Background
In the world, colorectal cancer (CRC) ranks third in terms of prevalence and second in terms of mortality [
1,
2]. The incidence of CRC is increasing annually, owing to recent changes in lifestyle and dietary structures [
3]. Early detection, timely diagnosis, and radical surgery are crucial for the successful treatment of CRC. However, early-stage clinical symptoms are not often apparent. Most CRC patients are diagnosed at an advanced stage where surgery is not an option. Presently, the gold standard screening methods for CRC are colonoscopy and tissue biopsy [
4]; however, these methods are not practical for large-scale screening for CRC owing to their high cost and poor patient compliance [
5]. Moreover, CRC is commonly detected using blood-based biomarkers, such as carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), and carbohydrate antigen 19-9 (CA19-9) [
6,
7]. However, the diagnostic performance of these biomarkers is unsatisfactory owing to low diagnostic sensitivity or specificity [
8]. As a result, a blood biomarker for the clinical diagnosis of CRC is urgently needed.
N
6-methyladenosine (m
6A) is the most prevalent and conserved transcriptional modification [
9,
10]. A methyltransferase complex composed of METTL3, METTL14, and WTAP catalyzes this modification. Additionally, FTO and ALKBH5 are m
6A demethylases that regulate the reversibility of the m
6A modification. Dysregulated m
6A-related genes have been reported in CRC cells [
11]. Low
FTO expression in patient-derived CRC cell lines increases m
6A levels in mRNAs, resulting in enhanced tumorigenicity and chemoresistance in vivo [
12]. ALKBH5 plays an antitumor role in CRC by increasing the stability of FOXO3 by attenuating the level of its m
6A modification, and FOXO3 targets miR-21 and promotes the expression of SPRY2, providing a new direction for CRC therapy [
13]. Moreover, the levels of m
6A and METTL3 were increased in CRC tissues, and high m
6A or METTL3 levels predict poor prognosis [
14]. Additionally, CRC tumorigenesis can be facilitated by the activation of the glycolysis pathway by m
6A methylation [
15]. Thus, the modification of m
6A is crucial in the development and progression of CRC.
Patients with cancer, including gastric cancer [
16], breast cancer [
17], and non-small-cell lung carcinoma [
18], have elevated levels of m
6A in their peripheral blood (PB). The levels of m
6A in PB correlate with tumor stage and can be used as diagnostic biomarkers. Moreover, these patients exhibit lower expression of m
6A demethylase genes
FTO and
ALKBH5 in PB. However, the diagnostic potential of the m
6A modification for patients with CRC has not been investigated.
In the current study, we aimed to investigate the diagnostic potential of levels of PB RNA m6A levels for CRC by comparing the same in HCs and patients with CRC. In addition, we demonstrated in vitro that CRC cells could regulate the levels of m6A in PB mononuclear cells (PBMCs) and detected the levels of PB RNA m6A in a mouse model of CRC.
Materials and methods
Sample collection
PB samples were collected from 78 patients with CRC and 44 HCs without a history of primary or chronic diseases at the Fujian Union Hospital using EDTA tubes. All patients with CRC were diagnosed based on histopathology via endoscopic examination or biopsy, and PB samples were obtained at diagnosis before surgery or radio/chemotherapy. The demographic characteristics of patients with CRC and HCs are listed in Table
1 and Additional file
1: Table S1. Ethical approval (2021QH036) was granted by the Ethics Committee of Fujian Union Hospital.
Table 1
Correlation between the levels of m6A and clinicopathological characteristics in CRC
Age |
≤ 60 | 36 | 0.2884 ± 0.01005 | 0.3547 |
> 60 | 42 | 0.301 ± 0.008999 |
Gender |
Female | 32 | 0.3019 ± 0.01148 | 0.4092 |
Male | 46 | 0.2905 ± 0.008122 |
Clinical stage |
I | 13 | 0.2707 ± 0.008552 | 0.1692 |
II | 20 | 0.3164 ± 0.01441 |
III | 31 | 0.2889 ± 0.01018 |
IV | 12 | 0.2879 ± 0.01869 |
T classification |
T1–T2 | 11 | 0.2773 ± 0.01684 | 0.3121 |
T3–T4 | 55 | 0.2974 ± 0.008176 |
N classification |
N0 | 25 | 0.2869 ± 0.01146 | 0.4016 |
N1–N2 | 42 | 0.2997 ± 0.0095 |
N classification |
N0–N1 | 47 | 0.2956 ± 0.009102 | 0.8516 |
N2 | 19 | 0.2925 ± 0.01294 |
M classification |
M0 | 45 | 0.2922 ± 0.009224 | 0.2933 |
M1 | 20 | 0.2753 ± 0.01174 |
CEA (ng/mL) |
< 5 | 50 | 0.2844 ± 0.008085 | 0.0380 |
≥ 5 | 26 | 0.3143 ± 0.01196 |
CA19-9 (ng/mL) |
< 35 | 61 | 0.2871 ± 0.007505 | 0.0252 |
≥ 35 | 15 | 0.3254 ± 0.01456 |
After blood collection, 1 mL of EDTA + blood was treated with 3 mL erythrocyte lysate (Cat. No. R1010; Solarbio) twice to obtain leukocytes, then 1 mL of Trizol reagent (Cat. No. 15596-026; Invitrogen) was added to stabilize the RNA, and the samples were preserved at − 80 ℃ until RNA extraction.
