Background
Colorectal cancer (CRC) is the third most common cancer worldwide, with 1–2 million new cases diagnosed annually and the fourth leading cause of cancer-related death, with 700,000 deaths reported every year [
1]. By 2030, the burden of CRC is speculated to rise by 60% to include 2.2 million new cases and 1.1 million CRC-related deaths [
2]. In recent years, radiotherapy is known as a standard preoperative treatment approach to reduce local recurrence, exhibiting promoted apoptosis in response to radiotherapy [
3]. However, CRC cells often develop the resistance to radiotherapy, which remains an intractable problem in therapeutic effect and represents a major obstacle to reduce the death of CRC cells [
4]. It is reported that cancer-associated fibroblasts (CAFs), recruited from local tissue-resident fibroblasts or pericryptal fibroblasts and distant fibroblast precursors, is involved in therapeutic resistance in CRC cells [
5]. Currently, CAFs serve as a target in the anti-cancer therapy due to its contribution to tumorigenesis and malignant behavior [
6]. Therefore, our research interests arouse considering the possible mechanism of CAFs in CRC.
It is interesting to note that CAFs could secret exosomes to CRC cells, thus facilitating the progression and metastasis of CRC [
7]. Exosomes are attractive targets for cancer treatments due to their small sizes (40 ~ 100 nm) and great impacts on cells [
8]. Accumulating evidences have reported that stromal cells-secreted exosomes in the tumor microenvironment play a vital role in cancer progression through the transfer of their cargo, encompassing proteins, and messenger RNAs (mRNAs), and microRNAs (miRNAs), to cancer cells [
9,
10]. CAFs-derived exosomes (CAFs-exo) are transferred to CRC cells with elevation in miRNA levels, contributing to proliferation and chemoresistance of CRC cells [
11]. miRNAs refer to small non-coding RNA molecules, which act as a regulator in cell proliferation, apoptosis and tumor growth [
12]. miRNAs are also implicated in some critical biological processes, including radioresistance [
13]. Some miRNAs, such as miR-31, exert great effects on CRC cells resistant to radiotherapy in CAFs by regulating CRC cell proliferation and apoptosis [
14]. miR-93 is demonstrated to have impacts on cell proliferation and tumor progression in breast cancer [
15], while its role in radioresistance of CRC cells has not been reported. Moreover, Forkhead box protein A1 (FOXA1), a founding member of FOX family of transcription factors, is also proved to participate in the CRC progression [
16,
17]. FOXA1 can bind to the promoters of more than 100 genes to influence signaling pathways and cell cycle in human cancers [
18], while its specific mechanism in CRC cells resistant to radiotherapy remains largely unknown. Based on the literature and findings, we proposed the hypothesis that CAFs-exo may transfer miR-93-5p to CRC cells. As FOXA1 was predicted to be a target of miR-93-5p by online prediction analyses, we speculated that miR-93-5p could mediate radioresistance in CRC cells by targeting FOXA1. Hence, the current study aims to validate if the aforementioned hypothesis was valid and to further explore the mechanisms by which exosomal miR-93-5p affects the radioresistance in CRC cells through regulation of FOXA1 expression.
Materials and methods
Ethics statement
The study was approved by the Ethics Committee of the Affiliated Tumor Hospital of Zhengzhou University and the written informed consent was obtained from all patients. All animal experiments were in line with the Guide for the Care and Use of Laboratory Animal by the National Institutes of Health.
Study subjects
CRC tissue samples were collected from 75 patients (46 males and 29 females; aged 55–76 years with a mean age of 63.16 ± 5.98 years) who received surgical resection in the Affiliated Tumor Hospital of Zhengzhou University from August 2016 to October 2018. During the operation, 75 pairs of tumor tissues and adjacent normal tissues were harvested and immediately washed with phosphate buffer saline (PBS) containing 20% antibiotics. The tissues were then digested with type I collagenase (Sigma-Aldrich Chemical Company, St Louis, MO, USA) and hyaluronidase (Sigma-Aldrich Chemical Company, St Louis, MO, USA) to isolate NFs and CAFs [
7].
