Primary breast cancer and corresponding adjacent healthy tissues were obtained from 136 women patients with breast cancer (age range,42–75 years old) following surgical resection at The Third Clinical Hospital of Hebei Medical University (Shijiazhuang, China) between July 2019 and December 2020. Data on the clinicopathological characteristics of each patient were obtained from the hospital records and pathological diagnosis, The patients did not receive chemotherapy, radiotherapy, endocrine therapy or other associated treatment before operation. The present study was approved by the Ethics Committee of The Third Clinical Hospital of Hebei Medical University (approval no. 2021-034-5) and all patients provided written informed consent prior to participation.

MSP was performed to detect the methylation status of LDH-C4. Briefly, the genomic DNA was extracted from tissues using the phenol chloroform method. The tissues were put into the EP tube and lysis buffer β-mercaptoethanol and protease K were added in a 56°C water bath. DNA extract was added, mixed well and centrifuged (11,433 × g; 7 min; 4°C). The supernatant was collected and added to double the volume of isopropanol at- 20°C for 10 min, and centrifuged again (11,433 × g; 7 min; 4°C). The supernatant was removed and 70% ethanol was added into the precipitation, mixed well and centrifuged (11,433 × g; 7 min; 4°C). The precipitation was collected and dried, ddH2O was added and the concentration was measured using ultra-micro ultraviolet spectrophotometer (Shanghai Ruiyue Experimental Equipment Co., Ltd.). Subsequently, the DNA was modified and purified using a hydrogen sulfite modification kit containing a DNA polymerase (Shanghai Yubo Biological Technology Co., Ltd.). Subsequently, a total of 3 µl modified DNA was used as a template for PCR amplification, which was performed using the following reaction conditions: Initial denaturation at 95°C for 4 min, followed by 35 cycles of denaturation at 95°C for 45 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec, and a final extension step at 72°C for 10 min. The PCR products were analyzed using 2% agarose gel electrophoresis and by UV gel electrophoresis imaging and a gel image analyzer. All PCR reactions were repeated in triplicate. The methylated (M) and unmethylated (U) primers of LDH-C4 were as follows: (M) forward, 5′-TCTGGGGTGTAGCGGTCGTC-3′ and reverse, 5′-GCCCACATACTAAATCACGCG-3′; and (U) forward, 5′-GTAGTTTGGGGTTGAGTGGTTGTT-3′ and reverse, 5′-CACCCCCAATACATAATCACAACA-3′. Normal placental DNA modified by CpG methyltransferase was used as the positive control for MSP amplification. Deionized water was used as the blank control.

Paraffin-embedded tissue sections (4 µm) were deparaffinized in xylene and rehydrated using a graded alcohol series. The sections were subsequently washed with PBS (pH 7.2) for 5 min and heated in a microwave oven for 5 min at 65°C in 10 mmol/l sodium citrate buffer (pH 6.0) for antigen retrieval. The sections were subsequently washed with PBS and immersed in 0.3% hydrogen peroxide in methanol for 20 min at room temperature. After washing with PBS, the sections were incubated in 1:10 normal goat serum (5%; Shanghai Yanjin Biotechnology Co., Ltd.) at room temperature in a humidified chamber for 45 min to prevent non-specific immunoglobulin binding. The sections were subsequently incubated with rabbit anti-DNMT1 (cat. no. ab19905), anti-DNMT3a (cat. no. ab226261) and anti-DNMT3b (cat. no. ab2851) polyclonal antibodies (all 1:500; Abcam) at 4°C overnight; the primary antibody was replaced with normal IgG to serve as the control. The sections were then thoroughly washed with PBS three times, and incubated with goat anti-rabbit polyclonal antibody (1:100; cat. no. ZB-2010; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at 37°C for 30 min, followed by washing 3 times with PBS. Then, the horseradish peroxidase (HRP)-conjugated streptavidin working solution was added and incubated C for 30 min. A streptavidin-biotinylated HRP-based detection system (Shanghai Hexing Biological Technology Co., Ltd.) was used to determine the specific binding of each antibody. Sections were subsequently counterstained with hematoxylin at room temperature for 5 min and prepared for visualization using a light microscope in high power field.

The expression was determined by the intensity of the positive cells using ImageJ software (v1.8.0; National Institutes of Health). Briefly, the area of positive staining was scored using the following scale:0–2, negative expression;3–7, positive staining (of those,3–4, weak positive expression; and 5–7, strong positive expression). The staining intensity was graded as follows: 0, no staining; 1, mild staining; 2, moderate staining; and 3, intense staining. The staining intensity and positive staining scores were multiplied together, and scores <4 indicated negative expression, while scores ≥4 indicated positive expression.