RNA isolation
Total RNA was extracted using the TRIzol reagent. The integrity of RNA was evaluated using agarose gel electrophoresis. RNA yield and purity were measured using the NanoDrop 1000 (Gene Company Limited, Hong Kong, China).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Quantitative RT-PCR was performed using the PerfectStart Green qPCR SuperMix (Cat. No. AQ601-04; TransGen Biotech, Beijing, China) and a 7500 Real-Time PCR System (Thermo Fisher Scientific). The cycling parameters were as follows: 94 ℃ for 30 s, followed by 40 cycles of 94 ℃ for 5 s, 60 ℃ for 15 s, and 72 ℃ for 10 s. To calculate the ΔCq values, the Cq value for the human actin beta [
ACTB] reference gene and the mice actin beta [
Actb] gene was subtracted from the original Cq value. Normalization of targeted gene expression in each independent sample was performed using the reference (
ACTB) gene. The primers for the target genes are listed in Additional file
1: Table S2. The 2
−ΔΔCt method was used to calculate the absolute expression.
Quantification of m6A in PB RNA
The EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric; EpiGentek, Farmingdale, NY, USA) was used to measure m6A levels in total RNA by the manufacturer’s instructions. First, 80 µL binding solution was added to the assay well, and then 200 ng RNA. Following a 90-min incubation at 37 ℃, the plates were washed thrice, and the assay wells were sequentially treated with diluted solutions of the primary and detection antibodies and the enhancer. Subsequently, the color reaction was initiated by adding the developer and stop solutions to each well, and the absorbance was measured at 450 nm wavelength. Finally, the standard curve was generated to determine the m6A levels.
Cell lines and cell experiments
The human CRC cell line (HCT116) was obtained from the Chinese Academy of Sciences (Beijing, China). SW480 and MC38 murine colon adenocarcinoma cells were kindly provided by Dr. Mi Zhang of the Basic Medicine of Fujian Medical University (Fuzhou, China). PBMCs were isolated from healthy human PB using the human PB lymphocyte separation medium. SW480 and HCT116 cells were cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 1%Pen/Strep (Thermo Fisher Scientific), and PBMCs were cultured in human PB lymphocyte particular medium in an incubator at 37 ℃ and 5% CO2.
Six-well Transwell chambers (0.4-µm pores, Corning Transwell; Corning Inc, Corning, NY, USA) were used for the co-culture assay. A total of 5 × 105 HCT116 or SW480 cells were seeded in the lower Transwell chamber, and 1 × 106 PBMCs were seeded in the upper chamber of the co-culture system for 48 h. PBMCs were collected separately for qRT-PCR and ELISA analyses.
Animal study
C57BL/6 male mice (6–8-week-old) were purchased from the Sibeifu Animal Center (Beijing, China). All animal experiments were conducted following the protocols approved by the Fujian Medical University of Medicine Policy on the Care and Use of Laboratory Animals. MC38 colon carcinoma cells were injected in 100 µL phosphate-buffered saline (PBS) subcutaneously into the right flank of C57BL/6 mice (n = 14, male) to construct an MC38 murine colon carcinoma cancer model (MC38 cancer model). C57BL/6 mice were also injected with 100 µL of PBS into their right flank as the control group (n = 8). Mice were euthanized 15 day after cells/PBS injection or if the longest dimension of the tumor was as large as 1.5 cm within 20 day. Retroorbital blood was obtained from the mice immediately following euthanasia using EDTA tubes.
Statistical analysis
Statistical analysis was conducted using IBM SPSS Statistics 20.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA). Differences between groups were analyzed using an unpaired 2-tailed parametric Student’s t-test for normally distributed data. Otherwise, the nonparametric Mann–Whitney U tests were used to analyze the data. Data were analyzed using a one-way analysis of variance followed by Bonferroni-corrected posthoc tests to allow for comparison of more than two groups. A receiver operating characteristic (ROC) curve was plotted to determine the diagnostic value of the biomarker. The standard deviations are represented by error bars. Statistical significance was set at P < 0.05.
