Exosomal miR-224-5p from colorectal cancer cells promotes malignant transformation of human colon normal cells by promoting cell proliferation through downregulation of CMTM4

Interactions between malignant cells and neighboring normal cells is important for carcinogenesis. In addition, cancer cell-derived exosomes have been shown to promote the malignant transformation of recipient cells, but the mechanisms remain unclear. The level of miR-224-5p in colorectal cancer (CRC) cell-derived exosomes was determined by RT-qPCR assay. In addition, PKH26 dye-labeled exosomes were used to assess the ecacy of the transfer of exosomes between SW620 and CCD 841 CoN cells.


Conclusion
These ndings suggested that SW620 cell-derived exosomal miR-224-5p could promote malignant transformation and tumorigenesis in vitro and in vivo via downregulation of CMTM4, suggesting that miR-224-5p might be a potential target for therapies in CRC.

Background
Colorectal cancer (CRC) is one of the most common digestive tract malignancies with high morbidity and mortality [1,2]. The development and progression of CRC are regulated by a number of factors, such as dietary behaviors, chronic intestinal in ammation, aging, smoking and mutations [3,4]. It is estimated that around 1 million people will be affected by CRC every year, accompanied by overt metastatic disease [2]. Recently, several methods including radiotherapy, and chemoradiotherapy have been used for the treatment of CRC for years [5][6][7]. However, the prognosis of patients with CRC is still unsatisfactory [5][6][7]. Therefore, identifying speci c biomarkers is of great importance in order to improve early diagnosis and investigate novel treatment strategies for patients of CRC.

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MicroRNAs (miRNAs) have been found to be involved in the development of CRC [8]. Accumulating evidences have shown that miRNAs exert a vital role in regulating CRC progression through acting as tumor suppressors and oncogenes [8,9]. Exosomes are small (30-100 nm) extracellular vesicles that carry different nucleic acids including miRNAs [10,11]. In addition, exosomes serve as key mediators in cell-to-cell communication, often to prepare a premetastatic niche, remodel the extracellular environment and escape immune surveillance [11,12]. Recently, cancer cell-secreted exosomes have been reported to in uence tumor microenvironment and promote cancer cell growth, invasion and angiogenesis [13][14][15].
Meanwhile, cancer cell-secreted miRNAs can be delivered to recipient cells via exosomes, then perform a key regulatory role in migration and invasion of CRC cells [16,17].
Herein, in this study, we found that CRC cell-derived exosomal miR-224-5p can be transferred to human colon normal cells, promoting the malignant transformation of CCD 841 CoN cells (human colon normal cells) by targeting CMTM4. These ndings may provide a novel target for the treatment of CRC.

Materials And Methods
Data collection and differential expression analysis CRC-related datasets (GSE18392, GSE115513 and GSE126093) were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed miRNAs (DEMs) between CRC tissues and adjacent normal tissues were identi ed using R language. The miRNAs with P<0.05 and |log2 (FC)|>2 were selected as the signi cantly DEMs. The overlapping DEMs were identi ed using a Venn diagram from three datasets (GSE18392, GSE115513 and GSE126093). In addition, TCGA dataset was used to determine the association between the overall survival of patients with CRC and miR-224-5p level.

Cell culture
Human colon normal cell line (CCD 841 CoN), CRC cell lines (HT-29, HCT116, SW620 and SW480) and 293T cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences. Cells were maintained in DMEM medium containing 10% FBS and cultured in a humidi ed incubator containing 5% CO 2 at 37°C.
In addition, the sequence of CMTM4 was ampli ed by PCR and then subcloned into pcDNA3.1 vector to generate pcDNA3.1-CMTM4 overexpression (CMTM4-OE) plasmids. The pcDNA3.1-NC and CMTM4-OE plasmids were transfected into CCD 841 CoN cells by using Lipofectamine 2000 reagents. Later on, 48 h after transfection, the transfected cells were selected with neomycin (Thermo Fisher Scienti c).

Cell viability assay
The transfected cells were plated onto 96-well plates (about 5,000 per well). At 0, 24, 48 or 72 h posttransfection, CCK-8 reagent (10 μl, MedChem Express) was added into each well, and cells were then incubated for 2 h. After that, the absorbance of each well was detected using a microplate reader at 450 nm.
Immuno uorescence assay Cells were treated with 4% paraformaldehyde for 20 min and incubated with 5% BSA for 1 h. Then, cells were incubated with primary antibodies anti-Ki67 at 4°C overnight. Later on, cells were incubated with a corresponding secondary antibody for 1 h. Subsequently, images were captured using a uorescence microscope. Nuclei were stained by DAPI.

Transwell assay
Transwell migration or invasion assays were performed using transwell chambers uncoated or coated with Matrigel. Cells (2 × 10 4 cells/well) were seed onto the upper chamber of each insert (Corning). In addition, the lower chambers were loaded with DMEM (600 μL) containing 10% FBS. After 24 h of incubation, the cells on the lower surface were stained with 0.1% crystal violet. Subsequently, a uorescence microscope was used to observe migrated or invasive cells.

