: 5′-TCAACTGGTGTCGTGGAG-3′; U6 forward primer: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, reverse primer: 5′-CGCTTCAGAATTTGCGTGTCAT-3′. Fold change was calculated by relative quantification method (2−△△Ct method). The data were representative of three independent experiments.


### 2.8. Western Blot

Cells were harvested in designate time and washed with PBS. The concentration of total proteins isolated from cells was detected by BCA protein assay kit (Thermo Fisher Scientific, USA). The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with specific primary antibodies overnight at 4°C after being blocked with 5% skim milk for 2 h at room temperature. The indicated primary antibodies included rabbit anti-CD63, rabbit anti-CD9, rabbit anti-Hsp70, rabbit anti-α-SMA, and rabbit anti-GAPDH. The membranes were then washed with TBST three times and incubated with a horseradish peroxidase- (HRP-) conjugated secondary antibody goat anti-rabbit IgG for 1 h at room temperature. Ultimately, all proteins were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.). All antibodies used here were ordered from Abcam.


### 2.9. Cell Transfection

As for cell transfection, miR-224-5p mimic (100μm), and its negative control NC mimic (100 μm) purchased from RiboBio (Guangzhou, China) were used for miR-224-5p overexpression in 769-P cells. After the transfection, cells were incubated for 48 h at 37°C to conduct follow-up experiments.


### 2.10. Cell Apoptosis Assay

Cell apoptosis was measured using Annexin V-FITC cell apoptosis assay kit (Beyotime, China). Cells were trypsin-treated (BD Biosciences, USA) and washed with cold PBS. Cells were centrifuged and then resuspended in 195μl of 1× Annexin V loading buffer after the supernatant was removed. Subsequently, cells were incubated with 5 μl Annexin-V/FITC, followed by 10 μl PI for 15 min at room temperature in the dark. The results were analyzed by CellQuest software (BD Biosciences, NJ, USA) of FACS Calibur flow cytometry (Becton Dickinson, CA, USA).


### 2.11. Cell Proliferation Assay

The cell proliferation was measured using CCK-8 kit (Dojindo, Japan) according to the information provided by the manufacturer. At indicated time points (0, 24, 48, and 72 h), 10μl CCK-8 was supplemented to each well, and cells were incubated for additional 2 h at 37°C. A spectrophotometer (Bio-Rad, USA) was then used to analyze the optical density (OD) at 450 nm.


### 2.12. Migration and Invasion Examination

The migration and invasion assays were performed using Transwell chambers (0.8μm pore size; Merck Millipore, USA). For Transwell migration assay, the cells (1×105) were suspended in 200 μl serum-free medium and plated onto the upper chamber, whereas DMEM with 10% FBS which served as attractant was added into the lower chamber. For Transwell invasion assay, the upper chamber was precoated with 50 μl Matrigel (Corning, USA) and dried for 4 h at 37°C. Measurement of cell migration or invasion was carried out 24 h postculture. The cells that migrated or invaded to the lower chamber were processed with 4% paraformaldehyde followed up 1% crystal violet. At last, cell number was counted for ability assessment in four fields selected at random under an optical microscope (100x).


### 2.13. Statistical Analysis

Analysis for the data in this study was carried out with GraphPad Prism 6.0 (La Jolla, CA), and all experiments were repeated in triplicate. Data presentation was done usingmean±standarddeviation. Between-group variance was assessed via Student’s t-test or one-way ANOVA. Statistical significance was defined when p value less than 0.05, while highly statistical significance was defined in the case of p value less than 0.01.


## 2.1. Bioinformatics Analysis

Normal tissue and cancer tissue samples of 12 ccRCC patients in GSE109368 dataset were obtained from GEO database (http://www.ncbi.nlm.nih.gov/geo). Gene differential analysis (∣logFC∣>2, padj<0.05) based on the obtained samples was performed via applying the “edgeR” package, and differentially expressed miRNAs (DEmiRNAs) were obtained. Relevant literature was searched to identify the target miRNA, and the expression of the exosomal miRNA of interest was localized using the EVmiRNA database (http://bioinfo.life.hust.edu.cn/EVmiRNA).