Discussion
Early diagnosis is crucial for improving CRC prognosis. Current invasive or noninvasive screening methods fail to achieve satisfactory early screening results owing to their limitations. Although multiple randomized controlled trials have shown that guaiac-based fecal occult blood testing and sigmoidoscopy reduce CRC mortality, their effectiveness in reducing CRC mortality may be diminished if patients do not repeat screening [
19‐
22]. The serological markers CEA and CA199, currently used to diagnose CRC, have low specificity and sensitivity, both individually and in combination [
23,
24]. Therefore, identifying simple and novel early diagnostic biomarkers for CRC is urgently required. In the current study, we observed that PB RNA m
6A levels could effectively differentiate patients with CRC from HCs. The diagnostic value of PB RNA m
6A levels was significantly improved when combined with the biomarkers CEA or CA199.
To date, few studies have investigated the diagnostic value and underlying mechanism of PB RNA m
6A levels in patients with CRC. Our study observed that PB RNA m
6A levels in patients with CRC were significantly increased compared with those in HCs, which is consistent with the findings of Xie et al. [
25]. However, our results showed no significant differences in PB RNA m
6A levels in patients with CRC with or without distant metastasis. The growth and progression of cancer are regulated by crosstalk between writers, readers, and erasers of m
6A [
26]. Although m
6A levels may vary in different tumors, previous reports have suggested that PB RNA m
6A levels in patients with gastric cancer [
16], non-small cell lung carcinoma [
17], breast cancer [
18], and rheumatoid arthritis [
27] were much higher than those in the corresponding HCs. Therefore, further investigating the diagnostic value of PB RNA m
6A levels in other tumors and diseases is crucial.
Our results indicated that PB RNA m
6A levels had a promising diagnostic potential for CRC. The AUC of m
6A (0.886) for discriminating patients with CRC from HCs was the highest, followed by those of CEA (0.825) and CA199 (0.671). Moreover, CEA and CA199 in combination with PB RNA m
6A improved the AUC to 0.935 in patients with CRC. Previous studies and meta-analyses have demonstrated that some miRNAs and circRNAs could serve as potential biomarkers for CRC; however, the AUC of the RNAs was not considered high. For example, a meta-analysis including six studies revealed that the AUC of miR-92a in the diagnosis of CRC was 0.772 [
28]. Another meta-analysis, including 18 studies involving 2021 individuals, reported that the AUC of circRNA in the diagnosis of CRC is 0.81 [
29]. In addition, our results showed that PB RNA m
6A levels in postoperative CRC patients were decreased following surgery, indicating its potential as a biomarker for postoperative monitoring. However, further studies using more clinical samples are required to confirm whether it can be used as a potential follow-up biomarker. Overall, our results indicate that PB RNA m
6A levels can act as a better diagnostic biomarker for CRC than the currently used biomarkers.
In the current study, we observed that the expression of both
FTO and
ALKBH5 was decreased in patients with CRC compared with that in HCs. There may be a relationship between the increased PB RNA m
6A levels in patients with CRC and the downregulation of
FTO and
ALKBH5. Moreover, co-culture with CRC cells results in an increase in m
6A levels and a decrease in
FTO and
ALKBH5 expression in PBMCs. A previous study has reported that imbalanced regulation of m
6A strongly confers immune destruction and tumor evasion [
30]. Consistently, in our study, PB RNA m
6A levels in the MC38 cancer model were significantly increased and were accompanied by a decrease in the expression of
FTO and
ALKBH5. These results indicate that increased PB RNA m
6A levels induced by CRC may be owed to the decreased expression of
FTO and
ALKBH5.
Our study has some limitations as no significant correlation was observed between PB RNA m
6A levels and expressions of
FTO or
ALKBH5 in the total RNA of PB cells. The possible reasons for the lack of correlation could be: (1) other unidentified methylases and demethylases that require further exploration [
31] are also involved in the regulation of m
6A; (2) the regulation of m
6A in the total RNA of PB cells may require the participation of methyltransferases; (3) interactions between methyltransferases and demethylases and their regulatory factors may also contribute to the changes in m
6A level. Therefore, changes in PB RNA m
6A levels may not solely depend on the expression of the demethylases investigated in this study. Further investigation is needed to understand the mechanism behind the upregulation of m
6A levels and the downregulation of demethylases induced by tumors.
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