A biomarker for tumor detection

Abstract

The application relates to the technical field of disease detection, in particular to a biomarker for tumor detection, wherein the biomarker comprises at least one of mir-195-3p, mir-10b-5p, mir-133a-3p, mir-195-5p and mir-155-3p. The serum biomarker panel (mir-10b-5p, mir-133a-3p, mir-195-5p, mir-195-3p and mir-155-3p) composed of five miRNAs determined in the application provides a hope for early, non-invasive and accurate diagnosis of BC.

Description

Technical Field

[0001] This invention relates to the field of disease detection technology, and more specifically to a biomarker for tumor detection. Background Technology

[0002] Breast cancer (BC), a highly prevalent malignant tumor, seriously endangers women's health and lives. According to data from the International Agency for Research on Cancer (IARC) in 2022, the global incidence of breast cancer is second only to lung cancer, with nearly 2.3 million new cases and over 660,000 new deaths. The choice of treatment and prognosis for BC patients are closely related to early detection. In recent years, the survival rate of BC patients has significantly improved with the continuous development of screening and treatment methods; however, the 5-year overall survival rate for advanced-stage patients is only 20%. Early diagnosis of BC and further guidance for treatment and improvement of prognosis are key tasks in current BC research. Commonly used auxiliary examination methods in clinical practice include breast ultrasound, mammography, and tissue biopsy, with tissue biopsy currently being the preferred method for obtaining molecular information about tumors. Given the limitations of current early screening methods, it is necessary to develop new examination methods and apply them in clinical practice.

[0003] MicroRNAs (miRNAs) are single-stranded non-coding RNA sequences of 20-22 nucleotides in length, widely distributed in humans, animals, and plants, and involved in RNA-mediated gene silencing. With increasing understanding of miRNAs, research has revealed that abnormal miRNA gene expression can cause functional disorders leading to diseases, especially cancer-related diseases, and they have high reference value in distinguishing normal tissues from tumor subtypes. Furthermore, due to their relatively simple molecular structure, extremely high tissue specificity and stability, and ease of quantification and amplification, miRNAs have become highly promising potential biomarkers.

[0004] Extensive evidence has demonstrated that miRNAs play a crucial role in the carcinogenesis of various cancers, including clear cell renal cell carcinoma, oral squamous cell carcinoma, and prostate cancer. Studies have shown that miRNAs play important roles in the biological functions of cancer cells, such as proliferation, differentiation, apoptosis, and invasion, becoming a key factor in regulating cancer development. The role of specific miRNAs as biomarkers for tumor diagnosis and prognosis is attracting increasing attention from researchers, providing new insights for cancer diagnosis and treatment.

[0005] Breast cancer (BC), one of the most common types of malignant tumors, is experiencing a year-on-year increase in both incidence and mortality. In its early stages, breast cancer often does not present with obvious symptoms, making it difficult to distinguish from benign breast diseases. This study aims to identify a specific group of miRNAs in serum as a non-invasive biomarker for early detection of BC. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker for tumor detection.

[0007] Current research has confirmed that miRNA expression may be associated with the occurrence and prognosis of breast cancer, but the impact of many miRNAs on its development remains poorly understood. This study aims to construct a specific miRNA combination model to explore its value in diagnosing breast cancer (BC) and to help identify BC patients in a timely manner.

[0008] The technical problem solved by this invention is achieved by the following technical solution:

[0009] In a first aspect, the present invention provides a biomarker for tumor detection, the biomarker comprising at least one of miR-195-3P, miR-10B-5P, miR-133A-3P, miR-195-5P, and miR-155-3P.

[0010] In some embodiments of the present invention, the biomarker includes:

[0011] mir-155-3p; and

[0012] At least one of mir-195-3p, mir-10b-5p, mir-133a-3p, and mir-195-5p.

[0013] In some embodiments of the present invention, the biomarkers include miR-10b-5p, miR-133a-3p, and miR-195-5p.

[0014] In some embodiments of the present invention, the biomarkers include miR-10b-5p, miR-133a-3p, miR-155-3p, and miR-195-5p.

[0015] In some embodiments of the present invention, the biomarkers include mir-195-3p, mir-10b-5p, mir-133a-3p, mir-195-5p, and mir-155-3p.

[0016] The reagents used for RT-qPCR detection of the five miRNAs were routinely purchased and sourced from: Reverse Transcription Kit (R323-01, Novizan, China).