## 2.2. Isolation of Human Primary Fibroblasts

A total of three patients undergoing partial or radical nephrectomy in Tangshan Gongren Hospital from December 2018 to October 2019 were included in this study. Tissue samples from the above patients including tumor and adjacent normal tissues were collected, and the normal part should be at least 3 cm away from the tumor margin. All patients met the criteria as follows: (1) all patients were pathologically diagnosed with ccRCC; (2) none of the patients had lymphatic metastasis or distant metastasis; (3) none of the patients had preoperative radiotherapy or chemotherapy; (4) all patients had signed informed consent prior to the beginning of the study, and this study was approved by the Ethics Committee of Tangshan Gongren Hospital.Freshly resected tissue samples were minced into approximately 1 mm3 cube chunks, and these chunks were then digested with collagenase. Cells were centrifugated at 1000 rpm for 10 min after being filtrated through a 75 μm filter. Next, Dulbecco’s modified Eagle medium (DMEM) which was supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA) as well as appropriate 100 μg/ml streptomycin and 100 U/ml penicillin (Gibco, Thermo Fisher Scientific, Inc.) was used for cell culture, and the culture condition was set to 5% CO2 and 37°C. After about 10 days, a uniform array of fibroblasts formed around cell debris. Fibroblasts that had been passaged more than 10 times were used for subsequent experiments.


## 2.3. ccRCC Cell Culture

ccRCC cell line 769-P (ATCC®CRL-1933) provided by American Type Culture Collection (Manassas, VA, USA) was placed in 10% FBS-supplemented DMEM for culture in an incubator containing 5% CO2 at a temperature of 37°C, with an addition of appropriate 100 μg/ml streptomycin and 100 U/ml penicillin (Gibco, Thermo Fisher Scientific, Inc.).


## 2.4. Exosomes Extraction

The Hieff™ exosome rapid isolation kit (Yeasen, Shanghai, China) was employed to extract exosomes, and the operation was in accordance with the manufacturer’s protocol. Firstly, fibroblasts were washed with phosphate-buffered saline (PBS) at 4°C when they were growing to 80% in confluence and then subjected to centrifugation at 2500 ×g lasting 10 min to isolate cell debris and dead cells in medium. Subsequently, 10 ml treated medium was collected into a new centrifuge tube mixed with 2.5 ml extraction reagent, standing for 2 h. Thereafter, the mixture was centrifuged at 10,000 ×g for 1 h at 4°C. The precipitate was collected for follow-up resuspension in 100μl PBS loading buffer. After that, the solution was transferred to an EP tubular and centrifuged again (12,000 ×g, 2 min, 4°C) to collect the supernatant with exosomes.


## 2.5. Transmission Electron Microscopy

The isolated fraction with exosomes was exposed to HEPES buffer (Invitrogen), after which an aliquot of the suspension was removed onto the carbon-coated grid of the JEOL JEM-2100 transmission electron microscope (100 kV). Thereafter, the grid was transferred into fresh PBS (Hyclone) for wash (3 times, 5 min per), fixed in 3% glutaraldehyde (Sigma-Aldrich) for 10 min, washed by dH2O three times (5 min per), and finally, exposed to 2% uranyl acetate (Sigma-Aldrich). The sample was then observed under the transmission electron microscope after several minutes of drying.


## 2.6. Coculture

Exosomes were mixed with PKH67 dye after resuscitated in loading buffer and incubated for 5 min at room temperature. The samples were then ultracentrifuged at 100,000 ×g for 1 h to remove the free dye with the addition of PBS plus 5% FBS. Stained exosomes were then cocultured with 769-P cell line for 6 h and fixed with 4% paraformaldehyde for 20 min at 25°C. At last, whether PKH67-labled exosomes are present in 769-P cells was determined via a fluorescence microscope.


