The lncRNA MAGI2-AS3 in peripheral blood mononuclear cells: a valuable biomarker for diagnosis and prognosis prediction of breast cancer

GEPIA

The GEPIA database (http://gepia.cancer-pku.cn/) is an online network that encompasses normal samples and tumor tissue sourced from TCGA and GTEx (Tang et al., 2017). We utilized this database to analyze the expression levels of MAGI2-AS3 in breast cancer tissues. The remaining default settings of the operating system were accepted.

Kaplan-Meier plotter

The Kaplan-Meier plotter (http://kmplot.com/analysis/) was employed to evaluate how genes impact survival by analyzing cancer samples from various cancer types (Nagy et al., 2018). We also made use of the Kaplan-Meier plotter to analyze the relationship between MAGI2-AS3 expression and the survival of breast cancer patients. The remaining default settings of the operating system were adopted.

Patients and sample collection

All 100 peripheral blood samples from breast cancer patients were sourced from Zibo Central Hospital. These patients were suspected of having breast cancer at initial diagnosis and had not received any treatment before blood collection. In accordance with the approval of the Institutional Review Board of Zibo Central Hospital (approval number: 202102005), written informed consent was waived for this study. As detailed in the attached “Proof of Exemption from Written Consent for Human Studies,” the peripheral blood collection in this research is a common minimally invasive clinical operation with minimal risks to subjects, such as mild pain, slight bleeding at the puncture site, and a very low-probability of local infection, which are no more than the minimal risks in daily medical examinations. Also, strict privacy protection measures were implemented, including anonymizing all collected blood samples. Therefore, verbal informed consent was obtained during blood collection, and patient anonymity was ensured. Blood samples (5 ml) were collected from fasting participants in the morning using 21-gauge BD Vacutainer needles and transferred to sterile 15-ml polypropylene centrifugation tubes (CF430052; Corning, Corning NY, USA).

PBMCs were isolated from both patients and 100 age-matched healthy female volunteers (controls) via density gradient centrifugation with Ficoll-Hypaque (10771; Amersham Pharmacia Biotech, Uppsala, Sweden). All experiments in this study, including sample collection, RNA isolation, qRT-PCR analysis, and data processing, were conducted at the Center of Translational Medicine, Zibo Central Hospital. This facility provided the necessary equipment and environment for the research, ensuring the accuracy and reliability of the experimental results. Briefly, the process involved gently mixing an equal volume of isopycnic Ficoll-Hypaque with venous blood. The mixture was then centrifuged at 1,500 rpm for 30 min at 20–22 °C to collect the middle white membrane layer. Isolated PBMCs were quickly frozen in liquid nitrogen vapor for about 10 min and then transferred to a −80 °C freezer for storage until use (Fig. 1). The collected peripheral blood samples and isolated PBMCs were not fixed during the entire experimental process.

Patient clinical characteristics, including age, tumor size, estrogen receptor (ER) expression, progesterone receptor (PR) expression, human epidermal growth factor receptor-2 (Her-2) expression, tumor-node-metastasis (TNM) stage, and histologic grade, were retrieved from the corresponding pathology reports of the Pathology Division. The data was cross-checked for accuracy by two independent researchers. CA15-3 was detected using electrochemiluminescence methods in the clinical laboratory at Zibo Central Hospital, following the manufacturer’s instructions for the Roche Cobas e601 analyzer. Quality control samples with known CA15-3 concentrations were run in parallel with patient samples to ensure the reliability of the assay. The study was approved by the Institutional Review Board of Zibo Central Hospital (approval number: 202102005) and adhered to the Declaration of Helsinki.

RNA isolation

Total RNA from PBMCs was extracted using RNAiso Plus (9108; TAKARA Biotech, Beijing, China) following the manufacturer’s protocol. Chloroform (496189; Sigma-Aldrich, Burlington, MA, USA), isopropanol (100272; Merck, Darmstadt, Germany), and 75% ethanol (prepared from absolute ethanol purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) were used during the extraction process. After thawing the frozen PBMCs on ice, an appropriate volume of RNAiso Plus was added for cell lysis. Chloroform was then added, and the mixture was centrifuged. The upper aqueous phase containing RNA was transferred, and isopropanol was used to precipitate RNA. The RNA pellet was washed with 75% ethanol, air-dried, and dissolved in RNase-free water.

RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The A260/A280 ratio of all samples ranged from 1.8–2.0, indicating high-quality RNA. The average RNA yield was 50 μg per 1 × 10⁶ PBMCs, with a range of 21–75 μg per 1 × 10⁶ PBMCs across all samples. RNA integrity was evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and the RNA Integrity Number (RIN) of each sample was above 7.0. To eliminate potential genomic DNA contamination, RNA samples were treated with DNase I (EN0521; Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. To assess the integrity of the RNA and the effectiveness of DNA contamination removal, agarose gel electrophoresis was performed on a 1% agarose gel containing ethidium bromide (E7637; Sigma-Aldrich, Burlington, MA, USA) at 120 V for 30 min. Visual inspection of the gel during the experiment indicated that the RNA samples showed clear 28S and 18S rRNA bands, suggesting intact RNA. Additionally, no visible DNA bands were detected, which confirmed the absence of DNA contamination in the RNA samples after DNase I treatment.

qRT-PCR

Reverse transcription was carried out using the PrimeScript RT reagent Kit with gDNA Eraser (RR047A; TAKARA Biotech, Beijing, China). The 20-μl reaction system contained 1 μg of total RNA, 4 μl of 5×PrimeScript Buffer 2, 1 μl of PrimeScript RT Enzyme Mix I, 1 μl of gDNA Eraser, and RNase-free dH₂O. The reaction conditions were 42 °C for 30 min for reverse transcription and 85 °C for 5 s to inactivate the enzyme. The resulting cDNA was stored at −20 °C.

qRT-PCR was performed using SYBR Premix Ex Taq II (RR820A; TAKARA Biotech, Beijing, China) on an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The 20-μl reaction system included 10 μl of SYBR Premix Ex Taq II, 0.8 μl of each forward and reverse primer (10 μM), 2 μl of cDNA template, and 6.4 μl of ddH₂O. All of the primers used are listed in Table 1.

The thermocycling parameters were 95 °C for 30 s of initial denaturation, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Melting curve analysis was conducted at the end to verify product specificity, showing a single peak. A no-template control (Cameron et al., 2017) was included in each run, and no amplification was detected in NTC wells.

In our relative quantification study, although the main goal was to analyze the relative expression levels of MAGI2-AS3 and β-actin, we still generated standard curves using serial dilutions (10⁸–10³ copies/μl) of plasmids containing the target gene sequences. These standard curves were essential for evaluating the performance of our qRT-PCR assays. For MAGI2-AS3, the standard curve had a slope of −3.32, a y-intercept of 37.25, an R² value of 0.996, and a PCR efficiency of 98.6%. For β-actin, the slope was −3.30, the y-intercept was 37.40, the R² value was 0.997, and the PCR efficiency was 99.2%. The high R² values demonstrated a reliable linear relationship between the template concentration and the Ct values, validating the reproducibility of our experimental procedures. The PCR efficiencies within an appropriate range further attested to the effectiveness of our reaction conditions.

We determined the linear dynamic range of our qRT-PCR assays to be 10³–10⁸ copies/μl. This range was significant as it defined the concentration interval within which we could accurately detect relative changes in gene expression. Samples with target gene expression levels falling within this range could be effectively analyzed for relative quantification.

The limit of detection (Perozo et al., 2002) for both MAGI2-AS3 and β-actin was found to be 10³ copies/μl. This LOD represented the lowest concentration at which we could confidently detect the target genes. In the context of relative quantification, this information was valuable for ensuring that the detected gene expression changes were reliable and not influenced by background noise.

Although inhibition testing is an important aspect of qRT-PCR quality control, the primary objective of this study was to rapidly explore the potential value of MAGI2-AS3 in the diagnosis and prognosis prediction of breast cancer. Our focus was on analyzing the expression differences of MAGI2-AS3 in peripheral blood mononuclear cells between breast cancer patients and healthy controls, as well as its associations with clinicopathological parameters and patient survival outcomes. Given the research priorities and resource allocation, we prioritized our efforts on the key experiments and data analysis. Although inhibition testing is valuable, it was not essential for achieving the core goals of this study, and its results would have relatively minor direct impacts on the research conclusions. Therefore, we did not perform inhibition testing in this study.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) and SPSS version 17 (IBM, Armonk, New York, USA). Student’s t-test was used for comparing two groups, and one-way ANOVA was applied for multiple-group comparisons. The chi-square test was used to analyze the correlation between MAGI2-AS3 expression and clinicopathological variables. The log-rank test was used for comparing patient survival curves, and the receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of MAGI2-AS3. A p-value < 0.05 was considered statistically significant. Outliers were identified using Grubbs’ test. Data were normalized using the 2^−ΔΔCt method with β-actin as the reference gene. Each sample was analyzed in three biological replicates and three technical replicates. The coefficient of variation (CV) was calculated to assess repeatability (intra-assay variation) and reproducibility (inter-assay variation). The intra-assay CV for MAGI2-AS3 expression was less than 5%, and the inter-assay CV was less than 10%. A power analysis was conducted using G*Power software. Based on an expected effect size, a significance level of α = 0.05, and a power of 1-β = 0.8, the sample size of 100 in each group was determined to be sufficient for detecting significant differences.

MAGI2-AS3 is downregulated in breast cancer

Using the GEPIA database, we found that MAGI2-AS3 expression was significantly downregulated in breast cancer tissues compared to normal tissues (Figs. 2A and 2B). Additionally, we analyzed the prognostic potential of MAGI2-AS3 using the Kaplan-Meier plotter database. Reduced MAGI2-AS3 expression was significantly associated with unfavorable overall survival (OS) outcomes in breast cancer patients (Fig. 2C).

