Background
Lung cancer is one of the most common malignant tumors. The number of newly diagnosed patients is approximately 1.8 million annually, and about 1.6 million patients die of lung cancer every year [
1,
2]. Despite advancements in the diagnosis and treatment of lung cancer, patient prognosis is still poor, primarily because the pathogenesis of the disease has not been fully elucidated. Lung adenocarcinoma (LUAD) is the most common type of lung cancer and has a very poor prognosis, with a five-year survival rate below 20%. Similar to other tumors, the occurrence of LUAD is caused by multiple factors, including the environment, living habits, occupational exposure, and personal factors [
3]. At present, abnormal expression of genes is considered to be the basis of the occurrence and progression of LUAD. These genes often regulate cell proliferation, survival, and radio- and chemotherapy resistance [
4‐
6].
Radiotherapy is often used for the treatment of solid tumors. Compared with other treatment methods, radiotherapy has evident economic advantages [
7‐
9]. Moreover, the combination of radiotherapy and other treatments usually has a better clinical outcome than individual modalities [
7‐
9]. However, radiation treatment also causes an adaptive response of LUAD, which facilitates the development of acquired radioresistance of LUAD. Radioresistance undermines the effectiveness and efficiency of radiotherapy, resulting in treatment failure in many cases [
10,
11]. Studies have found that irradiation especially at low dosages induces the up-regulation of survivin and hypoxia inducible factor in cancer cells. These adaptive responses contribute to the survival of cancers after exposing to irradiation [
12,
13]. There is also evidence suggesting that radioresistance is usually associated with the overexpression of DNA repair factors (e.g., ATM) and antioxidant enzymes such as Mn-superoxide dismutase [
14,
15]. Nevertheless, many scholars believe that the adaptive response to irradiation and consequently radiotherapy resistance are regulated by complex interactions between multiple genes and/or proteins. Therefore, these processes may depend on molecular and pathological processes that have not yet been identified.
6-methyladenine (m6A) is the most common RNA methylation modification in eukaryotic cells, which is regulated by m6A methyltransferases, m6A demethylases, and RNA binding proteins [
16], and these are termed m6A writers, erasers, and readers, respectively. Studies in LUAD cell lines, A549 and H1299, have found that methyltransferase-like 3 (METTL3), a core m6A writer, influences the m6A levels of numerous target genes, some of which are oncogenes, such as epidermal growth factor receptor (EGFR) and Hippo pathway effector molecule TAZ [
17]. Loss- and gain-of-function studies have further confirmed that METTL3 promotes the proliferation and invasion of lung cancer cells [
17]. Du et al. found that microRNA-33a inhibits
METTL3 gene expression, leading to inhibition of lung cancer cell growth [
18]. These results suggest that m6A modification of
METTL3 is closely related to lung cancer progression.
A class of small endogenous non-coding single-stranded RNAs, essentially consisting of miRNAs, plays important roles in mRNA expression by regulating their degradation [
19‐
21]. Studies have found that the expression of many miRNAs in LUAD cells is aberrant after irradiation, which disrupts mRNA expression. Therefore, miRNAs, such as miR-181, miR-221, miR-222, and miR-26b, have also been associated with multiple hallmarks of cancer cells, including radiosensitivity, proliferation, migration, and invasion [
19‐
21].
Numerous researchers have studied changes in mRNA, miRNA, and mRNA m6A levels in LUAD cells after irradiation [
17,
22‐
24]. Based on these studies, we screened out VANGL1 and found that the upregulation of VANGL1 is associated with increased
VANGL1 mRNA m6A levels and decreased miR-29b-3p abundance in LUAD in response to irradiation. Moreover, we revealed that VANGL1 attenuated the detrimental effect of irradiation probably through regulating DNA repair signaling. This study hence added a novel understanding of the mechanism underlying the adaptive response of LUAD to irradiation.