## 2.7. qRT-PCR

Total RNA was extracted from cells or exosomes using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.). cDNA was synthesized using PrimeScript RT reagent kit (Takara, Japan) according to the manufacturer’s instructions. miRNA cDNA was synthesized with the miRcute Plus miRNA First-strand cDNA synthesis kit (Tianjin, China). qRT-PCR was performed on Applied Biosystems ABI 7500 real-time PCR system (Thermo Fisher Scientific, Inc.) using FastStart Universal SYBR Green Master (Rox) reagent kit (Roch, Basel, Switzerland). Normalization for miRNA expression was done using U6 expression. miR-224-5p forward primer: 5′-CTGGTAGGTAAGTCACTA-3′, reverse primer: 5′-TCAACTGGTGTCGTGGAG-3′; U6 forward primer: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, reverse primer: 5′-CGCTTCAGAATTTGCGTGTCAT-3′. Fold change was calculated by relative quantification method (2−△△Ct method). The data were representative of three independent experiments.


## 2.8. Western Blot

Cells were harvested in designate time and washed with PBS. The concentration of total proteins isolated from cells was detected by BCA protein assay kit (Thermo Fisher Scientific, USA). The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with specific primary antibodies overnight at 4°C after being blocked with 5% skim milk for 2 h at room temperature. The indicated primary antibodies included rabbit anti-CD63, rabbit anti-CD9, rabbit anti-Hsp70, rabbit anti-α-SMA, and rabbit anti-GAPDH. The membranes were then washed with TBST three times and incubated with a horseradish peroxidase- (HRP-) conjugated secondary antibody goat anti-rabbit IgG for 1 h at room temperature. Ultimately, all proteins were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.). All antibodies used here were ordered from Abcam.


## 2.9. Cell Transfection

As for cell transfection, miR-224-5p mimic (100μm), and its negative control NC mimic (100 μm) purchased from RiboBio (Guangzhou, China) were used for miR-224-5p overexpression in 769-P cells. After the transfection, cells were incubated for 48 h at 37°C to conduct follow-up experiments.


## 2.10. Cell Apoptosis Assay

Cell apoptosis was measured using Annexin V-FITC cell apoptosis assay kit (Beyotime, China). Cells were trypsin-treated (BD Biosciences, USA) and washed with cold PBS. Cells were centrifuged and then resuspended in 195μl of 1× Annexin V loading buffer after the supernatant was removed. Subsequently, cells were incubated with 5 μl Annexin-V/FITC, followed by 10 μl PI for 15 min at room temperature in the dark. The results were analyzed by CellQuest software (BD Biosciences, NJ, USA) of FACS Calibur flow cytometry (Becton Dickinson, CA, USA).


## 2.11. Cell Proliferation Assay

The cell proliferation was measured using CCK-8 kit (Dojindo, Japan) according to the information provided by the manufacturer. At indicated time points (0, 24, 48, and 72 h), 10μl CCK-8 was supplemented to each well, and cells were incubated for additional 2 h at 37°C. A spectrophotometer (Bio-Rad, USA) was then used to analyze the optical density (OD) at 450 nm.


## 2.12. Migration and Invasion Examination

The migration and invasion assays were performed using Transwell chambers (0.8μm pore size; Merck Millipore, USA). For Transwell migration assay, the cells (1×105) were suspended in 200 μl serum-free medium and plated onto the upper chamber, whereas DMEM with 10% FBS which served as attractant was added into the lower chamber. For Transwell invasion assay, the upper chamber was precoated with 50 μl Matrigel (Corning, USA) and dried for 4 h at 37°C. Measurement of cell migration or invasion was carried out 24 h postculture. The cells that migrated or invaded to the lower chamber were processed with 4% paraformaldehyde followed up 1% crystal violet. At last, cell number was counted for ability assessment in four fields selected at random under an optical microscope (100x).


## 2.13. Statistical Analysis

Analysis for the data in this study was carried out with GraphPad Prism 6.0 (La Jolla, CA), and all experiments were repeated in triplicate. Data presentation was done usingmean±standarddeviation. Between-group variance was assessed via Student’s t-test or one-way ANOVA. Statistical significance was defined when p value less than 0.05, while highly statistical significance was defined in the case of p value less than 0.01.