The lncRNA MAGI2-AS3 was downregulated in the PBMCs of breast cancer patients, and its low expression was associated with a shorter overall survival

The expression levels of MAGI2-AS3 in the PBMCs of breast cancer patients and healthy females were measured. The RT-qPCR results demonstrated that breast cancer patients had significantly lower MAGI2-AS3 levels (Fig. 3A). Distant metastasis (DM) represents a crucial and often ominous stage in the progression of breast cancer. It refers to the spread of cancer cells from the primary tumor in the breast to distant parts of the body, most commonly the lungs, liver, bones, or brain. This metastatic spread significantly complicates treatment and reduces patients’ survival prospects. In the context of our study, we specifically focused on the differences in MAGI2-AS3 expression levels between patients with and without DM. This choice was driven by the need to explore the potential of MAGI2-AS3 as a biomarker for predicting breast cancer metastasis. By analyzing these differences, we aimed to gain a deeper understanding of the molecular mechanisms involved in the metastatic process. Such insights could be instrumental in the early detection of metastasis and the development of more targeted and effective treatment strategies, ultimately improving patient outcomes. After defining the importance of DM and our research goal, we delved into the analysis and obtained the following findings. As expected, we found that MAGI2-AS3 was expressed at lower levels in the PBMCs of breast cancer patients with DM compared to those without DM (Fig. 3B). Based on the expression level of MAGI2-AS3 in PBMCs, breast cancer patients were classified into two groups. Kaplan-Meier analysis revealed that low MAGI2-AS3 expression was significantly associated with shorter OS in breast cancer patients, indicating its potential prognostic value (Fig. 3C).

Associations between the MAGI2-AS3 expression level in PBMCs and clinicopathologic variables of breast cancer patients

The associations between MAGI2-AS3 expression and the clinical characteristics of breast cancer patients were explored using the chi-square test. MAGI2-AS3 expression was strongly correlated with tumor size, TNM stage, and histologic grade. There was no significant correlation between MAGI2-AS3 expression and other clinical indices, such as patient age, ER status, PR status, and HER-2 status (Table 2). The strong correlations between MAGI2-AS3 expression and tumor size, TNM stage, and histologic grade hold significant clinical implications. Tumor size is an important factor reflecting the progression of breast cancer. The significant correlation with MAGI2-AS3 expression indicates that this lncRNA may serve as a supplementary marker for evaluating tumor burden. For instance, patients with larger tumors often show lower MAGI2-AS3 expression levels, which might be associated with more advanced disease and a worse prognosis. This finding suggests that MAGI2-AS3 could potentially assist clinicians in better assessing the severity of breast cancer at an early stage. TNM stage plays a crucial role in determining the appropriate treatment plan for breast cancer patients. The correlation between MAGI2-AS3 expression and TNM stage implies that it could be integrated into the current staging system. This integration might help in more accurately stratifying patients, especially those at the boundaries of different stages. For example, for patients with early-stage breast cancer but low MAGI2-AS3 expression, more aggressive treatment strategies could be considered to prevent disease recurrence and metastasis. Histologic grade is an indicator of the malignancy of cancer cells. The relationship between MAGI2-AS3 expression and histologic grade provides valuable insights into the biological behavior of breast cancer. Lower MAGI2-AS3 levels in tumors with higher histologic grades may be related to increased cell proliferation, invasion, and metastatic potential. This information can guide pathologists in making more precise prognostic judgments and contribute to further research on the molecular mechanisms of breast cancer progression. Although no significant correlations were found between MAGI2-AS3 expression and other clinical indices such as patient age, ER status, PR status, and HER-2 status, it is possible that there are underlying associations in specific patient subgroups. Future research with larger sample sizes and more in-depth subgroup analyses is needed to fully explore these potential relationships. This could lead to a more comprehensive understanding of the role of MAGI2-AS3 in breast cancer and potentially uncover new therapeutic targets or prognostic markers.

Diagnostic performance of MAGI2-AS3 expression in PBMCs from breast cancer patients

In a further analysis of the diagnostic value of MAGI2-AS3 in PBMCs, we constructed a ROC curve to investigate the diagnostic value of MAGI2-AS3 in the PBMCs of breast cancer patients. The ROC curve indicated that when comparing patients with controls, MAGI2-AS3 had an area under the curve (AUC) of 0.886 (Fig. 4A). We also constructed an ROC curve using CA15-3 data. Compared to the control group, CA15-3 had an AUC value of 0.751 (Fig. 4B). The analysis of the ROC curve showed that the diagnostic efficiency of MAGI2-AS3 was higher than that of CA15-3. These results suggested that MAGI2-AS3 has the potential to serve as a diagnostic marker for breast cancer. Moreover, the combination of MAGI2-AS3 in PBMCs and serum CA15-3 exhibited better diagnostic performance for breast cancer (AUC = 0.940) than a single marker (Fig. 4C).