Background
Colorectal cancer (CRC) incidence ranks third in the world affecting over 1 million new cases per year [
1]. Early diagnosis and effective treatment such as endoscopic resection have improved the survival of patients, but the prognosis of advanced CRC patients remains poor. Genetic and microenvironment factors have been identified as critical factors for tumors, causing the complex lethality and heterogeneity of CRC [
2,
3]. However, the discovery of effective biomarkers and molecular targets for therapy are crucial.
Extracellular vesicles (EVs) are microvesicles derived from different types of cells and play critical roles in cellular communication by transporting information cargo, including proteins, mRNAs and microRNAs (miRNAs) [
4]. Numerous researches have reported that tumor-derived EVs, which are enriched in the body fluids of cancer patients, promoted tumor angiogenesis, metastasis or chemotherapy resistance in CRC [
5‐
8].
miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression. Although miRNAs are closely associated with cancer development, there is a limited understanding of their multiple mechanisms in tumor-derived EVs. Previously, the functions of extracellular miRNA to recipient cells have been extensively studied that normal fibroblasts or macrophages could be activated to cancer-associated cells by tumor-derived extracellular miRNA, further promoting tumor progression in different molecular pathways [
9,
10]. These pathways also include an emerging hallmark of malignancy “metabolic reprogramming”. For examples, Yan et al. uncovered that extracellular miR-105 derived from cancer cells mediated metabolic reprogramming of stromal cells to promote tumor growth [
11].
However, the regulatory EV secretion net-work in donor cells such as cancer and cancer-associated cells remains obscure. Mounting evidence suggests that the suppression of cancer-derived EVs could be a novel method for tumor therapy [
12]. Recent studies have reported that miRNA could regulate EV secretion to keep itself steady-state level. For instance, miR-26a has been found to be related to EV secretion, resulting in the inhibition of prostate cancer progression [
13]. Besides, the relationship between EV secretion and reprogrammed metabolism in cancer cells has not been identified before.
In our study, we demonstrated that let-7a miRNA, as a classical tumor suppressor, let-7a miRNA was upregulated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We hypothesized that let-7a could regulate EV secretion in CRC. We identified the effect of let-7a on EV secretion, which downregulated the intracellular levels of let-7a via directly targeting SNAP23, a controller of the docking and release of multi-vesicular bodies (MVBs). Furthermore, we found a novel mechanism of let-7a, as an important regulator of cancer cell metabolism [
14], by regulating mitochondrial oxidative phosphorylation (OXPHOS) that are involved in EV secretion. Thus, we show that let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS by targeting SNAP23.
Methods
Cell lines and clinical tissue specimens
The human CRC cell lines SW48, SW480, HT29, SW620 and normal colon epithelial cell line FHC were grown in DMEM supplemented with 10% fetal calf serum (FCS). After the thawing of the parental stock, all the cells were passaged within 6 months. Human serum and tissue specimens were collocated from CRC patients and healthy controls (HC) in Shanghai General Hospital. All experimental procedures were approved by the Ethics Committee of Shanghai General Hospital.
EV isolation and identification
EVs from the culture medium were isolated by a differential ultracentrifugation (UC) [
13,
15]. Cells were transplanted into 10 cm plates and changed DMEM with 10% EV-depleted FCS. After 48 h, the culture medium was collected and centrifuged at 2000×g for 15 min to remove contaminating cells. Subsequently, the supernatant was filtrated through 0.22 μm filter (Millipore, Billeria, MA). Then, the medium was centrifuged for 120 min at 120,000×g to pellet the enriched EVs (Optima L-100XP, Beckman, USA). The pelleted EVs were washed with PBS and centrifuged at 120,000 for another 120 min. For miRNA detection in serum samples, EVs were isolated from serum samples using UC or Exoquick™ Reagent precipitation (System Biosciences, Mountain View) according to these methods [
16,
17]. Briefly, 750 μl of serum was diluted with phosphate-buffered saline (PBS) and centrifuged by UC, or 250 μl of serum was mixed with precipitation solution, centrifuged and collected for subsequent RNA extraction.
EVs to be observed by transmission electron microscopy were suspended in 2.5% glutaraldehyde at 4 °C overnight. On the next day, vesicles were dropped in carbon-coated copper grids, stained with uranylacetate and imaged with a microscope (H7500 TEM, Japan). EVs were determined by Nanosight particle tracking analysis (Merkel Technologies Ltd., Israel, NTA 3.2 Dev Build 3.2.16). The concentrations of EV release were normalized as particle/cell to obtain net EV secretion rates.