Cell culture
Human normal intestinal epithelial cells (HIEC) and human CRC cells lines, HT-29, SW480, and LoVo were purchased from American Type Culture Collection (Manassas, VA, USA). All cell lines underwent incubation in the Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone Company, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies Corporation, Gaithersburg, MD, USA) and 0.2% penicillin and streptomycin. CAFs and NFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium containing 10% FBS. Cells were then cultured in a 5% CO2 incubator (thromo3111, Jinan Beisheng Medical Devices Co., Ltd., Jinan, Shandong, China) at 37 °C.
Immunofluorescence staining
CAFs and NFs cells were seeded into 6-well plates coated with polylysine, followed by fixation in 4% polyformaldehyde at room temperature for 30 min and incubation with blocking buffer (Beyotime Institute of Biotechnology, Shanghai, China) at 37 °C for 60 min. The samples were incubated with specific primary antibody, rabbit antibodies to α-SMA (ab32575, 1: 200), and FAP (ab53066, 1: 50), FSP1 (ab124805, 1: 500) at 4 °C overnight. All of the above antibodies were purchased from Abcam Inc. (Cambridge, UK). Subsequently, the cells were cultured with fluorescent secondary antibodies, donkey anti-rabbit antibody to Alexa Fluor 594 (A21202, 1: 400) or donkey anti-mouse antibody to Alexa Fluor 488 (A21207, 1: 400), which were provided by the Life Technologies Corporation (Gaithersburg, MD, USA). After incubation avoiding light exposure for 1 h, cells were stained with 4′, 6-diamidino-2-phenylindole (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 5 min and photographed under a High Content Screening Imaging System (ImageXpress Micro 4, Molecular Devices, San Jose, CA, USA). Nuclear translocation of TGFB3 was detected using the same procedure mentioned above. The antibodies used were rabbit antibody to TGFB3 (ab15537, 5 μg/ml) and donkey anti-rabbit antibody to Alexa Fluor 594 (A21202, 1: 400).
Isolation of exosomes
CAFs and NFs cells were cultured in 6-well plates, respectively. Cells in each well were then cultured with 2 mL serum-free DMEM/F12 medium for 2 h, when the confluence reached 80–90%. The exosomes were harvested from CAFs-culture medium (CM) or NFs-CM by filtration through a 0.22 μm filter, followed by ultracentrifugation at 100000×g for 90 min. The concentrated material underwent centrifugation at 100000×g (4 °C) for 60 min. The resulting pellet was re-suspended and pelleted again. The final pellet was re-suspended in a small volume of PBS. Exosomes were stored at − 20 °C until further use.
Transmission electron microscope (TEM)
After the ultracentrifugation of exosomes, the precipitation was fixed with the mixture of 2% polyformaldehyde and 2.5% glutaraldehyde at 4 °C for 1 h, and with 1% osmic acid for 1.5 h. Following dehydration with gradient alcohol, immersion in epoxy resin overnight and embedding, the samples were polymerized at 35 °C, 45 °C, and 60 °C for 24 h and sectioned. The sections were stained with lead-uranium and observed under a TEM (H-600, Hitachi, Tokyo, Japan).
Nanoparticle tracking analysis (NAT)
Size distributions and quantification of exosomes were determined by measuring the rate of Brownian motion using a Nanoparticle tracer analyzer (Malvern, Malvern, UK). The diluted samples at concentration of (1–9) × 108 cells/ml were detected by the machine. The appropriate background gray level was selected by the operation software. The particle trajectory was recorded and the concentration and particle size distribution of the diluted samples were output. The concentration of exosomes in the original solution was calculated by dilution ratio.
Western blot analysis
The total protein was extracted using radio immunoprecipitation assay (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) containing phenylmethylsulfonyl fluoride. Total proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto the polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane underwent incubation with the primary antibodies, rabbit antibodies to CD63 (ab118307, 1: 50), CD81 (ab109201, 1: 1000), TSG101 (ab125011, 1: 1000), GRP94 (ab13509, 1: 1000), FOXA1 (ab151522, 1: 500), TGFB3 (ab227711, 1: 5000), TGF-β1 (ab92486, 2 μg/mL), Smad3 (ab40854, 1: 1000), p-Smad3 (ab63403, 1: 2000) and GAPDH (ab8245, 1: 10000) at 4 °C overnight. Subsequently, the membrane was supplemented with horseradish peroxidase-labeled Immunoglobulin G (IgG; ab205719, 1: 2000) as the secondary antibody for incubation for 1 h and visualized using an enhanced chemiluminescence kit (BB-3501, Amersham Pharmacia Biotech, Chicago, IL, USA). All of the aforementioned antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). Afterwards, the samples were photographed using the IS gel image analysis system and analyzed using the Image J.