## 3. Results

### 3.1. Fibroblasts of ccRCC Patients Can Secret Exosomes

Primary CAFs and normal fibroblasts (NFs) were isolated from 12 ccRCC patients who were included in this study, and the expression ofα-smooth muscle actin (α-SMA) was used to distinguish them from other cells. It was found that α-SMA was expressed in both NFs and CAFs, and the expression of α-SMA was significantly higher in CAFs (Figure 1(a)). Transmission electron microscope was used to further determine the size of the exosomes that we isolated from primary CAFs and NFs. It was noted that the diameter of the exosomes was ranged from 40 nm to 120 nm (Figure 1(b)), which is within the normal range of exosomes. Subsequently, the exosome surface marker proteins HSP70, CD9, and CD63 in exosomes secreted by NFs and CAFs were detected by Western blot, respectively, and these proteins were found to be expressed in both groups of exosomes (Figure 1(c)). Based on the size of the exosomes and the presence of marker proteins, we substantiated that exosomes were successfully isolated from fibroblasts.Figure 1
Verification of fibroblasts and their exosomes. (a) Western blot was employed to test the expression ofα-SMA in CAFs and NFs; (b) representative images of exosomes isolated from NFs and CAFs; (c) Western blot was used to analyze the expression of exosome marker proteins Hsp70, CD9, and CD63.
(a)(b)(c)


### 3.2. Exosomes Derived from CAFs Promote the Proliferation, Migration, and Invasion of ccRCC Cells and Inhibit Cell Apoptosis

Due to the fact that exosomes play a critical role in tumor microenvironment (TME), we designed to study the potential effect of the exosomes derived from CAFs on ccRCC cell properties. Before examination, the exosomes isolated from CAFs or NFs were firstly cocultured with 769-P cancer cells. After 24 h, relevant assays were performed for function assessment. As revealed by the data from CCK-8 and flow cytometry, the viability of 769-P cells cocultured with CAF-derived exosomes was stronger compared to cells cocultured with NF-derived exosomes (Figure2(a)), while cell apoptosis was much higher in the group with NF-derived exosomes (Figure 2(b)). Additionally, cell migratory and invasive abilities were also noted to be strengthened in the group of cells with exosomes derived from CAFs, as judged by Transwell assay (Figures 2(c) and 2(d)). Therefore, we can conclude that CAF-derived exosomes play an active role in ccRCC cell proliferation, migration, and invasion but a suppressive role in cell apoptosis.Figure 2
CAF-derived exosomes promote proliferation, migration, and invasion and inhibit apoptosis of ccRCC cells. (a, b) The proliferation and apoptosis of 769-P cells cocultured with CAF- or NF-derived exosomes were measured after 24 h of incubation; (c, d) Transwell assay was conducted to assess the migration and invasion of 769-P cells cocultured with CAF- or NF-derived exosomes.∗p<0.05.
(a)(b)(c)(d)


### 3.3. Exosomes Can Transfer miR-224-5p from CAFs to ccRCC Cells

To explore the underlying mechanism of CAF-derived exosomes mediating ccRCC progression, we focused on miRNA dysregulation in CAF-derived exosomes. As differential expression analysis revealed based on GEO database, 24 differentially upregulated and 33 downregulated miRNAs (Figure3(a)) were obtained in GSE109368 dataset. miR-224-5p was observed significantly upregulated in tumor tissue (Figure 3(b)). Moreover, literature showed that exosomal miR-224 serves as a prognostic biomarker for ccRCC [21]. Combined with the finding in 3.2, we hypothesized that CAF-derived exosomes might be rich in miR-224-5p and can deliver miR-224-5p to ccRCC cells to affect ccRCC cell phenotypes.Figure 3
Exosomes can transfer miR-224-5p from CAFs to ccRCC cells. (a) Volcano plot of DEmiRNAs in normal and cancer samples of GSE109368 dataset (red dot represents upregulated miRNAs, green dot represents downregulated miRNAs); (b) box plots of the expression of miR-224-5p in normal and cancer samples from GSE109368 dataset; (c) the expression of miR-224-5p in NF- and CAF-derived exosomes; (d) the expression of miR-224-5p in 769-P cells cocultured with CAF- or NF-derived exosomes; (e) CAF-derived exosomes were able to be internalized by 769-P cell line (scale: 50μm). ∗p<0.05.
(a)(b)(c)(d)(e)In order to confirm the above speculation, we made a comparative analysis in miR-224-5p expression in CAF- and NF-derived exosomes. miR-224-5p was found to be significantly highly expressed in CAF-derived exosomes in comparison with that in NF-derived exosomes by qRT-PCR (Figure3(c)). Then, we cocultured CAFs-exosomes or NFs-exosomes with 769-P cells and found that miR-224-5p expression in cells cultured with CAFs-exosomes was significantly elevated (Figure 3(d)), which suggested that 769-P cells cocultured with CAF-derived exosomes were responsible for the increase of miR-224-5p expression. To determine whether miR-224-5p in CAF-derived exosomes could enter and affect the activity of ccRCC cells, we used PKH67 dye to label CAF-derived exosomes and then conducted coculture with 769-P cells, finding that PKH67 fluorescence could be detected in 769-P cells (Figure 3(e)). In brief, these findings illustrate that miR-224-5p can be transferred from CAF-derived exosomes to ccRCC cells.


