Background
Reactive oxygen species (ROS) are generated during regular electron transport in the mitochondria or other cellular metabolic pathways. Cellular macromolecules, including nucleic acids, proteins and lipids, are always at high risk of being oxidized by ROS and thus lead to functional or structural abnormalities. Oxidative damage to nucleic acids is the most hazardous owing to the alteration of gene information caused by base mismatch to oxidative nucleotide, such as 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxodGTP). Accumulation of 8-oxodGTP in genomes can result in mutagenesis or cell death, and is eventually minimized by the functions of nucleoside diphosphate linked moiety X-type motif 1 (NUDT1), also known as MutT homolog-1 (MTH1) with its activity of 8-oxo-dGTPase [
1]. NUDT1 can hydrolyze 8-oxodGTP into 8-oxodGMP or 8-oxodGDP, then sanitize from the nucleotide pools [
1].
With high proliferative properties and various microenvironmental factors, cancer tissues are exposed to high oxidative pressure levels and thus accumulate a high level of 8-oxo-dGTP in their nucleotide pools [
2‐
5]. As a result, cancer cells up-regulate NUDT1 to eliminate excessive 8-oxo-dGTP [
5‐
7], indicating that increased expression of NUDT1 in cancer cells may be detrimental for cancer patients. Overexpression of NUDT1 may have several protective functions for cancer cells by hydrolyzing 8-oxo-dGTP or other oxidized nucleotides produced by endogenous elevated ROS or by therapy-induced ROS, thus contributing to malignant phenotypes, poor prognosis and resistance to therapy in cancer patients. It has been reported that NUDT1 is overexpressed in various cancers [
8,
9]. And some positive correlations have been observed between the expression levels of NUDT1 and the prognosis of cancer patients [
10‐
12]. Recent studies also demonstrate that NUDT1 is indispensable for the oncogenic RAS-mediated transformation and proliferation in tumorigenic cells, because oncogenic RAS can induce ROS formation [
13‐
15]. Since normal tissues are in regular cell cycles and are with low oxidative pressure, the actions of NUDT1 are unnecessary. Based on the protective properties of NUDT1 against oxidative stress in cancer cells and the nonessential role of it in normal cells, NUDT1 inhibitors have been developed as potential anti-cancer drugs [
2,
16].
Gastric cancer (GC) is the fourth most common cancer and the third lethal cancer worldwide [
17]. The morbidity and mortality of GC in China is much higher than others, both rank second merely after lung cancer [
18]. Target therapy is a promising strategy in the cancer treatment, however, the effective target used in the treatment of GC is limited. Hence, many scholars focus on the mechanisms of GC oncogenesis to discover novel targets. It is noteworthy that GC is also in high oxidative stress, so GC cells theoretically require the overexpression of NUDT1 to survive. However, the mechanism underlying the dys-regulation of NUDT1 in cancer, particularly in GC remains unknown.
MicroRNAs (miRNAs) are significant modulators in carcinogenic pathways by specific mRNA cleavage or translational repression of target genes. Whether miRNAs play an important role in the dys-regulation of NUDT1 in GC is unidentified. The tumor suppressor role of miR-485-5p was discovered in several cancers [
19‐
21]. A circulating non-coding RNA panel, including miR-485-5p, was reported to act as an early detection predictor of non-small cell lung cancer [
22]. Down-regulation of miR-485-5p was also found in GC tissues and involved in predicting prognosis in GC patients [
23,
24]. However, the molecular mechanism that miR-485-5p regulate tumorigenesis in GC remains largely unspecified. Bioinformatics tools showed that NUDT1 might be a target of miR-485-5p. Hence, this study was carried out to investigate the actual role of miR-485-5p and its relationship with the ROS scavenger NUDT1 in the oncogenesis of GC.
Methods
Human tissues and clinical cohorts
Fresh GC tissues and paired adjacent noncancerous tissues were obtained from patients undergoing a radical surgery at the Tianjin Medical University Cancer Institute and Hospital. Tissue fragments were immediately frozen in liquid nitrogen at the time of surgery. Formalin-fixed, paraffin-embedded (FFPE) sections of GC specimens and paired adjacent noncancerous specimens were derived from 40 GC patients with complete clinicopathological and follow-up information who underwent radical surgery from October 2009 to December 2009 at the Tianjin Medical University Cancer Institute and Hospital. Tumor tissues were histopathologically verified adenocarcinoma and noncancerous tissues were confirmed negative. All aspects of the study were approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital and informed consent was obtained before surgery.
A total of 407 GC patients with mRNA expression profiling and 491 GC patients with miRNA expression profiling were included from The Cancer Genome Atlas (TCGA) database.