[0017] The nucleotide sequences of the five miRNAs are shown in Table 1:

[0018]

[0019] Table 1

[0020] In some embodiments of the present invention, the biomarkers include mir-195-3p, mir-10b-5p, mir-133a-3p, mir-195-5p, and mir-155-3p in blood.

[0021] In some embodiments of the present invention, the tumor is breast cancer, gastric cancer, lung cancer, esophageal cancer, colorectal cancer, pancreatic cancer, or liver cancer.

[0022] In some embodiments of the present invention, the tumor is breast cancer.

[0023] This invention offers the following advantages: This study mainly includes a screening phase, a testing phase, and a validation phase. In the screening phase, eight miRNAs associated with breast cancer (BC) were selected for analysis through literature review. In the testing phase, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to select five miRNAs with the most significant expression differences from 15 BC patients and 15 benign breast cancer controls for the next phase. In the subsequent validation phase, RT-qPCR was used to evaluate these five miRNAs obtained from serum samples of another 75 BC patients and 50 benign control patients. Receiver operating characteristic (ROC) curves and area under the curve (AUC) were used to evaluate the diagnostic ability, specificity, and sensitivity of the candidate miRNAs. Finally, an optimal diagnostic combination model with high sensitivity and specificity was constructed using these five miRNAs.

[0024] The expression levels of miRNAs mir-195-3p, mir-10b-5p, mir-133a-3p, mir-195-5p, and mir-155-3p in the serum of patients with benign bronchitis (BC) were significantly lower than those in benign controls, while the expression level of mir-195-3p was higher than that in benign controls. To assess their diagnostic potential for BC, we used receiver operating characteristic (ROC) curve analysis. The analysis showed that all five miRNAs exhibited strong diagnostic ability, with areas under the curve (AUC) > 0.8. To further improve diagnostic accuracy, we constructed a final diagnostic combination model consisting of these five miRNAs, which showed good diagnostic value, with an AUC of 0.948.

[0025] The serum biomarker panel consisting of five miRNAs identified in this study (mir-10b-5p, mir-133a-3p, mir-195-5p, mir-195-3p, and mir-155-3p) offers hope for early, non-invasive, and accurate diagnosis of BC. Attached Figure Description

[0026] Figure 1 The differential expression diagram of the eight candidate miRNAs in section 2.2 is shown.

[0027] Figure 2 Differential expression and ROC curves of five candidate miRNAs in section 2.3.

[0028] Figure 3 for Figure 3 The ROC curve for evaluating model 25 after combining 5 miRNAs.

[0029] Figure 4 The target gene prediction, GO functional annotation, and KEGG pathway enrichment analysis of five miRNAs were performed using miRWalk3.0.

[0030] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0031] As described in this invention, the term "prevention" refers to preventing the occurrence of disease and / or preventing the recurrence of disease. Detailed Implementation

[0032] Detection of breast cancer using 5 miRNAs

[0033] 1 Experimental Methods

[0034] 1.1 Participants

[0035] This study included 90 patients with breast cancer (BC) and 65 benign control volunteers. All recruited patients visited the Department of Breast Surgery at the First Affiliated Hospital of Xinjiang Medical University between March 2023 and March 2024. All BC patients were diagnosed by preoperative biopsy or postoperative histopathology. None of the patients included in this study had received any clinical treatment prior to serum sample collection.

[0036] All enrolled patients had primary BC without metastasis or other cancers. All benign control volunteers had no history of acute or chronic diseases or other tumors.

[0037] 1.2 Blood Sample Collection and Processing

[0038] Serum samples from patients and controls were obtained using the following collection and sampling procedures: After enrollment, all participants had 5–10 mL of circulating blood collected on an empty stomach in the morning. The blood was placed in sterile anticoagulant tubes and centrifuged at 3000 rpm for 10 min at room temperature. The supernatant was transferred to microcentrifuge tubes and stored at −80°C for subsequent experiments.

[0039] 1.3 Research Design

[0040] The study was conducted in four phases.

[0041] First, through extensive literature review, eight differentially expressed miRNAs that were significantly associated with BC expression were identified and screened as candidate biomarkers.

[0042] Secondly, these candidate biomarkers were further tested and validated. In the testing phase, quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed on 15 BC patients and 15 benign control groups to assess the relative serum levels of candidate miRNAs. The top 5 miRNAs showing the most significant differences in this phase were selected for further study.

[0043] Then, this study expanded the sample size, repeating the above steps using serum samples from 75 BC patients and 50 benign control patients to validate the results of the previous stage. The expression levels and diagnostic capabilities of candidate miRNAs were determined, and a diagnostic combinatorial model composed of candidate miRNAs was developed to improve diagnostic sensitivity and specificity.