Materials and Methods
Clinical study
The clinical study was approved by the Ethics Committee of Harbin Medical University, and all patients involved provided written informed consent. This study selected a total of 36 patients with unresectable stage III or IV LUAD (age range: 31–67 years) from January 2018 to May 2019. LUAD tumor staging was performed according to the International Classification of Diseases for Oncology. All these patients had not received chemotherapy, immunotherapy, and tumor surgical resection before radiotherapy. In addition, these patients had no other serious diseases before they were diagnosed with LUAD. Thoracic radiotherapy to the planning gross tumor volume (pGTV) (60-66 Gy/30‐33F/6‐7w) was delivered to patients. Patients received conventionally fractionated radiotherapy, namely 1.8-2.0 Gy irradiation each time per day, and five times each week for 6–7 weeks. Approximately 5 mL of blood was drawn before and 30 min after the final irradiation, and plasma was separated to detect miR-29b-3p levels by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR).
Cell culture and irradiation
A549 and H1299 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). A549 cells were grown in High Glucose Dulbecco’s Minimal Essential Medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, Utah, USA) and 1% penicillin/streptomycin (Invitrogen Life Technologies). H1299 cells were grown in HEPES-containing RPMI 1640 medium (HyClone) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Both A549 and H1299 cells were cultured at 37 °C in a humidified incubator containing a mixture of 95% air and 5% CO2.
A549 and H1299 cells were plated in 3.5 cm dishes and incubated in culture medium until 70–80% confluency was attained. Cells were next cultured in medium without FBS and exposed to 6 MV X-ray irradiation at a dose of 2 Gy, a dose determined based on preliminary experiments in which the cells were subjected to 1, 2, 4, and 6 Gy. Seventy-two hours after irradiation, the rates of cell proliferation and apoptosis were measured by CCK-8 assays and Alexa Fluor 488‑Annexin V /propidium iodide (PI) double-staining tests.
Cell transfection
Tumor cells were seeded at a density of 2.0 × 105 cells per well in a 6 cm dish. Lentivirus packaging VANGL1-shRNA (GenePharma, Co., Ltd. Shanghai, China) was transduced into cells to generate VANGL1-stable knockdown cell lines. Briefly, the medium was replaced with virus-containing supernatant supplemented with 10 ng/mL polybrene (Sigma-Aldrich). After 48 h, the medium was refreshed. Cells were selected by incubation with 4 mg/mL puromycin (InvivoGen). To knock-down IGF2BP2 and 3, or to over-express METTL3 and VANGL1, cells were transfected with IGF2BP2-shRNA and IGF2BP3-shRNA (GenePharma), or METTL3 and VANGL1 expression vector (pEGFP-C1; Invitrogen Life Technologies), using Lipofectamine™ 2000 (Invitrogen Life Technologies), following the procedures recommended by the manufacturers.
The inhibitors and mimics of miR-29b-3p, miR-15b-5p and miR-200a were synthesized by GenePharma, Co., Ltd. (Shanghai, China). These inhibitors were added individually to the culture media at a final concentration of 100 nM and transfected into cells using Lipofectamine™ 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. These mimics were transfected into cells using the similar method, then these cells were exposed to 2 Gy irradiation.
Quantitative polymerase chain reaction (qPCR)
Total cellular RNA was obtained using Trizol reagent (Invitrogen Life Technologies). Complementary DNA (cDNA) was synthesized using a First-Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania) and 1 µg of RNA template. qPCR was performed with a 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using a SYBR® Green PCR kit (Applied Biosystems). The PCR primers are shown in Table
1. U6 small nuclear RNA (U6 snRNA) and β-actin were used as an internal control to calculate the relative expression levels of miRNAs and mRNA, respectively.