Co-culture of CRC cells and exosomes
The exosomes were labeled with PKH67 (Sigma-Aldrich Chemical Company, St Louis, MO, USA) to monitor the interaction between CAFs-exo and NFs-derived exosomes (NFs-exo) with SW480 cells. After co-culture with PKH67-labeled CAFs-exo and NFs-exo for 24 h in a 5% CO2 incubator at 37 °C, SW480 cells were observed with the use of a Nikon Eclipse Ti confocal laser scanning microscope.
Cell treatment
All plasmids were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, Guangdong, China). SW480 cells were suspended in serum-free RPMI 1640 medium and seeded in 6-well plate. The transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Following a 24 h transfection, cells were used for the follow-up experiments. Mimic-NC and miR-93-5p mimic plasmids were transfected into CAFs by the same method. After transfection for 24 h, the exosomes were isolated from CAFs. Next, exosomes were co-cultured with SW480 cells or transfected SW480 cells for the subsequent experiments.
Irradiation in vitro
Cells in each group were cultured in disposable T25 culture flasks (5 × 106 cells/flask) in a 5% CO2 incubator at 37 °C for 16 h. Prior to irradiation, the culture flasks were filled with culture medium, and the condensate plate was used as medium to set up a built-up area (1.5 cm). Cells were irradiated with medical electron linear accelerator. The total dose was 6 Gy and the dose rate was 5 Gy/min. Source-axis distance was 100 cm, and culture continued for 48 h following irradiation.
RNA isolation and quantitation
Total RNA was extracted from cells and tissues using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Next, total RNA of mRNA and lncRNA was reversely transcribed into cDNA using a reverse transcription kit (RR047A, Takara Bio Inc., Otsu, Shiga, Japan), and the total RNA of miRNA was reversely transcribed into a cDNA using miRNA First Strand cDNA Synthesis (Tailing Reaction) kit (Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China). According to the instructions provided on the SYBR® Premix Ex Taq™ II (Perfect Real Time) kit (DRR081, Takara Bio Inc., Otsu, Shiga, Japan), reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted for mRNA and lncRNA using a real-time PCR instrument (ABI 7500, ABI, Foster City, CA, USA). The general negative primers of miRNAs and the upstream primers of internal reference U6 were provided in the miRNA First Strand cDNA Synthesis (Tailing Reaction) kit. Other primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) (Table
1). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were considered as the internal references. The expression ratio of target gene between the experimental and control groups was calculated using the 2
-ΔΔCt method.
Table 1Primer sequences for RT-qPCR
miR-93-5p | GCAGCAAACTTCTGAGACAC | GTGCAGGGTCCGAGGTATTC |
FOXA1 | GCAATACTCGCCTTACGGGCT | TACACACCTTGGTAGTACGCC |
TGFB3 | ATGCCAAAGAAATCCATAAATTC | GAAGCGGAAAACCTTGGAGGTA |
TGF-β1 | CACCATCCATGACATGAACC | TCATGTTGGACAACTGCTCC |
Smad3 | ACGCAGAACGTGAACACCAA | GCTGTGAAGCGTGGAATGTC |
GAPDH | ACAACCTTTGGTATCGTGGAAGG | GCCATCACGCCACAGTTTC |
Cell counting kit-8 (CCK-8)
After receiving their corresponding treatments in the study, SW480 cells were harvested and seeded into 96-well plates (1 × 105 cells/mL, 100 μL) for a 24-h incubation with 5% CO2 at 37 °C. To test the cell viability, each well was added with 10 μL CCK-8 reagent at 24 h, 48 h, and 72 h and incubation was carried out. After 4 h, the optical density (OD) value was measured at the wavelength of 450 nm using a microplate reader.