Statistical analysis was performed using SPSS 22.0 software (IBM Corp.). Enumeration data were expressed using rate. The association between DNMT expression or the methylation status of the promoter region of LDH-C4 and clinicopathological risk factors was statistically evaluated using a χ² or Fisher's exact test. The correlation between the methylation status of the LDH-C4 promoter and the expression levels of DNMTs was detected using Spearman's rank correlation analysis. The overall survival of patients was estimated using the Kaplan-Meier method, and statistical differences between the groups were determined using a log-rank test. Univariate and multivariate analyses of overall survival according to prognostic factors were analyzed using Cox regression analysis. P<0.05 was considered to indicate a statistically significant difference.

Using the MSP method and primers designed to amplify the promoter region of the LDH-C4 gene, it was revealed that a marked proportion of breast cancer tissues had low levels of methylation compared with adjacent healthy tissues. In total, 59 of the 136 (43.38%) breast cancer tissues samples contained methylation patterns in the promoter region of the LDH-C4 gene, while 121 of the 136 (88.97%) corresponding adjacent healthy tissue samples contained methylation in the promoter region of the LDH-C4 gene (Fig. 1).

To determine whether the methylation status of the LDH-C4 promoter region was correlated with the protein expression levels of DNMTs, their expression levels were analyzed in breast cancer and corresponding adjacent healthy tissues using IHC analysis. As shown in Fig. 2, DNMT1, DNMT3a and DNMT3b expression levels were observed in the nuclei of breast cancer cells. DNMT1, DNMT3a and DNMT3b were positively expressed in 11.76, 9.56 and 16.18% of adjacent healthy tissues, respectively, while DNMT1, DNMT3a and DNMT3b positive expression was found in 35.29, 41.91 and 39.71% of breast cancer tissues, respectively.

Clinical correlation between DNMT expression and the methylation status of the promoter region of LDH-C4 and the clinicopathological factors of patients with breast cancer. To further explore the possible effects of the methylation of LDH-C4 and DNMT expression in breast cancer, the correlation between the methylation status of the promoter region of LDH-C4 and DNMT expression, and the clinicopathological factors of patients with breast cancer was determined. As shown in Table I, the methylation status of the promoter region of LDH-C4 was not associated with age, menopausal status, tumor size, tumor histology, TNM stage or antigen Ki-67 (Ki-67) expression (Ki-67 is associated with poor clinicopathological factors (14) in breast cancer), but was significantly associated with histological grade, estrogen receptor (ER), progesterone receptor (PR) and HER-2 status, and lymph node metastasis (P<0.05). The expression of DNMT1 was not associated with age, menopausal status, tumor size, tumor histology, TNM stage or Ki-67 expression, but was significantly associated with ER, PR and HER-2 status, histological grade and lymph node metastasis (P<0.05). The expression of DNMT3a was not associated with age, menopausal status, tumor size, tumor histology, TNM stage, and PR or HER-2 status (P>0.05), but was significantly associated with histological grade, ER and Ki-67 expression, and lymph node metastasis (P<0.05). The expression of DNMT3b was not associated with age, menopausal status, tumor size, tumor histology, TNM stage, or PR, HER-2, ER or Ki-67 expression, but was significantly associated with histological grade and lymph node metastasis (P<0.05). These data suggested that hypomethylated profiles of the LDH-C4 promoter and DNMT expression may indicate a poor prognosis of patients with breast cancer.

As shown in Table II, χ² analysis revealed that the unmethylated level of LDH-C4 in breast cancer tissues was inversely correlated with the expression of DNMT1 (r=−0.273; P=0.018), DNMT3a (r=−0.216; P=0.032) and DNMT3b (r=−0.298; P=0.010). These results indicated that DNMT1, DNMT3a and DNMT3b expression may play an important role in the demethylation of LDH-C4.

To determine the association between DNMT expression and patient survival, or the methylation status of the promoter region of LDH-C4 and patient survival, Kaplan-Meier curve analysis for overall survival was performed. Kaplan-Meier analysis revealed that patients with a demethylated LDH-C4 promoter exhibited a shorter survival time and a poorer prognosis compared with those patients with a methylated LDH-C4 promoter (P<0.05; Fig. 3A). Patients with high expression levels of DNMT1, DNMT3a and DNMT3b exhibited a shorter survival time and worse prognosis compared with those with low expression levels of DNMTs (Fig. 3B-D). Moreover, a demethylated LDH-C4 promoter and high DNMT expression levels predicted an unfavorable prognosis (Fig. 3E-G).

To further investigate the prognostic factors for poor breast cancer outcomes, univariate and multivariate analyses were performed. The results of the univariate analysis revealed that overall survival was significantly associated with the methylation of the LDH-C4 promoter, and DNMT1, DNMT3a and DNMT3b expression, histological grade, ER, PR and HER-2 status, and lymph node metastasis (Table II). Multivariate analysis revealed that a demethylated LDH-C4 promoter, high expression of DNMT3a and DNMT1, histological grade and lymph node metastasis were independent prognostic factors for patients with breast cancer (Table III).