Cancer Res Treat.  2021 Apr;53(2):399-408. 10.4143/crt.2020.870.

Upregulated N6-Methyladenosine RNA in Peripheral Blood: Potential Diagnostic Biomarker for Breast Cancer

• Xiao H
• Fan X
• Zhang R
• Wu G

• KMID: 2514922
• DOI: http://doi.org/10.4143/crt.2020.870

Abstract

Purpose
An effective biomarker for the diagnosis of breast cancer (BC) and benign breast diseases (BBD) is crucial for improving the prognosis. We investigated whether N6-methyladenosine (m⁶A) can be a diagnostic biomarker of BC.
Materials and Methods
We detected the contents of peripheral blood m⁶A in 62 patients with BC, 41 patients with BBD, and 41 normal controls (NCs) using the colorimetric method. The relative expression of the m⁶A regulated genes methyltransferase-like 14 (METTL14) and fat mass and obesity-associated (FTO) was analyzed using quantitative real-time polymerase chain reaction.
Results
m⁶A in peripheral blood RNA was significantly higher in patients with BC than that in patients with BBD (p < 0.001) or the NCs (p < 0.001). m⁶A was closely associated with the disease stage (from stage 0 to stage I-IV, p=0.003). The receiver operating characteristic curve of m⁶A contained an area under the curve (AUC) value of 0.887 in BC, which was greater than that of carcinoembryonic antigen (CEA) or carbohydrate antigen 153 (CA153). The combination of m⁶A, CEA, and CA153 improved the AUC to 0.914. The upregulated and downregulated mRNA expression of METTL14 and FTO, respectively, might contribute to the increase of m⁶A in patients with BC. m⁶A combined with METTL14 and FTO improved the AUC to 0.929 with a specificity of 97.4% in the peripheral blood of patients with BC.
Conclusion
The peripheral blood RNA of m⁶A might be a valuable biomarker for the diagnosis of BC.

Keyword

Breast neoplasms; Benign breast diseases; N6-methyladenosine; Biomarker; Peripheral blood RNA

Figure

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  Fig. 1 Quantification and statistical analysis of N6-methyladenosine (m6A) from peripheral blood RNA in patients with breast cancer (BC), patients with benign breast diseases (BBD), normal controls (NCs), and noncancerous groups (NGs). m6A in peripheral blood RNA from 62 patients with BC, 41 patients with BBD and 41 NCs (A), or 82 patients from the NGs (B). Bars represent the mean±standard deviation of the results from replicate measurements.

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  Fig. 2 Statistical analysis of N6-methyladenosine (m6A) in peripheral blood RNA with different clinical characteristics in patients with breast cancer. m6A in total peripheral blood RNA of patients with breast cancer at different clinical stages (6 stage 0, 10 stage I, 31 stage II, 6 stage III, and 2 stage IV) (A) and those with no metastasis or regional lymph node metastasis (B). Bars represent the mean±standard deviation of the results from replicate measurements.

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  Fig. 3 Diagnostic value for peripheral blood RNA of N6-methyladenosine (m6A) alone or combination with carcinoembryonic antigen (CEA) and carbohydrate antigen 153 (CA153) to diagnose patients with breast cancer (BC). Receiver operating characteristic (ROC) curve analysis for m6A in the diagnosis of BC (A) and cutoff value of m6A for normal controls (NCs) and patients with BC (B). ROC curve analysis of m6A compared and combined diagnosis with CEA and CA153 (C) and cutoff value analysis for the combination of m6A, CEA, and CA153 in NCs and patients with BC (D). AUC, area under the curve.

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  Fig. 4 Methyltransferase-like 14 (METTL14) and fat mass and obesity-associated (FTO) mRNA expression in peripheral blood RNA in patients with breast cancer (BC), and the expression of FTO and their association with overall survival in tumor tissue. Quantitative real-time polymerase chain reaction analysis of METTL14 (A) and FTO (B) mRNA expression levels in the peripheral blood of normal controls (NCs) and patients with BC. (C) Analysis of the expression level of FTO from the StarBase database in NCs and patients with BC. (D) Correlation of the expressions with the overall survival analysis from a Kaplan-Meier plot. Bars represent the mean±standard deviation of the results.

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  Fig. 5 Methyltransferase-like 14 (METTL14), fat mass and obesity-associated (FTO), and their combination diagnostic value along with N6-methyladenosine (m6A). Receiver operating characteristic curve analysis for METTL14 and FTO alone or combined diagnosis with m6A (A) and cutoff value analysis for the combination of m6A, METTL14, and FTO in normal controls (NCs) and patients with breast cancer (BC) (B). AUC, area under the curve.

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  Fig. 6 Study design and the dynamic N6-methyladenosine (m6A) modification of peripheral blood RNA in breast cancer (BC). (A) Flowchart depicting the study design for m6A of peripheral blood RNA biomarker in BC; (B) the schematic illustration for dynamic m6A modification of peripheral blood RNA in BC. BBD, benign breast diseases; FTO, fat mass and obesity-associated; METTL14, methyltransferase-like 14; NC, normal control; qRT-PCR, quantitative real-time polymerase chain reaction.

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