Background
Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality [
1,
2]. More than 60% of CRC patients have initiated the metastatic process by the time of diagnosis [
3]. Although there are multiple tests available for CRC screening, each method has its own limitations in terms of sensitivity and specificity. To the best of our knowledge, carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA19–9) are well established tumor markers with low sensitivity and specificity for early detection of CRC [
4]. Hence, ideal CRC-specific biomarkers are urgently required to improve the current CRC diagnostic strategies.
Exosomes, membrane vesicles of endocytic origin ranging in size from 30 to 150 nm approximately, are emerging as key players in intercellular communication between cancer cells and their microenvironment [
5]. A distinct feature of exosomes is that they efficiently carry and deliver molecular signatures (proteins, lipids, RNA and DNA) to recipient cells [
6,
7]. In cancer development, exosomes are described as functional mediator of cancerous malignant alteration in recipient cells [
8]. This intercellular communication is known to be involved in various pathophysiological processes including cell proliferation, migration, apoptosis, treatment resistance and metastasis [
9‐
12], tumor innervation [
13] and angiogenesis [
14]. Exosomes also potentially participate in the development and progression of CRC. A recent study provides a novel notion that miR-200c and miR-141 contain in exosomes of mesenteric vein plasma could predict colon cancer patients with poor prognosis [
15]. Exosomes derived from bone marrow-derived mesenchymal stem/stromal cells also enlarge the population of colon cancer stem cells by treating colon cancer cells (HCT-116, HT-29 and SW480) [
16]. Therefore, it is important to explore the mechanisms by which exosomes derived from CRC cells regulate CRC progression, especially the metastatic process.
Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides that have limited or no protein-coding capacity [
17]. Exosome-derived lncRNAs have been detected in a wide range of bodily fluids due to active cellular secretion [
18]. Although ribonuclease is present in the blood, lncRNA nevertheless exists stably due to the protection of exosomes and microvesicles. Previous studies have demonstrated that exosome-derived lncRNAs affect tumor growth, metastasis, invasion, and prognosis by regulating the tumor microenvironment [
19]. By traveling to cells through exosomes, lncRNAs could create a microenvironment suitable for the metastasis of tumor cells. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is an evolutionarily highly conserved lncRNA that lacks open reading frames. It plays essential roles in tumor development and is highly expressed in several tumors [
20]. In CRC, inhibition of MALAT1 suppresses CRC progression and metastasis and improves the sensitivity of cancer cells to 5-FU [
21]. Moreover, MALAT1 regulates the miR-106b-5p expression and further mediates the mobility of SLAIN2-related microtubules by functioning as a competing endogenous RNA, which results in the progression of CRC [
22]. However, whether exosome-derived MALAT1 affects the malignant behavior of CRC cells by interacting with microRNAs (miRNAs) and mediating tumor metastasis is rarely reported.
In the present study, our findings provide further evidence that exosomal MALAT1 contributes to CRC progression and regulates FUT4 expression by sponging miR-26a/26b via fucosylation and PI3K/Akt pathway, which may provide novel insights into the function of exosomal MALAT1 in CRC.
Materials and methods
Clinical samples and cell culture
45 CRC tissues (24 with distant metastasis and 21 without metastasis) were collected from the First Affiliated Hospital of Dalian Medical University between 2015 and 2018. The study and its informed consent have been examined and certified by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University (YJ-KY-FB-2016-16). Every patient was definitively identified as having CRC based on the clinicopathologic findings. None of the patients had received chemotherapy and/or radiotherapy prior to surgery.
Human CRC cell lines LoVo, HCT-8, SW620, and SW480 were obtained from KeygenBiotech Co. Ltd. (Nanjing, China). Cells were cultured in 90% DMEM/L-15 (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin–streptomycin (HyClone, Logan, Utah, USA) at 37 °C with 5% CO2.
Exosomes isolation from cell culture supernatants
Exosomes were collected from different CRC cells cultured in exosome-depleted fetal bovine serum (ultracentrifuged at 120,000 g overnight). Briefly, supernatant was collected from cells at approximately 80–85% confluence. After centrifugation of cells at 1000 g for 20 min, the supernatants were then centrifuged at 12000 g for 30 min to eliminate cells and debris. Finally, exosomes were obtained after centrifugation for 2 h at 120,000 g and then washed twice with a large volume of phosphate buffered saline (PBS). This protocol specifically collects exosomes and excludes large vesicles. The exosome proteins recovered were measured using the Bradford assay (Bio-Rad).