Flow cytometry assay
Cell apoptosis was detected using an Annexin-V-FITC apoptosis detection kit. Brie y, 1 × 10 5 cells were stained with Annexin-V-FITC (5 μl) and PI (5 μl) staining solution for 15 min in darkness. Subsequently, cell apoptosis was analyzed by a ow cytometer.

Exosome isolation and characterization
The conditioned media (CM) of SW620 cells was collected. Later on, exosomes were isolated using the GETTM Exosome Isolation Kit (GeneExosome technologies). Nanoparticle-tracking analysis (Particle Metrix, Meerbusch, Germany) was applied to determine the size of exosomes. Next, a transmission electron microscopy (TEM) was used to visualize the morphology of exosomes as described previously [18].
Exosome labeling and uptake SW620 cell-derived exosomes were mixed with PKH26 dye for 30 min. After that, PKH26-tagged exosomes were added into CCD 841 CoN cells and incubated for 24 h. Subsequently, CCD 841 CoN cells that uptake the labelled exosomes was observed by a uorescence microscope. Nuclei were stained by DAPI.

Western blot assay
Proteins were separated by 10% SDS-PAGE and then transferred onto a PVDF membrane. Later on, the membrane was incubated overnight at 4°C with primary antibodies against CMTM4, p-Akt, Akt, p-ERK, ERK, CD63, TSG101, β-actin, and then incubated with the corresponding secondary antibody at room temperature for 1 h. Subsequently, immune complexes were detected using the ECL reagents.

Animal studies
The BALB/c nude mice (4-5 weeks old) were purchased from the Vital River Laboratories (Beijing, China). SW620 cells (1 x 10 7 cells) were subcutaneously injected into left ank of nude mice. When the tumors reach about 200 mm 3 , mice were divided randomly into four groups: control, Exo-NC, Exo-miR-224-5p agomir and Exo-miR-224-5p antagomir. Then, mice were intravenously injected with PBS, Exo-NC, Exo-miR-224-5p agomir or Exo-miR-224-5p antagomir twice a week. The tumor size was measured with a vernier caliper every week and the volume was calculated by the following formula V = length x width 2 x 0.5. After 3 weeks of tumor cell implantation, the mice were sacri ced, and the tumors from different groups were removed and weighted. All animal experiments were approved by the Ethics Committee of the Harbin Medical University Cancer Hospital and performed following the procedures of National Institutes of Health guide for the care and use of laboratory animals.

TUNEL assay
Cell apoptosis in tumor tissues were assessed using an APO-BrdU™ TUNEL Assay Kit (Thermo Fisher Scienti c) according to the manufacturer's instructions.

Statistical analysis
All statistical analyses were performed using GraphPad Prism software (version 7.0, La Jolla, CA, USA). Differences between three or more groups were analyzed by One-way analysis of variance (ANOVA) and Tukey's tests. Data are presented as the mean ± standard deviation (S.D.). The differences were considered signi cant at *P < 0.05. All data were repeated in triplicate.

Identi cation of DEMs in CRC
To identify the DEMs between CRC tissues and adjacent normal tissues, R language was performed to analyze the expression pro les of miRNAs from three CRC-related datasets (GSE18392, GSE115513 and GSE126093). As shown in Fig. 1A, 1B and 1C, a total of 24, 44 and 417 DEMs were identi ed from the GSE18392, GSE115513 and GSE126093 respectively. In addition, 4 overlapping DEMs were identi ed in three datasets, including miR-31-5p, miR-135-5p, miR-183-5p and miR-224-5p (Fig. 1D). Zheng et al revealed that miR-224 level was increased in CRC tissues [19]. Meanwhile, in the TCGA dataset, high levels of miR-224-5p in patients with CRC are associated with a shorter overall survival, indicating that increased miR-224-5p levels may predict poor overall survival in patients with CRC (Fig. 1E).
CMTM4 is a binding target of miR-224-5p Evidences have shown that miRNAs can negatively regulate protein expression though binding to the 3′-UTR of their target mRNA [20,21]. To nd the target genes of miR-224-5p, ve bioinformatics tools (miRWalk, miRanda, miRDB, RNA22 and TargetScan) were used. We found that CMTM4 was one of the best candidates (Fig. 4A). Xue et al indicated that CMTM4 level was frequently downregulated in CRC tissues [22]. As predicted, a marked decrease in CMTM4 at mRNA level was found in SW620 and HCT116 cells (Fig. 4B). To validate that CMTM4 is a binding target of miR-224-5p, dual-luciferase reporter assay was used. As shown in Fig. 4C, miR-224-5p agomir notably reduced the luciferase activity of CMTM4-WT. Meanwhile, miR-224-5p agomir obviously decreased the expression of CMTM4 at mRNA level in SW620 cells, while miR-224-5p antagomir displayed the opposite results (Fig. 4D). These results indicated that CMTM4 is a direct binding target of miR-224-5p.
MiR-224-5p can be transferred from SW620 cells to CCD 841 CoN cells via exosomes Evidences have shown that cancer cell-derived exosomes exert a vital role in the process of malignant transformation [23,24]. In this study, exosomes were isolated from the CM of CCD 841 CoN cells and SW620 cells. TEM and NTA analysis demonstrated that exosomes secreted from CCD 841 CoN and SW620 cells had the characteristic size (40 to 100 nm) and cup-shaped morphology, and expressed the exosomal markers, TSG101 and CD63 (Fig. 5A, 5B and 5C), suggesting that exosomes were isolated from cells. Meanwhile, miR-224-5p level was increased in exosomes derived from SW620 cells compared with that in exosomes derived from CCD 841 CoN cells (Fig. 5D).