For soft agar colony formation assay, 2000 cells were seeded in 0.3% agar on a base of 2 mL 0.6% agar (Gibco, Carlsbad, CA, USA) in a 6-well plate. Culture dishes were transferred sequentially to a refrigerator at 4 °C for 10 min, and then to the cell culture incubator at 37 °C for 14 d. The colonies (more than 50 cells) were inspected and photographed under a microscope. Three parallel wells were set in the experiment, with the mean value obtained.
Flow cytometry
Single-cell suspensions were fixed in 70% precooling ethanol overnight at 4 °C, washed twice with PBS, and incubated with 1 mL propidium (PI, 50 mg/L)/RNAase (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 30 min under dark conditions. Cells were then evaluated using a flow cytometer (Gallios, Beckman Coulter, Shanghai, China) at 488 nm. To analyze apoptosis rates, the cell suspension was incubated with 10 μL Annexin V-fluorescein isothiocyanate (FITC) and 5 μL PI without light exposure for 15 min and were analyzed immediately with the use of a flow cytometry.
Dual luciferase reporter gene assay
Luciferase reporter vectors were constructed by inserting the three-prime untranslated regions (3’UTR) of FOXA1 downstream of the luciferase gene in a pGL3-control (Beijing Huayueyang Biotechnology Co., Ltd., Beijing, China). A site-specific mutation at the miR-93-5p binding site was created to make a target mutant form (mut) based on the FOXA1-wild type (wt). The correctly sequenced luciferase reporter plasmids of wt and mut were co-transfected with miR-93-5p mimic into the HEK-293 T cells.
The TGFB3 promoter luciferase reporter plasmid (PGL3-basic-TGFB3P) was inserted into the TGFB3 promoter sequence encoding − 45 ~ − 39, with PGL3-basic used as carrier, 5′-GACGTCA-3, which was constructed by Shanghai Generay Biotech Co., Ltd. (Shanghai, China). The PGL3-basic-TGFB3P plasmids were co-transfected with oe-NC and oe-FOXA1 plasmids into CRC cells, respectively. After transfection for 48 h, cells were lysed and incubated at 25 °C for 20 min.
A dual Luciferase Reporter Assay System kit (Promega Company, Madison, WI, USA) was used to detect the activity of firefly luciferase (M1) and of renilla luciferase (M2) in cells of each group. Luciferase activity of target gene and promoter was expressed as M2/M1.
Chromatin immunoprecipitation (ChIP) assay
The ChIP kit (Merck Millipore, Billerica, MA, USA) was used to detect the enrichment of FOXA1 in TGFB3 promoter region. According to the instructions, primers were designed based on the promoter sequence of TGFB3: 5′-TGCGCCCCCTCTACATTG-3′ and 5′ -GGTTCGTGGACCCATTTCC-3′, and synthetized by the Invitrogen (Shanghai, China).
Tumor xenografts in nude mice
Before animal experiments, a total of 1.5 × 106 SW480 cells were cultured in (1) serum-free medium, (2) serum-free medium containing 100 μg agomir-NC-exo or (3) serum-free medium containing 100 μg agomir-93-5p-exo. After 12 h, the SW480 cells were washed with PBS for the removal of excessive exosomes. A total of 30 specific-pathogen-free (SPF) BALB/c nude mice (aged 3–4 weeks) were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Nude mice were injected with the SW480 cells with different treatments as mentioned above. After 2 weeks, the tumors were 0.5 cm3 in size, and subjected to local X-ray irradiation (once every 2 days, 2 Gy/time, totally 3 times). Tumor volume was recorded every 3 days. After 15 days of irradiation, nude mice were euthanized using barbiturate overdose, and the tumors were isolated for immunohistochemistry.
Immunohistochemistry
Serial sections (4 μm) were cut from formalin-fixed and paraffin-embedded xenograft tissue samples for Immunohistochemistry in accordance with a streptavidin peroxidase kit (Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China). The sections underwent incubation with the rabbit antibodies to Bax (ab97779, 1: 500), Bcl2 (ab38898, 1: 5000), and goat anti-rabbit antibody to IgG (ab150077, 1: 500). Finally, the samples were observed and photographed under an inverted fluorescence Microscope (IX70, Olympus, Tokyo, Japan).