Electron microscopy and exosome size and density measurement
Exosome suspension was placed onto 200 mesh carbon-coated grids and allowed to be absorbed to the velamen for 3 min. Then grids were allowed to dry at room temperature for 1 min and stained for contrast using 3% phosphotungstic acid. The samples were viewed with a JEM-2000EX transmission electron microscope (JEOL, Japan) and images were taken in a suitable proportion. The size and density of exosomes were measured by Zetasizer Nano (Malvern, England). Briefly, exosome-enriched pellets were resuspended in 1 ml of 0.1 μm triplefiltered sterile PBS. Three recordings of 60s were performed for each sample. Collected data were analyzed with Zetasizer Nano software, which provided size distribution report by intensity.
Exosome labeling and macrophage trafficking in vitro
For exosome-tracking purposes, purified exosomes were fluorescently labeled using PKH67 (green) membranedye (Sigma-Aldrich, USA). Labeled exosomes were washed with PBS, re-collected by centrifugation at 12000 g for 30 min and then isolation with ExoQuick™. Labeled exosome pellets were resuspended in DMEM/L-15 medium and then added into receptor cell culture. After co-culturing for 3 h at 37 °C, the cells were washed with PBS for three times. Then the cells were fixed in 10% form aldehyde for 10 min and incubated with DAPI for 5 min. Images were obtained on a fluorescence microscope.
RNA extraction and quantitative real-time PCR
Total RNA was isolated from frozen tissues and CRC cell lines, using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA), and cDNA was synthesized using QuantiTect Reverse Transcription Kit (QIAGEN, valencia, CA, USA) according to the manufacturer’s specifications. The expression of miRNAs was determined by using mirVanaTM qRT-PCR microRNA Detection Kit (Ambion Inc., Austin, TX, USA). Relative quantities of each miRNA were calculated using the ΔΔCt method after normalization with endogenous reference U6-small nuclear RNA. MALAT1 and FUT4 mRNA was quantified with SYBR-Green-quantitative real-time PCR Master Mix kit (Toyobo Co., Osaka, Japan). The expression level of MALAT1 and FUT4 was determined by using Biosystems 7300 Real-Time PCR system (ABI, Foster City, CA, USA) and calculated using the ΔΔCt method after normalization with GAPDH.
Dual luciferase reporter gene assay
A pmirGLO Dual-Luciferase miRNATarget Expression Vector was purchased from GenePharma Co.Ltd. (Suzhou, China). Firefly luciferase functioned as primary reporter to regulate mRNA expression, and renilla luciferase was used as a normalized control. Co-transfection was conducted and the dual luciferase reporter assay system (Promega) was utilized. The mean luciferase intensity was normalized to renilla luciferase. Data were shown as the mean value ± SD and each experiment was performed thrice.
RNA immunoprecipitation (RIP) assay
RIP assay was performed using the Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA). Cells were collected and lysed in complete RIPA buffer containing a protease inhibitor cocktail and RNase inhibitor. Next, the cell extracts were incubated with RIP buffer containing magnetic bead conjugated with human anti-Ago2 antibody (Millipore) or mouse immunoglobulin G (IgG) control. The protein was digested with proteinase K, and subsequently, the immunoprecipitated RNA was obtained. The purified RNA was finally subjected to a qRT-PCR analysis to demonstrate the presence of the binding targets.
Western blot analysis and Lectin blot analysis
The tissues and cells were lysed in RIPA buffer with protease and phosphatase inhibitors (Roche, Beijing, China). Proteins (30 μg protein per lane) were resolved on 10% SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Pall Corporation). Following protein transfer, membranes were blocked for 1 h in PBS containing 5% non-fat dry milk and 0.1% Tween-20. Blots were then incubated overnight with primary antibody or LTL lectin (1:500) (Vector Laboratories, Burlingham, CA) at 4 °C. Membrane proteins were detected by HRP-conjugated secondary antibody (1:1000) or HRP-labeled Streptavidin (1:1000) (Beyotime Biotechnology, Shanghai, China). GAPDH was used as a control. The proteins were visualized and quantified using a chemiluminescence method (ECL Pus Western Blotting Detection System; GE Healthcare UK Ltd., Buckinghamshire, UK) and the ImageQuant LAS 500 (General Electric Co, USA).
Immunofluorescent staining
Paraffin-embedded sections (4 μm) were performed, and followed by antigen retrieval. The sections were washed in phosphate-buffered saline and incubated with primary antibodies including anti-FUT4 (1:150) at 4 °C overnight, and followed by an incubation with the secondary antibodies including Alexa Fluor 594-conjugated Goat Anti-Rabbit IgG (1:300) (Proteintech, Wuhan, China) and FITC-LTL lectin (1:500) (Vector Laboratories, Burlingham, CA) at 37 °C for 1 h. The sections were then counterstained with 4, 6 diamidino-2 phenyl-indole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) for nuclear staining. Images were taken by a Carl Zeiss fluorescence microscopy (Carl Zeiss, Hallbergmoos, Germany).