RNA extraction and qPCR
RNA was extracted using TRI reagent solution (Sigma) and RNeasy mini kit (Qiagen, Germany). RNA was also purified with miRCURY RNA Isolation Kits-Biofluids (Exiqon) from re-suspended EVs. Complementary DNA (cDNA) synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche for mRNA and Novabio for miRNA). qPCR assay was performed using ABI 7900 T PCR System (Applied Biosystems, Foster City, USA). The sequences of the primers used for detection are provided in Additional file
1: Table S1.
Immunoblotting
The cell and tissue lysates were extracted using RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (NCM Biotech, Suzhou, China). Equivalent amounts of protein were electrophoresed on SDS-PAGE gels followed by transferring to PVDF membranes (Millipore, Billeria, MA). Antibodies against SNAP23 (Proteintech, China), TSG101, CD9 (Abcam, USA), Calnexin, Lin28a, Cyclin-D1, c-Myc, PKM2, p-PKM2 (Tyr105) (Cell Signaling Technology, USA) and ATP5A1, SDHA, CYTb, COX1 (ABclonal, Wuhan, China) were used for immunoblotted. β-Actin (Proteintech, China) served as the loading control. Protein bands were visualized and analyzed using the Odyssey Imaging System (LI-COR, USA) and Image J software.
RNA interference and luciferase reporter assay
miRNA mimics, inhibitors and siRNAs were purchased from Shanghai GenePharma Company. According to the manufacturer’s instruction, an amount of 50 nM miRNA mimics, inhibitors or siRNAs were transfected into cells with Lipofectamine 3000 (Invitrogen, USA) to investigate the effect of let-7a on EV secretion.
Luciferase reporter assays were performed by co-transfecting HEK293 cells with let-7a mimic or ctrl with luciferase vectors (empty luciferase vector, luciferase vector containing wild-type target gene 3′-UTR or mutant-type target gene 3′-UTR) for SNAP23 using Lipofectamine 3000 and performing the Dual-Luciferase Reporter Assay System (Promega, USA) after 72 h.
Establishment of stable cells
Lentiviral vectors containing shRNA SNAP23 (sh-SNAP23) and overexpressing SNAP23 were obtained from Shanghai Qihe Company, with shRNA non-targeted control (sh-NT) and empty vector (Vector) as controls. The designed target sequences were showed in Additional file
1: Table S1. The transduced CRC cells were selected with 5 μg/ml puromycin (Sigma, USA).
Cell functional assays
Cell proliferation was assessed using a cell counting kit (CCK8; Dojindo, Japan). It was confirmed using a Gen5 microplate reader (BioTek, USA) by measuring the absorbance at 450 nm at 48 h. The EdU assay (RiboBio, Guangzhou, China) was also performed to detect the proliferation of CRC cells. Cell migration was determined by a wound healing assay. Cells were wounded by scratching lines with a pipette tip and then imaged after 48 h. Cell invasion was detected using transwell chambers (Corning, USA). Six hundred μl of DMEM containing 20% FCS was added in the lower chamber, and 100 μl of DMEM medium containing CRC cells was added to the upper chambers. The cells were incubated for 24 h and fixed with methyl alcohol and stained with 0.1% crystal violet.
Bioenergetic assays
We used the Seahorse analyzer (Seahorse Bioscience, USA) to measure the mitochondrial activity of CRC cells. Briefly, cells were seeded into Seahorse miniplates (96 wells) overnight. After which cells were washed and exchanged in Seahorse assay buffer (adjust the pH to 7.4) in a 37 °C non-CO2 incubator for 1 h prior to assay, pharmaceutical compounds including oligomycin, FCCP, antimycin and rotenone or glucose, oligomycin and 2-DG were prepared to stressor mix at optimized concentration. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were then determined by Wave software. The values of OCR and ECAR were normalized to the number of cells per well. Cellular ATP levels were detected using the Cell-Glo ATP assay (Promega, USA).
Fluorescence microscopy
Fluorescence microscopy was used to observe the subcellular localization and expression of SNAP-23 and mitochondrial in CRC cells. Cells were firstly added Mito-tracker™ Red CMXRos (Invitrogen™, USA) for 40 min. After it, cells were fixed with 4% paraformaldehyde for 15 min and then added with 0.2% Triton X-100 for 5 min and subsequently incubated with antibodies against SNAP-23 followed by incubation with Goat anti-Rabbit Secondary Antibody, Alexa Fluor 488 (Invitrogen™, USA). At last, the samples were imaged by confocal microscopy (Leica TCS SP8, Germany).