### 3.4. Overexpression of miR-224-5p Promotes the Proliferation, Migration, and Invasion of ccRCC Cells and Inhibits Cell Apoptosis

Since we had proved that miR-224-5p can be transferred from CAF-derived exosomes to ccRCC cells, we further analyzed whether miR-224-5p is involved in regulation of CAF-derived exosomes on ccRCC cell phenotypes. At first, miR-224-5p mimic or NC mimic was transfected into 769-P cells. Afterwards, CCK-8, Transwell, and flow cytometry assays were carried out, and the results showed that after miR-224-5p was highly expressed, the OD value at 450 nm was increased, indicating elevated cell viability (Figure4(a)), yet cell apoptosis was suppressed significantly (Figure 4(b)). Additionally, the numbers of cells that happened migration and invasion were remarkably increased upon miR-224-5p upregulation, demonstrating that cell migratory and invasive abilities both were highly enhanced (Figures 4(c) and 4(d)). Collectively, miR-224-5p may act as a promoter for the malignant progression of ccRCC.Figure 4
Overexpression of miR-224-5p promotes proliferation, migration, and invasion of ccRCC cells and inhibits cell apoptosis. (a) CCK-8 was performed to determine cell proliferation after miR-224-5p was overexpressed; (b) flow cytometry was used to detect cell apoptosis after miR-224-5p was overexpressed; (c, d) Transwell assay was performed to determine the invasion (c) and migration (d) of cells after transfection with miR-224-5p mimic or NC mimic (100x).∗p<0.05.
(a)(b)(c)(d)


## 3.1. Fibroblasts of ccRCC Patients Can Secret Exosomes

Primary CAFs and normal fibroblasts (NFs) were isolated from 12 ccRCC patients who were included in this study, and the expression ofα-smooth muscle actin (α-SMA) was used to distinguish them from other cells. It was found that α-SMA was expressed in both NFs and CAFs, and the expression of α-SMA was significantly higher in CAFs (Figure 1(a)). Transmission electron microscope was used to further determine the size of the exosomes that we isolated from primary CAFs and NFs. It was noted that the diameter of the exosomes was ranged from 40 nm to 120 nm (Figure 1(b)), which is within the normal range of exosomes. Subsequently, the exosome surface marker proteins HSP70, CD9, and CD63 in exosomes secreted by NFs and CAFs were detected by Western blot, respectively, and these proteins were found to be expressed in both groups of exosomes (Figure 1(c)). Based on the size of the exosomes and the presence of marker proteins, we substantiated that exosomes were successfully isolated from fibroblasts.Figure 1
Verification of fibroblasts and their exosomes. (a) Western blot was employed to test the expression ofα-SMA in CAFs and NFs; (b) representative images of exosomes isolated from NFs and CAFs; (c) Western blot was used to analyze the expression of exosome marker proteins Hsp70, CD9, and CD63.
(a)(b)(c)


## 3.2. Exosomes Derived from CAFs Promote the Proliferation, Migration, and Invasion of ccRCC Cells and Inhibit Cell Apoptosis