Cell immunofluorescence staining was conducted after fixing cells with 10% formaldehyde for 40 min, and permeabilized with 0.3% Triton X-100. 2% BSA was utilized to block the non-specific binding for 30 min at room temperature. Then cells were incubated with the primary antibodies and secondary antibodies as mentioned above.
Cell proliferation assay and focus formation assay
The Cell Counting Kit-8 (CCK-8; KeyGEN, Nanjing, China) and focus formation assay were conducted to determine the cell proliferation activity. Approximately 1 × 103 cells per well were transferred to 96-well plates with 100 μl of DMEM medium containing 10% FBS and cultured in a humidified incubator at 37 °C for 24, 48, 72 and 96 h. The absorbance at 450 nm was measured following the addition of 10 μL of CCK-8 solution at 37 °C for 2 h. There were 5 replicates for each group, and 3 independent experiments were performed.
For the focus formation assay, cells (1 × 103) were seeded in 6-well plates. The cultures were maintained in the DMEM containing 10% FBS, with medium changes every 3 days, until the appearance of foci from transformed cells was evident. Then the colonies were stained with 0.2% crystal violet, and foci were counted. Images of the colonies were obtained using a NIKON digital camera.
Wound healing assay
Cells were cultured in serum-free medium and grown to 100% confluence in 6-well plates. After scratching the cell monolayer with a sterile pipette tip, the cells were washed twice with 1% PBS. The wound closing procedure was observed and photographed at 0 and 24 h under an inverted phase-contrast microscope (Olympus Corporation, Tokyo, Japan).
Cell invasion assay
Cell invasion assay was performed using transwell inserts with polycarbonate membranes of 8.0-μm pore size (Corning Inc., NY) with ECMatrix gel (Chemicon) to form a continuous thin layer. In brief, 4 × 104 cells in serum-free medium were added into the upper chamber. Culture medium with 10% FBS was added into the lower chamber. The chamber was cultured in 37 °C with 5% CO2 for 24 h and fixed with methanol. After staining with 0.4% crystal violet for 30 min, cells were photographed (400×) and counted in 5 random fields. Each experiment was performed thrice.
Tumorigenicity assays in nude mice
4-week-old athymic male BALB/c nude mice were purchased from the Animal Facility of Model Animal Research Institute of Nanjing University (Nanjing, China). For xenograft models, the mice were randomly assigned to four groups. The mice in groups were inoculated subcutaneously with 1 × 107 SW480 cells, SW480 + Exo-SW620 cells, SW480 + Exo-siRNA-SW620 cells and SW480 + Exo-siMALAT1-SW620 cells in the right flank. The size of tumor was measured every 7 days. 28 days after inoculation, mice were sacrificed and tumors were isolated and weighed. Tumor volume was calculated as the following formula: (length × width2)/2. Tumors were dissected out for tissue slice or proteins analysis.
Lung and hepatic metastatic model were used to measure cells metastatic ability, and the mice were randomly assigned to four groups as mentioned above. In brief, 2 × 106 CRC cells in 0.2 ml PBS were injected into the tail vein of male BALB/c nude mice. At the beginning to show symptoms of dying after injection, tumor in lung metastasis was dissected out for tissue slice or proteins analysis. For liver metastatic model, nude mice were anaesthetized with pentobarbital sodium (Sigma, USA) by intraperitoneal injection. 1 cm incision was formed on the left side and the spleen was separated. Total of 2 × 106 CRC cells were suspended in PBS and then injected into the spleen with a 30-gauge needle. After 5–6 weeks, the mice were sacrificed, and the spleen and liver were dissected out for tissue slice or proteins analysis.
Statistical analysis
SPSS 17.0 software was used to analyze the experimental data. Each experiment was performed at least in triplicate and data were displayed as mean ± standard deviation (SD). Student’s t-test was used to compare the significant difference of two groups. The one-way analysis of variance (ANOVA) was used to determine the significant difference of multiple groups. P < 0.05 was considered to be statistically significant.
Discussion
Cancer-related deaths are primarily attributed to metastasis. Tumor microenvironment, a dynamic system mediated by intercellular communications, is responsible for tumor metastasis [
26]. Therefore, it necessitates the research of the interaction between tumor and stroma modulated by exosomes. However, the functional role of fucosylation associated with exosomes in cancer progression is largely unknown. This study supported a new function of CRC derived exosomes in the transfer of MALAT1 to promote CRC cell aggressiveness by regulating FUT4-associated fucosylation and PI3K/Akt/mTOR pathway.