Cell lines and culture
Human gastric cancer cell line SGC7901 and MGC803, normal gastric cell line GES-1 and embryo kidney epithelial cell line HEK-293T were cultured following instructions.
The miRNA target prediction
Luciferase reporter assay
Part of the wild and mutated 3′UTR of NUDT1 mRNA which contained the predicted miR-485-5p targeting regions were synthesized and inserted into the pMIR-REPORT plasmid. The β-galactosidase expression vector was used as a transfection control. For the luciferase reporter assays, 2 mg of firefly luciferase reporter plasmid, 2 mg of β-galactosidase vector, and equal doses (200 pmol) of mimics, inhibitors, or scrambled negative control RNA were transfected into the prepared cells. At 24 h after transfection, cells were analyzed using the Luciferase Assay Kit.
Cell transfection
GC cells were seeded in plates and performed transfection using Lipofectamine 2000 (Invitrogen, Life Technologies) according to the manufacturer’s instructions. The NUDT1 overexpressing plasmid and the control plasmid were generous gifts from Dr. Wang at the Tianjin Medical University Cancer Institute and Hospital. MiR-485-5p mimics and inhibitors, the siRNAs targeting NUDT1 along with control RNA were bought from Ribobio. Lentivirus to knockdown miR-485-5p was bought from shanghai Genechem Co., LTD. Equivalent doses (2 μg) of plasmids, or equal amounts (100 pmol) of miRNA mimics/inhibitors or siRNAs were transfected into each well. The cells were harvested at 24 h after transfection for RNA detection and at 48 h for protein analysis.
RNA isolation and quantitative RT-PCR
Total RNA was extracted using TRIzol Reagent following the manufacturer’s protocol. The expression level of miR-485-5p was analyzed by TaqMan miRNA probes. U6 snRNA was used as an internal control for miRNA, and the mRNA levels of NUDT1 were normalized to GAPDH. After the PCRs were accomplished, the cycle threshold (CT) data were calculated using fixed threshold settings, and the mean CT value was determined from triplicate PCR reactions. A comparative CT method was used and the relative levels of target genes normalized to control were calculated with the equation 2−ΔCT, in which ΔCT = CTgene − CTcontrol.
Protein extraction and western blotting
Cells were lysed in RIPA buffer. The total proteins were separated on SDS-PAGE gels and transferred to PVDF membrane. The membrane was blocked within 5% fat-free dried milk for 1 h and then incubated overnight at 4 °C with primary anti-NUDT1 (1:2000, Abcam, ab197028), or anti-GAPDH (1:5000, Santa Cruz, sc-293335), respectively. After incubation with secondary antibodies, the protein bands were visualized.
Cell proliferation assay
The proliferative ability of GC cells was determined by EdU proliferation assay, CCK8 assay and colony formation assay. For EdU proliferation assay, at 24 h after transfection, cells were incubated in 50 μM EdU for 5 h, and then fixed within 4% paraformaldehyde for 30 min. After treatment with 0.5% Triton X-100 for 10 min, cells were incubated in darkness with Apollo staining solution for 30 min, and nuclei were then stained with DAPI for another 30 min. For CCK8 assay, GC cells were collected at 12, 24, 36 and 48 h post-transfection, and 10 μL of CCK8 was added and incubated for 4 h. Absorbance was measured at a wavelength of 450 nm. For colony formation assay, transfected cells (500 cells per well) were seeded into 12-well plate, then fixed and stained after 7–10 days.
Cell migration assay
Wound healing assay and transwell migration assay were used to evaluate the motility of GC cells after transfection. When the cells attached, a wound healing assay was performed. Each well was scraped with a 10 μL pipette tip to create a linear region devoid of cells. The remaining cells were cultured in the medium with 1% serum. The wound closure was monitored at 0, 6, 12, 18, and 24 h after scraping. For transwell migration assay, 24-well chambers with 8-μm pore size polycarbonate membrane were used. Transfected cells (105 cells per well) were seeded into the upper chamber with 200 μL serum-free medium, and 600 μL complete medium containing 10% serum was added to the lower chamber as a chemo-attractant. After 24 h of incubation, nonmigratory cells on the upper chamber were removed slightly by cotton swabs, and the membranes were fixed with methanol and subsequently stained.
Immunofluorescence
At 48 h after transfection, cells were fixed within 4% paraformaldehyde for 30 min. After treatment with 0.5% Triton X-100 for 10 min, cells were incubated overnight at 4 °C with primary anti-8-oxo-dG (1:600, Abcam, ab62623). After incubation with appropriate secondary antibodies, nuclei were stained with DAPI.