Immunohistochemistry
Human CRC tissues were blocked and subsequently incubated with antibodies overnight at 4 °C, followed by incubation with the HRP-conjugated secondary detection antibodies (Dako Cytomation, USA) at room temperature for 30 min. The calculation of IHC staining scores was the same as the methods in our previous report [
18]. Briefly, the staining intensity was independently scored by 2 pathologists blinded to the clinical data (0, no color; 1, weak; 2, moderate; 3, strong), and the percentage of positive cells (0, < 5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%) was semi-quantitatively assessed. The final scores (0–12) were then calculated by multiplying these 2 values. For analysis of clinical parameters, patients were divided into 4 subgroups based on this final score (negative, 0–2; weak, 3–4; moderate, 5–8; and strong, 9–12).
Animal experiments
6-week-old female NSG mice were used for animal studies. All mice were injected into the back with 8.0 × 106 SW480 cells expressing sh-NT or sh-SNAP23 to establish a xenograft model. Upon tumor xenografts reaching a volume of ~ 30 mm3, 3 μg of EVs derived from sh-NT SW480 cells were intravenously injected into mice through the tail vein twice a week. Each group had 4 mice and tumor volumes were measured twice weekly. After 4 weeks, all mice were sacrificed and tumor weights were calculated. All animal experiments were approved by the Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine Animal Care and Use guidelines.
Pathway analysis of data from the Cancer genome atlas
RNA-seq data of 456 colon cancer tissues and miRNA-seq data of 444 colon cancer tissues from TCGA Data Portal were obtained in March 2019. Four hundred forty-one samples with both RNA-seq data and miRNA-seq data were used to apply pathway analysis. The median value of let-7a was used to divide the samples into two groups. CAMERA performs a competitive test to identify whether the genes in the set are highly ranked in terms of differential expression relative to genes not in the set. The limma package and Hallmark gene sets curated by MSigDB was used to apply CAMERA to perform pathway analysis. Gene sets with adjusted p-value below 0.05 were considered significantly enriched.
Statistical analysis
Each result was repeated from three independent experiments and presented as mean ± SEM. Two-tailed unpaired and paired Student’s t-test and x2-test were used to determine statistical comparisons, and p-value < 0.05 was considered statistically significant.
Discussion
It has been well established that miRNA let-7 family, first identified in
Caenorhabditis elegans, is essential pathological events in tumorigenesis and progression of various human cancers [
29]. As tumor suppressors, let-7 miRNAs inhibit oncogenes expression including c-Myc, K-ras, HMGA2 and cell cycle factors [
30]. Loss expression of let-7 relieves suppression of these oncogenes facilitating tumor growth and metastasis. However, the functional role of let-7 miRNA in EVs remains unclear.
In our study, we first determined that let-7a was markedly upregulated in the serum EVs of CRC patients compared to health groups. And we also determined that serum EV let-7a could be an effective blood marker for tumor detection, suggesting the clinical importance of our findings. However, we confirmed the reduced let-7a expression in CRC tissue, consistent with early results that let-7a is known to be downregulated in various human malignancies including CRC [
29,
31]. These results prompted us to seek the causes of the difference between let-7a expression levels in tissue and serum, which have never been reported before. A previous study found that the regulation of the intracellular levels of miR-200 family involved their secretion in EVs by protein kinase C to maintain miR-200 steady-state levels [
23]. Therefore, we assume a specific link of let-7a to EV secretion in tumors. To support this, we show the significant difference between intra- and extra-cellular expressions of let-7a in CRC cell lines. SW620 with high malignant degree decreased endogenous and increased exogenous expression of let-7a compared to SW480. Furthermore, our data indicated that let-7a regulated EV secretion in CRC cells (Fig.
2g, h). As mentioned above, let-7a not only inhibits tumorigenesis but also prevents the secretion of EVs.