Exo-miR-224-5p antagomir suppressed the growth of CRC cells in vivo
We further investigated the role of exosomal miR-224-5p on tumor growth in vivo. As shown in Fig. 8A, 8B and 8C, the tumor volume and tumor weight were signi cantly increased in the Exo-miR-224-5p agomir group, while the opposite results were observed in the Exo-miR-224-5p antagomir group. In addition, the level of miR-224-5p was upregulated in tumor tissues of tumor-bearing mice that were received Exo-miR-224-5p agomir; whereas mice that received Exo-miR-224-5p antagomir displayed the opposite results (Fig.  8D). Moreover, TUNEL assay indicated that Exo-miR-224-5p antagomir obviously induced cell apoptosis in tumor tissues (Fig. 8E and 8F). Meanwhile, the expression of CMTM4 was decreased and p-Akt and p-ERK protein expressions was increased in tumor tissues of mice that received Exo-miR-224-5p agomir compared with Exo-NC group; whereas mice that treated with Exo-miR-224-5p antagomir displayed the opposite results (Fig. 8G, 8H, 8I and 8J). Collectively, Exo-miR-224-5p antagomir could suppress the growth of CRC cells in vivo via upregulation of CMTM4.

Discussion
The development of human cancers is a complex process [25]. Tumor cells are the major driving force behind the development and progression of human cancers [26]. García-Olmo found that interactions between tumor cells and neighboring normal cells in the tumor microenvironment might be essential for tumor progression [27]. Local microenvironments have been found to exert a vital role in mediating intercellular communication between malignant cells and nonmalignant cells [28,29]. In addition, cancer cell-derived exosomes can act as communicative vectors, participating in remodeling the tumor microenvironment [30,31]. Importantly, cancer cell-derived exosomes have been shown to promote the malignant transformation of recipient cells, increasing cell migratory and invasive abilities [32]. Meanwhile, cancer cell-derived exosomes can carry different miRNAs, and these exosomal miRNAs can modulate the function of recipient cells via activation or inactivation of multiple cancer related pathways through transferring into recipient cells and mediating protein expression [33,34]. Thus, the discovery of novel circulating biomarkers of CRC may help to improve diagnosis or treatment of CRC.
Currently, miR-224-5p has been identi ed to be dysregulated in human cancers, including CRC [19,35]. In this study, we found that miR-224-5p level was signi cantly upregulated in CRC cells, and downregulation of miR-224-5p could induce cell apoptosis and inhibit cell migration and invasion. Consistent with our present results, zheng et al showed that miR-224 overexpression promoted CRC cell proliferation and migration via targeting BTRC [19]. In addition, liang et al showed that downregulation of miR-224 could suppress the proliferation and trigger the apoptosis of adriamycin-resistant CRC cells [36]. Our data found that miR-224-5p may act as an oncogene for CRC.
For the rst time, we found that exosomal miR-224-5p is secreted by SW620 cells that could be internalized by normal colon cell line CCD 841 CoN, suggesting that SW620-secreted miR-224-5p can be delivered into CCD 841 CoN cells via exosomes. A series of functional experiments indicated that exosomal miR-224-5p derived from CRC cells could promote CCD 841 CoN cell proliferation, migration and invasion. Consistent with our present results, wei et al showed that miR-15b-3p can be transferred from GC cells to normal GES-1 gastric epithelium cells via exosomes, and then promoted GES-1 cell malignant transformation [37]. In addition, evidence has shown that inactivation of tumor-suppressor and activation of oncogene are considered as the key causes driving the progressive transformation of normal cells to malignant cells [26]. In the present study, we found that CMTM4 was a binding target of miR-224-5p. It has been shown that CMTM4 is a tumor suppressor in human cancers, including CRC [22,38]. Our data showed that exosomal miR-224-5p promoted the proliferation, migration and invasion of CCD 841 CoN cells via downregulation of CMTM4. These results suggested that SW620 cell-derived exosomal miR-224-5p could promote the malignant transformation of CCD 841 CoN cells via inactivation of a tumor suppressor CMTM4.

Conclusion
Collectively, our data showed that exosomes secreted from SW620 cells can deliver miR-224-5p into CCD Declarations Feng Wu made major contributions to the conception, design and manuscript drafting of this study. Jiani Yang, Guoyin Shang, Zhijia Zhang, Sijia Niu, Yang Liu, Jing Jing and Hongru Liu were responsible for data acquisition, data analysis, data interpretation and manuscript revision. Yu Fang made substantial contributions to conception and design of the study and revised the manuscript critically for important intellectual content. All authors agreed to be accountable for all aspects of the work. All authors read and approved the nal manuscript.