Table 1
The primers for PCR assay
VANGL1 | forward | 5'-ATGTCACAGTCCGTGTTCCA-3' |
reverse | 5'-CCAGAAGTGCCGAATCATTT-3' |
METTL3 | forward | 5'-TTGTCTCCAACCTTCCGTAGT-3' |
reverse | 5'-CCAGATCAGAGAGGTGGTGTAG-3' |
IGF2BP2 | forward | 5'-AGCTAAGCGGGCATCAGTTTG-3' |
reverse | 5'-CCGCAGCGGGAAATCAATCT-3' |
IGF2BP3 | forward | 5'-ACGAAATATCCCGCCTCATTTAC-3' |
reverse | 5'-GCAGTTTCCGAGTCAGTGTTCA-3' |
beta-actin | forward | 5'-CATGTACGTTGCTATCCAGGC-3' |
reverse | 5'-CTCCTTAATGTCACGCACGAT-3' |
miR-29b-3p | forward | 5'-ACACTCCAGCTGGGTAGCACCATTTGAAATC-3' |
reverse | 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTG AGAACACTGA-3' |
miR-15b-5p | forward | 5’-TAGCAGCACATCATGGTTTACA-3’ |
reverse | 5’-TGCGTGTCGTGGAGTC-3’ |
miR-200a-3p | forward | 5ʹ-TAACACTGTCTGGTAACGATGT-3’ |
reverse | 5ʹ-CATCTTACCGGACAGTGCTGGA-3’ |
U6 snRNA | forward | 5'-CTCGCTTCGGCAGCACA-3' |
reverse | 5'-AACGCTTCACGAATTTGCGT-3' |
Western blot analysis
Cells and tissues were solubilized in lysis buffer (NO. 20–188; Sigma-Aldrich) and the solution was boiled for 5–10 min. The protein samples were separated by SDS-PAGE, following which the proteins were transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and then incubated with primary antibodies directed against VANGL1 (dilution 1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Ki67 (dilution 1:1000, Santa Cruz Biotechnology), caspase-3 (dilution 1:1000, Santa Cruz Biotechnology), BRAF (dilution 1:500, Santa Cruz Biotechnology), TP53BP1 (dilution 1:500, Santa Cruz Biotechnology), RAD51 (dilution 1:500, Santa Cruz Biotechnology), and β-actin (dilution 1:1500, Santa Cruz Biotechnology) for 2 h at room temperature. The blots were developed using peroxidase-conjugated anti-mouse IgG secondary antibody (Santa Cruz Biotechnology, Inc.), and the proteins were visualized by enhanced chemiluminescence (Amersham Biosciences, NJ, USA). β-actin was used as a loading control.
Cell viability and apoptosis assays
Cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell viability assays. The cells (1 × 103) were seeded into 96-well plates. Optical absorbance was measured using an automated microplate reader (Tecan, NANOQUANT, Switzerland).
Cells were dual-stained with Annexin V‑Alexa Fluor 488 and propidium iodide (Kaiji Biological Inc., Nanjing, China), according to the manufacturer’s instructions. Briefly, 1 × 106 cells were seeded in 6 well plate after cells were underwent transfection and radiation treatments. The cells were trypsinized and single cells suspension was mixed with 5 µL Annexin V‑Alexa Fluor 488 followed by 5 µL PI solution in dark and incubated for 15 min. The percentage of apoptotic cells was analyzed using a dual laser flow cytometer (Becton Dickinson, San Jose, CA, USA) and estimated using ModFit LT software (Verity Software House, Topsham, ME, USA).
Cell invasion assay
Transwell chambers (Corning, New York, USA) were used to test cancer cell invasion, with 8 µm membrane filter inserts coated with Matrigel (BD Biosciences, California, USA). Briefly, the cells were trypsinized and suspended in serum-free medium. Next, 1 × 104 cells were added to the upper chamber, and the lower chamber was filled with medium containing 10% FBS. After 36 h of incubation, the cells that had invaded the lower chamber were fixed with 95% ethyl alcohol for 15–20 min and stained with hematoxylin for 10 min. Next, the cells were enumerated microscopically.
Immunofluorescence (IF) assay
Cells were fixed with 4% paraformaldehyde for 15 min and blocked with PBS containing 0.3% Triton X-100/5% BSA (w/v) for 1 h at room temperature, before incubation with antibody specific for γH2AX (1:500). Incubation with the secondary fluorescent-labeled antibody (Alexa Fluor 488, Invitrogen) was performed in the dark prior to microscopic analysis.