The biogenesis and release of EVs are regulated by a variety of molecules, which involves the transport of MVBs and their docking and fusion with the plasma membrane. It has previously been reported that Rab small GTPases, including Rab27a and Rab27b, participate in regulating different steps of EV secretion [
22,
32]. SNAP23 is a t-SNARE molecule mediating the fusion process that contributes to release intraluminal vesicles [
20,
33]. To investigate the target genes of let-7a that inhibit EV secretion, three individual gene sets were combined analyzed, and three candidate genes,
SNAP23, SYT7 and RAN15 were selected. Given that SNAP23 is widely expressed in cell types and plays a big role in various kinds of carcinomas [
20,
21], we choose SNAP23 to further investigation. Let-7a directly regulated SNAP23 was determined using a luciferase reporter assay. Next, we inhibited let-7a expression in SW480 cells expressing sh-SNAP23, that the enhanced ability of EV secretion was lost. Several studies have demonstrated that the suppression of cancer-derived EVs may have therapeutic value by inhibiting cancer proliferation and metastasis [
34,
35].
We further investigated the effects of SNAP23 to cell growth that downregulated SNAP23 expression suppressed the proliferation, migration and invasion of CRC cells. Importantly, sh-SNAP23 CRC cells with overexpressed let-7a had a more significant impact on the inhibition of cell growth. Furthermore, animal experiments showed that the tumor xenografts of NSG mice injected with EVs increased the tumor size and weight of the SNAP23-depleted SW480 xenografts. Besides, elevated SNAP23 expression in CRC tissue has been confirmed compared to adjacent normal samples. Overall, our results demonstrated that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.
Another key finding of this work was that let-7a suppressed mitochondrial OXPHOS in CRC cells dependently on SNAP23, as well as the regulation of EV secretion. The “Warburg effect”, a defining feature of cancer cells to employ a modified metabolic program with increased glycolytic activity and lactate secretion, even when oxygen is present, facilitates cell energy needs to support tumor growth [
36]. However, this enhanced aerobic glycolysis of cancers doesn’t occur from a consequence of defective mitochondrial respiration, with most cancers retaining mitochondrial function to facilitate the dynamic interplay between OXPHOS and glycolysis [
37]. Our previous study has reported that Toll-like receptor 2 could augment both OXPHOS and glycolysis to promote tumor growth [
18]. It has also been established that Lin28 and let-7 family enhanced the translation of mRNAs for several metabolic enzymes, thereby increasing not only OXPHOS but also glycolysis [
27]. In another study, let-7 was reported to facilitate glycolysis while inhibiting OXPHOS process in hepatoma cells [
14]. Therefore, a provocative question remains to be answered to understand more adequately the role of let-7-regulated mitochondrial metabolic reprogramming. Apart from it, the increased secretion of EVs is another phenomenon observed during tumorigenesis. But few studies have explored the link between the metabolic reprogramming and active EV secretion in tumor cells. We confirmed that let-7a significantly suppressed mitochondrial OXPHOS and ATP synthesis in CRC cells. Furthermore, the mechanism of let-7a-regulated OXPHOS was been found to be closely related to SNAP23. Although several studies have reported that let-7a could directly downregulated PKM2 [
38,
39], we found no obvious change of PKM2 with overexpression of let-7a in CRC cells (Additional file
5: Fig. S4c). Because SNAP23 could in turn downregulate the let-7a expression, we assumed that SNAP23 promoted OXPHOS by regulating the lin28/let-7a pathway. Previous studies suggest that Lin28a/Sdha axis could promote OXPHOS in macrophages [
28]. We found SNAP23 upregulated Lin28/SDHA axis in CRC cells. Interestingly, SNAP23 has been observed to locate in the mitochondrial membrane and facilitate the fatty acids into mitochondria for β-oxidation [
40‐
42]. SNAP23 might co-localize with plasma membrane and mitochondria, which clarifies the reason that the inhibition of it eliminates the effect of enhanced OXPHOS.
Since cancer cells released large amounts of intracellular let-7a miRNAs to extracellular fluids, the function of extracellular let-7a absorbed into recipient cells is still worth investigating. Several studies reported that extracellular let-7a suppressed the growth of tumor cells [
43,
44]. However, the tumor growth of sh-SNAP23 SW480 bearing mice was partially rescued with the injection of sh-NT SW480-derived EVs (Fig.
5). Let-7a was enriched in sh-NT CRC cells-derived EVs, but the uptake of EVs by sh-SNAP23 cells promotes tumor growth. Tumor cells might not only selectively secrete but also uptake of various types of EVs to keep alive. Let-7 family has been recently found to control both pro- and anti-inflammatory responses in macrophages by regulating the accumulation of the key metabolite genes [
45,
46]. It suggests that we could focus on the roles of extracellular let-7 in cellular metabolism and energy in immune cells or other tumor-associated cells. And the mechanisms of let-7a/SNAP23 pathway in tumor cells need to be more specific.
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