Immunohistochemistry assays
All sections were deparaffinized twice with xylene and rehydrated in a graded series of ethanol. The sections were performed heat mediated antigen retrieval with Tris/EDTA buffer, and incubated overnight at 4 °C with anti-NUDT1 antibody (1:100, Abcam, ab197028). The next day, the slides were incubated with second antibodies for 40 min at 37 °C and stained with the DAB system, then counterstained with hematoxylin, dehydrated, and coverslipped. Based on the degree of cell staining and the percentage of positive cells, the expression status of NUDT1 was quantified. The specimen was scored as 0, 1, 2 or 3 for no staining, light yellow staining, brown yellow staining or dark brown staining, respectively. According to the percentage of positive cells, ≤ 10, 11–25, 26–50 or ≥ 51% was recorded as the score of 0, 1, 2 or 3, respectively. Then the two scores multiplied to be the immunohistochemical staining index. The index of less than 4 was represented of the negative expression of NUDT1. Conversely, it was considered as the positive expression of NUDT1.
Statistical analyses
All statistical analyses were performed using IBM SPSS Statistics, Version 20.0. And all data were representative of at least three independent experiments. The Student’s t test was used for two-group comparisons. Clinicopathological categorical variables were compared using the χ2 test or Fisher’s exact test. Survival curves according to NUDT1 expression were estimated with the Kaplan–Meier method and compared using the log-rank test. Differences were considered statistically significant for P < 0.05.
Discussion
In our present study, miR-485-5p and NUDT1 levels showed opposite trends in 5 pairs of gastric carcinoma and noncancerous tissues. The overexpression of NUDT1 was related to later T stage and indicated poorer survival in GC patients. MiR-485-5p was verified to be an upstream regulator of NUDT1, and the dramatic knock-down of miR-485-5p in GC leads to upregulation of NUDT1, thus contributing to accelerated proliferation and increased migration of cancer cells.
NUDT1, acts as a nucleotide pool sanitizing enzyme, plays an indispensable part in surviving the oxidative stress in cancer cells. However, several researchers found distinct roles of NUDT1 [
25,
26], in which NUDT1 deficiency in certain cancer cell lines achieved by small RNA interference or genome editing does not result in any detrimental effects on these cells, indicating that NUDT1 may not be always indispensable for cancer cell survival under oxidative conditions. Hence, the authentic role of NUDT1 in oncogenesis needs more investigation. In accordance with the results of most previous studies, we found that the expression of NUDT1 was up-regulated at both mRNA and protein levels in GC. The oncogenic role of NUDT1 which we verified in the GC cells and GES-1 cells can provide another proof for the therapy targeting NUDT1. By far, some NUDT1 inhibitors have been discovered and have showed some favorable effects in vitro and in vivo [
16,
27‐
29].
Although miR-485-5p has been found to be down-regulated in both GC tissues and cell lines [
23,
24], its specific mechanism in the process of tumorigenesis remains unclear. We identified that miR-485-5p could directly bind to the 3′UTR of NUDT1 mRNA, then lead to its clearance in GC. MiRNA-mediated repression or degradation of mRNA transcripts is one of the essential modes of post-transcriptional regulation. It has been under investigated how the miRNA-mRNA interaction decides whether to degrade the mRNA or repress its translation. Researchers concluded that the short length of 3′UTR [
30] or the A/U-rich 3′UTRs [
31] were positively associated with the degradation of mRNA. The 3′UTR of NUDT1 mRNA is short, and it may be one reason why miR-485-5p can lead to the clearance of NUDT1 mRNA. The miRNA-NUDT1 pathway has been investigated previously in lung cancer [
32]. It was pointed out that miR-145 could inhibit cell proliferation and was in the negative regulation of NUDT1 expressions at both mRNA and protein levels [
32]. Since one miRNA can target multiple genes and one gene can be regulated by several miRNAs, identification of the complete miRNA-gene pathway is necessary and can provide more insights into the exploration of new therapeutic targets. The inhibitory effects of miR-485-5p on GC cells can clue to the treatment based on miR-485-5p.
It is the first time to detailedly describe the expression pattern and biological role of NUDT1 in GC. And the correlation between NUDT1 expression and survival of GC patients has not been identified previously. The miR-485-5p/NUDT1 axis is verified to be involved in the carcinogenesis of GC, however, additional cohorts and in vivo experiments are needed.
Authors’ contributions
DJ performed most of the experiments, analyzed data, and wrote the manuscript. ZH reviewed and edited the manuscript. LS, WX and YH performed some experiments. BY and JS designed the experiments and edited the manuscript. All authors read and approved the final manuscript.