M6A-IP-qPCR assay
M6A-IP-qPCR was performed following the previous study [
25]. RNA was extracted and treated with DNase I to remove genomic DNA contamination. The RNA was fragmented by ultrasound for 10 s. The mRNA (~ 3 µg) as an input was then incubated with protein A magnetic beads conjugated with an anti-m6A antibody (Abcam). The magnetic beads were washed with 0.5 mL RIP Wash Buffer (Abcam), and RNA-antibody complexes were treated with protease, following which only RNA was left after the m6A antibody was digested. The precipitated mRNA and input RNA was subjected to conventional qPCR to detect the expression of VANGL1. VANGL1 m6A level = VANGL1 expression in the precipitated mRNA / its expression in the input × 100%.
m6A dot blot assay
Total poly(A) + RNA was isolated and denatured at 65 °C within 5 min. Next, the samples were divided into subgroups of 400, 200 and 100 ng, and transferred to a nitrocellulose membrane with a Bio-Dot Apparatus (Bio-Rad, USA). The membrane was UV crosslinked for 5 min, blocked with 5% nonfat milk and incubated with specific m6A antibody (1:1000, Abcam) overnight at 4 °C. Dot-blots were incubated with HRP-conjugated anti-mouse immunoglobulin G (IgG) for 1 h before being visualized using an imaging system (Bio-Rad). The membrane was stained with 0.02% methylene blue (Sangon Biotech, Wuhan, China), which was performed to ensure consistency between the different groups.
mRNA and protein stability assays
Actinomycin D (Sigma) was added to a concentration of 5 mg/mL to inhibit intracellular RNA synthesis. At stipulated time points, total RNA from the cells was obtained using TRIzol reagent (Invitrogen). RNA quantities were determined using RT-qPCR.
Cycloheximide (CHX, Sigma) was added to a concentration of 20 mg/mL to inhibit intracellular protein synthesis. At stipulated time points, total protein from the cells was collected for western blot assay.
Dual-luciferase reporter assay
The putative binding site of miR-29b-3p at VANGL1 3′-UTR together with 100 nt upstream and downstream from the binding site were cloned and further inserted into into the pmirGLO vector (XhoI and NotI restriction enzyme sites; Promega, Madison, WI, USA) to construct VANGL1 wild type (WT) reporter. In addition, the putative binding site of miR-29b-3p at VANGL1 3′-UTR was mutant to construct VANGL1 mutant type (MT) reporter. A549 cells were seeded onto 12-well plates with the density of 1 × 104 cells/well and transfected with the wild or mutant reporters together with either miR-29b-3p mimics or NC using Lipofectamine 2000 (Invitrogen). Cells were harvested at 48 h and the activity of firefly luciferase was normalized to that of renilla luciferase.
Immunoprecipitation (IP) assay
Total protein was extracted and then incubated with Protein A/G Plus‑Agarose beads (Thermo Fisher Scientific) conjugated with an anti-BRAF antibody (Santa Cruz Biotechnology). After incubation at 4˚C overnight, the mixture was centrifuged at 500 × g for 5 min at 4˚C. The supernatant was discarded and the IP products were washed three times with PBS. The targeted proteins were finally detected using western blot assays.
Tumor xenograft in nude mice
All animal experiments were approved by the Ethics Committee for Animal Research of Harbin Medical University Cancer Hospital (protocol number: 2017-029). Nude mice (4–5 weeks old, male) were purchased from the Central Animal Facility of Harbin Medical University. Two hundred milliliters of A549 cells (1 × 106) were injected into the left flank of the back of each mouse. When the average tumor size reached approximately 50 mm3, the tumor-bearing nude mice were exposed to 2.0 Gy X-irradiation for each time. The same treatment for each group was repeated 3 times (the interval time was 5 days). Tumor volumes were measured and calculated according to the formula V = (length × width2)/2 (mm3) every 3 days. All mice were sacrificed on day 25 and the weights of tumors were measured. Primary tumors were excised, formalin-fixed, and paraffin-embedded for subsequent immunohistochemistry assays.