Statistical analysis
The data was analyzed using SPSS 21.0 software (IBM Corp., Armonk, NY, USA). The measurement data was expressed as mean ± standard deviation. The comparison of measurement data conforming to normal distribution and homogeneous variance with paired design between two groups was conducted by paired t-test. The comparison of measurement data conforming to normal distribution and homogeneous variance with unpaired design between two groups was conducted using the unpaired t-test. The comparison among multiple groups was assessed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The comparisons of data at different time points were performed by the repeated measures ANOVA, followed by Bonferroni’s post hoc test. The relationship between two indicators was analyzed using the Pearson correlation analysis. p value < 0.05 was indicative of statistical significance.
Discussion
It is known that the radioresistance of CRC cells leads to the failure of radiotherapy and toxic impacts of ionizing radiation [
20]. Recent evidence has already demonstrated that miRNAs played an important role in radio-induced apoptosis and the radioresistance of CRC cells [
21]. Exosomes were derived from various types of cells and contained parent cells-secreted miRNAs, which were involved in cancer therapies [
22]. Therefore, our study investigated the role of CAFs-exo carrying miR-93-5p in radioresistance of CRC cells. Collectively, CAFs-exo carrying miR-93-5p functioned as a facilitator in radioresistance of CRC cells via their promotion in cell proliferation and colony formation and disruption in apoptosis of CRC cells through downregulating FOXA1 expression.
One important finding in our study was that miR-93-5p was highly expressed in CRC tissues and cells, while FOXA1 was poorly expressed. The aberrant expression of miRNAs is implicated in the development and progression of CRC [
23]. Elevated miR-10b expression is found in CRC, which is correlated with the poor prognosis of patients with CRC [
24]. miR-93 expression is reported to be increased in non-small lung cancer tissues [
25]. A bioinformatics website in combination with a dual luciferase reporter gene assay validated that miR-93-5p cold target FOXA1, which was negatively regulated by miR-93-5p. FOXA1 could act as a tumor suppressor in human cancers, and its expression is associated with the prognosis of patients with cancers [
17]. FOXL1 is also demonstrated to be downregulated in gallbladder cancer tissues and cells [
26]. However, the decreased FOXL1 expression in CRC has not been reported. These evidences support that miR-93-5p was upregulated while FOXA1 was downregulated in CRC, and miR-93-5p displays a negative correlation with FOXA1.
In addition, we found that CRC cells could endocytose exosomes derived from CAFs which contained robust miR-93-5p. Exosomes have been previously described as secreted microvesicles that carry proteins, mRNAs and miRNAs by means of bodily fluids, which stimulate immune responses and accelerate communication among cells [
27]. Ren et al. confirmed that CAFs could transfer exosomes to CRC cells, affecting tumor progression [
7]. Meanwhile, various cell types-derived exosomal miRNAs were found to be correlated with metastatic niche preparation and tumor growth interference [
28]. Exosomes derived from CAFs containing abundant miR-21 could be transferred into CRC cells [
11], which supports our findings that CAFs show elevated miR-93-5p expression and deliver it to CRC cells through exosomes.
Furthermore, the data in the current study implied that exosome-mediated transfer of miR-93-5p from CAFs promoted CRC cell viability and colony formation and inhibited apoptosis to induce radioresistance in CRC cells by downregulating FOXA1 and upregulating TGFB3. It has been confirmed that elevated miR-106b could induces cell radioresistance in CRC [
13]. CAFs transfer overexpressing miR-21 into CRC cells so as to rescue apoptosis and facilitate cell proliferation [
29]. Consistent with this finding, exosome-mediated transfer of miR-21 enhances CRC cell proliferation and chemoresistance to promote the progression of CRC [
11]. Moreover, the effects of FOXA1 in CRC cell proliferation and apoptosis have been revealed by an existing literature [
17]. It has been proved that upregulation of FOXL1 inhibits cell proliferation in vitro and tumorigenicity in vivo and stimulates the apoptosis in gallbladder cancer [
26]. In addition, FOXA1 is found to negatively regulate the transcription of TGFB3 in the current study. It has been indicated that knockdown of FOXA1 could upregulate TGFB3 to activate the TGF-β signaling pathway [
19]. Those mentioned above are partially consistent with the most crucial finding of the current study, whereby CAFs-exo carrying miR-93-5p were identified to induce CRC cells resistant to radiotherapy by promoting cell proliferation and suppressing apoptosis in CRC by downregulating FOXA1 via activation of TGF-β signaling pathway.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.