Due to the fact that exosomes play a critical role in tumor microenvironment (TME), we designed to study the potential effect of the exosomes derived from CAFs on ccRCC cell properties. Before examination, the exosomes isolated from CAFs or NFs were firstly cocultured with 769-P cancer cells. After 24 h, relevant assays were performed for function assessment. As revealed by the data from CCK-8 and flow cytometry, the viability of 769-P cells cocultured with CAF-derived exosomes was stronger compared to cells cocultured with NF-derived exosomes (Figure2(a)), while cell apoptosis was much higher in the group with NF-derived exosomes (Figure 2(b)). Additionally, cell migratory and invasive abilities were also noted to be strengthened in the group of cells with exosomes derived from CAFs, as judged by Transwell assay (Figures 2(c) and 2(d)). Therefore, we can conclude that CAF-derived exosomes play an active role in ccRCC cell proliferation, migration, and invasion but a suppressive role in cell apoptosis.Figure 2
CAF-derived exosomes promote proliferation, migration, and invasion and inhibit apoptosis of ccRCC cells. (a, b) The proliferation and apoptosis of 769-P cells cocultured with CAF- or NF-derived exosomes were measured after 24 h of incubation; (c, d) Transwell assay was conducted to assess the migration and invasion of 769-P cells cocultured with CAF- or NF-derived exosomes.∗p<0.05.
(a)(b)(c)(d)


## 3.3. Exosomes Can Transfer miR-224-5p from CAFs to ccRCC Cells

To explore the underlying mechanism of CAF-derived exosomes mediating ccRCC progression, we focused on miRNA dysregulation in CAF-derived exosomes. As differential expression analysis revealed based on GEO database, 24 differentially upregulated and 33 downregulated miRNAs (Figure3(a)) were obtained in GSE109368 dataset. miR-224-5p was observed significantly upregulated in tumor tissue (Figure 3(b)). Moreover, literature showed that exosomal miR-224 serves as a prognostic biomarker for ccRCC [21]. Combined with the finding in 3.2, we hypothesized that CAF-derived exosomes might be rich in miR-224-5p and can deliver miR-224-5p to ccRCC cells to affect ccRCC cell phenotypes.Figure 3
Exosomes can transfer miR-224-5p from CAFs to ccRCC cells. (a) Volcano plot of DEmiRNAs in normal and cancer samples of GSE109368 dataset (red dot represents upregulated miRNAs, green dot represents downregulated miRNAs); (b) box plots of the expression of miR-224-5p in normal and cancer samples from GSE109368 dataset; (c) the expression of miR-224-5p in NF- and CAF-derived exosomes; (d) the expression of miR-224-5p in 769-P cells cocultured with CAF- or NF-derived exosomes; (e) CAF-derived exosomes were able to be internalized by 769-P cell line (scale: 50μm). ∗p<0.05.
(a)(b)(c)(d)(e)In order to confirm the above speculation, we made a comparative analysis in miR-224-5p expression in CAF- and NF-derived exosomes. miR-224-5p was found to be significantly highly expressed in CAF-derived exosomes in comparison with that in NF-derived exosomes by qRT-PCR (Figure3(c)). Then, we cocultured CAFs-exosomes or NFs-exosomes with 769-P cells and found that miR-224-5p expression in cells cultured with CAFs-exosomes was significantly elevated (Figure 3(d)), which suggested that 769-P cells cocultured with CAF-derived exosomes were responsible for the increase of miR-224-5p expression. To determine whether miR-224-5p in CAF-derived exosomes could enter and affect the activity of ccRCC cells, we used PKH67 dye to label CAF-derived exosomes and then conducted coculture with 769-P cells, finding that PKH67 fluorescence could be detected in 769-P cells (Figure 3(e)). In brief, these findings illustrate that miR-224-5p can be transferred from CAF-derived exosomes to ccRCC cells.


## 3.4. Overexpression of miR-224-5p Promotes the Proliferation, Migration, and Invasion of ccRCC Cells and Inhibits Cell Apoptosis