Immunohistochemistry assays
Formalin-fixed and paraffin-embedded tissues of the tumor xenografts were deparaffinized and rehydrated. Tissues were treated for 20 min at 100 °C in an autoclave for antigen retrieval, and then blocked with a blocking reagent (Protein Block Serum-Free, Dako Cytomation, Glostrup, Denmark) to avoid non-specific interactions. Next, the sectioned tissues were incubated with anti-Ki67 and anti-active caspase-3 antibodies (Santa Cruz Biotechnology) overnight at 4 °C, followed by horseradish peroxidase (HRP)-labeled anti-rabbit IgG (Histofine, Simple stain MAX-PO; Nichirei, Tokyo, Japan) for 30 min at room temperature. The sections were treated with 3, 3′-diaminobenzidine tetrahydrochloride solution.
Statistical analysis
Results are presented as the mean ± S.D. of three independent experiments. One-way analysis of variance (ANOVA) with post-hoc Dunnett’s testing was used for multiple comparisons between each group (SPSS13.0 software, IBM, USA). Paired or unpaired two-tailed t-tests were used for comparisons between two groups. Significant differences were established at P < 0.05.
Discussion
This study found that VANGL1 expression in LUAD cells was dose-dependently increased by irradiation, while increased TRIAP1, DRG1 and SEMA3A expression after 1 Gy irradiation was not observed after higher doses of irradiation. The knock-down of VANGL1 significantly increased radiation-induced injury in LUAD cells, suggesting that VANGL1 mitigates the detrimental effect of irradiation on LUAD. VANGL1 is a transmembrane protein. It is well recognized that VANGL1 interacts with the C-terminal domain of a tumor metastasis suppressor, KAI1, therefore VANGL1 is also termed KITENIN (
KAI1 C-terminal
interacting
tetraspa
nin). The cancer-promoting effects of VANGL1 on cell proliferation and invasion have been identified in colorectal and glial malignancies [
27,
28]. According to our
in vitro and
in vivo studies, we suggested that upregulated VANGL1 protects LUAD cells from radiation-induced injury. Therefore, VANGL1 is a potential target to improve the efficiency of radiotherapy.
In this study, we observed that the m6A levels of
VANGL1 mRNA were increased by irradiation in a dose-dependent manner. Xiang et al. found that methylation at the 6 position of adenosine (m6A) in RNA is rapidly induced at sites of DNA damage in response to ultraviolet irradiation [
24]. However, cells with METTL3 knock-down show delayed repair of ultraviolet-induced DNA damage and elevated sensitivity to ultraviolet, which suggests the importance of m6A modification in ultraviolet-responsive DNA damage response. To determine the effects of m6A modification on VANGL1 expression, we changed the expression of m6A writers and readers. Over-expressed METTL3 promoted
VANGL1 mRNA stability and subsequent protein levels in LUAD cells after irradiation. However, these promoting effects were reversed after IGF2BP2 and IGF2BP3 were knocked-down. These results suggested that METTL3, IGF2BP2 and IGF2BP3 collectively promoted VANGL1 expression by elevating mRNA stability.
m6A readers are a group of proteins that recognize m6A-modified RNAs and then regulate multiple processes involving RNA, such as RNA stability, translation, splicing and transport. IGF2BP2 and IGF2BP3 play important roles in mRNA stability [
29,
30]. It has been identified that IGF2BPs recruit target transcripts to cytoplasmic protein-RNA complexes (mRNPs). This transcript ‘caging’ into mRNPs allows mRNA transport and transient storage, which facilitates transcripts to encounter the translational apparatus and shields them from endonuclease attacks or miRNA-mediated degradation [
29,
30]. A study in colorectal carcinoma showed that higher expression of METTL3 promotes m6A levels in
SOX2 transcripts. m6A-modified
SOX2 is subsequently recognized by IGF2BP2, which prevents
SOX2 mRNA degradation [
29]. Previous studies have also found that METTL3 overexpression stimulates m6A modification of hepatoma-derived growth factor (
HDGF) mRNA. m6A reader IGF2BP3 further enhances
HDGF mRNA stability, resulting in increased HDGF expression. HDGF contributes to the proliferation and metastasis of gastric cancer [
31]. In addition, IGF2BP3 promotes
CERS6 mRNA stability, which is associated with increased expression of CERS6 and a number of malignant behaviors of breast cancer mediated by CERS6 [
30]. This study found that over-expressed METTL3 mitigated the toxic effect of irradiation on LUAD cells. Knock-down of IGF2BP2 and IGF2BP3 reversed the effects of METTL3 on LUAD cells. Since METTL3, IGF2BP2 and IGF2BP3 are involved in the regulation of VANGL1 expression, their effects on radiosensitivity are probably associated with VANGL1.