In recent years, many reports have convincingly demonstrated an important function of exosomes. Intercellular exchange of molecules via exosomes has been shown to be an effective mechanism of intercellular communication, especially within the tumor microenvironment [
27]. Here, exosome-depleted FBS was used in our research mainly considering its advantages in cell nutritional support, maintaining good cellular secretion activity and the enrichment of exosomes. The exosomes treatment obviously promoted CRC cells proliferation, migration and invasion, as well as lung and liver metastasis, xenograft tumor growth. In line with our observations, Zeng et al. demonstrated that exosomes derived from CRC cells dramatically induced vascular leakiness and enhances CRC metastasis in liver and lung of mice [
28].
Aberrant fucosylation and dysregulation of FUTs have been frequently found in human cancer [
29,
30]. FUT4 could catalyze α1, 3-fucosylation that is particularly involved in a variety of pathological processes and in cancer biology [
31]. Importantly, cancer-related CD15/FUT4 is overexpressed in most of metastatic colorectal cancer (mCRC) patients and participates in cetuximab or bevacizumab mechanisms of resistance in mCRC patients [
32]. Similarly, we identified the profiles of FUT4 and α1, 3-fucosylation in mCRC patients and exosomes could regulate FUT4 expression and fucosylation level of recipient cells in CRC metastasis process, but not by directly transmitting FUT4 mRNA.
Growing evidence has indicated that the lncRNA MALAT1 contributes to tumor development in several types of human cancers, such as lung cancer and colorectal cancer [
33,
34]. The elevated expression of MALAT1 plays important role in tumor cell progression. MALAT1 gene mutation was recently found in CRC and MALAT1 overexpression induced the invasion of SW480 cells [
35]. Based on our results of clinical specimens and Oncomine and TCGA database, MALAT1 showed a higher level in tumor tissues and was connected with metastasis of CRC patients. In addition, competitive endogenous RNA (ceRNA) was reported that formed a large-scale regulatory network across the transcriptome. MALAT1 was overexpressed in six CRC cell lines and regulated the metastasis and invasion of CRC cells via targeting miR-20b-5p [
21]. However, exosome-derived MALAT1 modulating CRC progression by interacting with miRNAs has not been addressed.
Exosomes potentially promote the malignancy of tumor cells by transferring oncogenic lncRNAs to induce tumor formation and metastasis [
36]. Exosomal LncRNAs are transcribed to regulate the expression of oncogenes and tumor suppressor genes in tissue- and cell-specific manners [
17]. Ren et al. quantitatively detected that carcinoma-associated fibroblasts promoted the stemness and chemoresistance of CRC by transferring exosomal lncRNA H19 [
15]. Here, MALAT1 was proved to reside in the lumen area of CRC exosomes and exosomal MALAT1 derived from metastatic CRC cells could be transferred and regulate the expression of FUT4 in recipient cells, as a competing endogenous RNA for miR-26a and miR-26b, which regulated malignant traits of primary CRC cells. Furthermore, exosomes from siMALAT1-SW620 had no effect on promoting SW480 cells proliferation, migration and invasion, as well as lung and liver metastasis, xenograft tumor growth. On the other hand, the cell proliferation, migration and invasion were also attenuated in CRC cells treated with miR-26a/26b mimic or FUT4 inhibitor in presence of exosomal MALAT1. These results suggested that MALAT1/miR-26a/ 26b/FUT4 axis played an important role in exosome-mediated CRC progression and inhibition of this regulatory axis could attenuate the effect of exosome-induced CRC metastasis.
As a key oncogenic signaling pathway, PI3K/AKT/mTOR pathway plays a pivotal role in various cancers, including colorectal cancer [
24,
37]. More researchers clarify the molecular mechanism of this signaling pathway involved in cancer proliferation, metastasis and chemoresistance [
38,
39]. Based on our work, exosomes activated the PI3K/AKT/mTOR pathway in CRC. SW480 and HCT-8 cells treated with exosomes derived from SW620 or LoVo cells showed increased phosphorylation levels of PI3K/AKT/mTOR. However, targeting exosomal MALAT1 could attenuate this cellular signaling pathway. In addition, exosome-induced activation of PI3K/AKT/mTOR pathway exhibited dose- and time-dependent effects. Treatment with LY294002 inhibited the phosphorylation of PI3K/Akt/mTOR of SW480 cells in presence of Exo-SW620. Moreover, altered FUT4 regulated PI3K/AKT/mTOR pathway in exosome-induced CRC progression. These results further revealed that exosomal MALAT1 might promote CRC development through regulating FUT4 expression and PI3K/Akt/mTOR pathway, which offered a promising therapy target for CRC patients.
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