Since we had proved that miR-224-5p can be transferred from CAF-derived exosomes to ccRCC cells, we further analyzed whether miR-224-5p is involved in regulation of CAF-derived exosomes on ccRCC cell phenotypes. At first, miR-224-5p mimic or NC mimic was transfected into 769-P cells. Afterwards, CCK-8, Transwell, and flow cytometry assays were carried out, and the results showed that after miR-224-5p was highly expressed, the OD value at 450 nm was increased, indicating elevated cell viability (Figure4(a)), yet cell apoptosis was suppressed significantly (Figure 4(b)). Additionally, the numbers of cells that happened migration and invasion were remarkably increased upon miR-224-5p upregulation, demonstrating that cell migratory and invasive abilities both were highly enhanced (Figures 4(c) and 4(d)). Collectively, miR-224-5p may act as a promoter for the malignant progression of ccRCC.Figure 4
Overexpression of miR-224-5p promotes proliferation, migration, and invasion of ccRCC cells and inhibits cell apoptosis. (a) CCK-8 was performed to determine cell proliferation after miR-224-5p was overexpressed; (b) flow cytometry was used to detect cell apoptosis after miR-224-5p was overexpressed; (c, d) Transwell assay was performed to determine the invasion (c) and migration (d) of cells after transfection with miR-224-5p mimic or NC mimic (100x).∗p<0.05.
(a)(b)(c)(d)


## 4. Discussion

Tumor progression is deeply influenced by the local microenvironment. CAFs are one of the most abundant and active cell types of TME [22]. The myofibroblast-like properties of CAFs, such as robust contractility and extracellular matrix (ECM) deposition, allow them to generate mechanical and chemical signals that support invasive tumor growth [23]. Functionally speaking, CAFs act as an important player that regulates tumor progression by delivering exosomes to their adjacent cells [24].In this study, we extracted exosomes from CAFs and studied the effect of CAF-derived exosomes on ccRCC cell behavior. CAF-derived exosomes were found to be able to facilitate the malignant cell phenotypes of ccRCC. Exosome-delivered miRNAs are associated with cancer cell communication, which makes tumor cells to “shape” and affects their environment [14]. Up to present, several exosomal miRNAs from different sources have been reported in ccRCC. For example, Song et al. [25] found that urine exosomal miR-30c-5p inhibits ccRCC progression by directly regulating the expression of HSPA5 in vivo and in vitro. Zhang et al. [26] reported that serum exosomal miR-210 and miR-1233 have good specificity and sensitivity as diagnostic tools for ccRCC. Wang et al. proved that exosomes derived from cancer stem cells transport miR-19b-3p into ccRCC cells and initiate epithelial-mesenchymal transition (EMT) to promote metastasis [27]. In this study, to determine the carcinogenic miRNA in CAF-derived exosomes, bioinformatics analyses and in vitro experiments were performed. miR-224-5p which was differentially upregulated in ccRCC tissue and cells was identified. Besides, miR-224-5p was highly expressed in CAF-derived exosomes and was able to be transferred from CAF-derived exosomes to ccRCC cells. Therefore, we believe that miR-224-5p participates in the regulation of CAF-derived exosomes on ccRCC cell phenotypes.The dysregulation of miRNAs in CAFs is an important factor that affects cell proliferation, invasion, and migration of various solid tumors [28]. Here, we designed miR-224-5p overexpressed 769-P ccRCC cells to discuss the potential promotive effect of exosomal miR-224-5p on ccRCC cell malignant behaviors, which was in agreement with that of miR-224-5p in renal cell carcinoma cells [29]. Furthermore, we also found that overexpression of miR-224-5p suppressed cell apoptosis. Collectively, these findings demonstrate that exosomes transfer miR-224-5p from CAFs into ccRCC cells to facilitate the malignant properties of ccRCC cells.To sum up, our research identified high expression of miR-224-5p in CAF-derived exosomes and clarified that the miR-224-5p could be delivered to ccRCC cells to participate in the regulation of the CAF-derived exosomes on ccRCC cell malignant properties. Given our findings, we believe that miR-224-5p is expected to become a novel diagnostic or prognostic biomarker for ccRCC. Our study not only puts forward a new insight into the mechanism underlying ccRCC progression but also brings hope for effective treatment of patients with ccRCC. However, limitations still exist. For example, we have not probed into the specific mechanism by which miR-224-5p affects ccRCC malignant progression. Besides, the effect of miR-224-5p from CAF-derived exosomes on ccRCC tumor growthin vivo has not been identified. For these deficiencies, we aim to identify relevant downstream target of miR-224-5p or signaling pathways and construct animal models in the future, to help support the role of miR-224-5p as a potential therapeutic target for ccRCC.



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*Source: 5517747-2021-05-25.xml*