The primary function of miRNAs is the induction of mRNA degradation; therefore, miRNAs are commonly associated with decreased abundance of mRNA. However, this study found that miR-29b-3p expression was decreased in LUAD cells and in serum of patients with LUAD after receiving irradiation. Using miRNA microarray technology, a number of studies have found that the expression of many miRNAs in LUAD cells and in serum of patients with LUAD are aberrantly perturbed upon irradiation [
23,
26]. This suggests that radiation influences the expression of some miRNAs. Tang et al. further found that miR-208a, which is increased by X-ray irradiation, induces radioresistance via targeting p21, with corresponding activation of the AKT/mTOR pathway in lung cancer cells [
26]. In contrast, miR-365 acts in a converse manner in the regulation of radiosensitivity of non-small cell lung cancer (NSCLC) cells. Upregulated miR-365 impairs the radioresistance of NSCLC cells by targeting CDC25A [
32]. This study confirmed that increased miR-29b-3p enhanced the toxic effect of irradiation on LUAD. VANGL1 was shown to be the target of miR-29b-3p. miR-29b-3p deficiency resulted in the upregulation of VANGL1. However, restoration of miR-29b-3p expression blocked the upregulation of VANGL1 in LUAD cells after irradiation. These results suggested that VANGL1 mediates the effects of miR-29b-3p on radiosensitivity of LUAD cells.
The cancer-promoting effects of VANGL1 have been associated with influencing the function of KAI1, ErbB4-c-Jun and Dishevelled (Dvl)-PKC signaling. However, the mechanisms by which VANGL1 affects the adaptive response of LUAD to irradiation are unclear. It is well-recognized that DNA is vulnerable to radiation. Radiation-induced DNA damage is able to trigger cell-cycle arrest and subsequent apoptosis, which is a key mechanism responsible for radiation cytotoxicity. However, a number of studies have revealed that radiation-induced DNA damage can be repaired through DNA repair systems. Disruption to DNA repair systems notably increases radiosensitivity of cancer cells, indicating that DNA repair systems bear the responsibility for cell radioresistance. Using IP analysis, this study revealed interactions involving VANGL1 and BRAF. This interaction facilitated the increased levels of BRAF stimulated by irradiation, because overexpression VANGL1 retarded the degradation of BRAF and irradiation-induced increase in BRAF was abolished with VANGL1 knock-down. BRAF is a member of the RAF serine/threonine protein kinases family. Three RAF proteins (A, B, and C) can form homodimers and heterodimers to activate MEK/ERK signaling, promoting cell proliferation. Therefore, elimination of BRAF restrains the growth of lung cancer cells [
33]. Mutant BRAF is observed in various types of cancer. BRAF mutations occur in 3–8% of patients with NSCLC, and result in increased or decreased RAF kinase activity [
34]. BRAF, especially the mutant type BRAF
V600E, has been found to enhance cancer radioresistance by promoting DNA repair by stimulating 53BP1 and RAD51 [
35]. This study found that depletion of VANGL1 reduced BRAF, 53BP1 and RAD51 expression after irradiation and severe DNA damage. Therefore, the role of BRAF/53BP1/RAD51 cascades in DNA repair probably mediates the protective effects of VANGL1 against irradiation in LUAD.
There is a deficiency in this study. We did not elucidate the mechanisms through which VANGL1 facilitated increased BRAF levels after irradiation. A possible reason for this is that interactions between VANGL1 and BRAF maintain the stability of BRAF protein, which protects BRAF from ubiquitination and targeted degradation. It is also possible that the promoting effect of VANGL1 on BRAF is VANGL1-mediated signals. Additional studies are warranted to identify the mechanisms underlying the effects of VANGL1 on BRAF after irradiation.
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