[0044] Finally, bioinformatics analysis was used to explore the potential biological functions of candidate miRNAs related to BC occurrence.

[0045] 1.4 RNA Extraction and Detection

[0046] cDNA synthesis was performed using a reverse transcription kit (R323-01, Novizan, China) on a thermocycle instrument (T100, Bio-Rad, USA). The reverse transcription conditions were 42℃ for 5 minutes, 37℃ for 15 minutes, and 85℃ for 5 seconds. Then, qPCR reactions were performed on an ABI QuantStudio 5 instrument with the following program: 95℃ pre-denaturation for 10 minutes, 95℃ denaturation for 15 seconds, and 60℃ annealing and extension for 1 minute, with 40 cycles of denaturation and extension. Each sample was performed in triplicate.

[0047] Next, the relative expression level of each transcript mRNA was calculated, using GAPDH as an internal reference gene for normalization, and 2... - ΔΔCTThe methods were analyzed (Livak and Schmittgen 2001). Two-way ANOVA or t-test statistical analysis was performed using GraphPad Prism software (version 8.0, San Diego, CA).

[0048] 1.5 Bioinformatics Analysis

[0049] The miRWalk database (http: / / mirwalk.umm.uni-heidelberg.de / ) was used to analyze the target genes of five miRNAs in the miRDB, mirTarBase, and TargetScan databases, and Venn diagrams were used to visualize the results. The main biological functions, predicted pathways, and related functions of the five target miRNAs were analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG).

[0050] 1.6 Statistical Analysis

[0051] Data from this study were analyzed using SPSS software. The Shapiro-Wilk normality test was used to test whether continuous variables conformed to a normal distribution. Categorical variables were expressed as percentages, while continuous variables (conforming to a normal distribution) were expressed as mean ± standard deviation. A p-value less than 0.05 was defined as statistically significant. To compare miRNA levels between BC and benign control samples, t-tests were used for continuous variables, and χ² tests were used for categorical variables. Receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) were used to assess the diagnostic performance of each candidate miRNA. These measures were used to evaluate the specificity, sensitivity, and overall diagnostic ability of each miRNA. In addition, we calculated the Youden index (J = sensitivity + specificity - 1) to identify the combination of miRNAs providing the highest sensitivity and specificity for diagnosis.

[0052] 2. Experimental Results

[0053] 2.1 Clinical and demographic characteristics of study participants

[0054] This study included 155 participants, comprising 90 patients with breast cancer (BC) and 65 volunteers with benign breast lesions. Table 1 shows the demographic and clinical characteristics of the participants during the testing and validation phases. The results showed that the mean age in the BC group was 49.9 ± 7.5 years, and the mean age in the benign control group was 44.5 ± 9.8 years. There were no significant differences in age and menopausal status among the participants. After expanding the sample, the mean age of the participants included in the study was 51.5 ± 7.0 years, and the mean age in the benign control group was 40.4 ± 9.5 years. They showed significant differences in age and menopausal status, which may be due to hormonal effects; reproductive factors such as age and menopausal status are particularly closely related to the occurrence of BC.

[0055] Table 1. General demographic characteristics of the participants included in the study

[0056]

[0057] 2.2 Confirmation of candidate miRNAs

[0058] Through extensive literature review and refinement, candidate miRNAs related to BC were selected as candidate miRNAs for subsequent studies in this research (Table 2). The miRNA precursor produces two complementary, functional mature miRNAs, generally derived from the 5' and 3' arms of the precursor, named "-5p" and "-3p" respectively, targeting different sites. Therefore, this study included eight complementary miRNAs for further investigation.

[0059] Table 2. Diagnostic value of different miRNAs in breast cancer patients

[0060]

[0061] To assess serum levels of eight selected candidate miRNAs, we performed RT-qPCR on a randomized subset of 15 BC patients and 15 benign controls. Figure 1 The differential expression map of 8 candidate miRNAs shows that our study found mir-10b-5p ( Figure 1 B), mir-133a-3p Figure 1 C), mir-155-3p ( Figure 1 E), mir-195-3p ( Figure 1 G) and mir-195-5p ( Figure 1 There were significant differences in the expression levels of H).

[0062] Based on these findings, further analysis focused specifically on these five miRNAs. Univariate analysis revealed that all five miRNAs included in the analysis were significantly associated with the occurrence of breast cancer (Table 3).

[0063] Table 3. Univariate analysis of the relationship between five miRNAs and BC occurrence.

[0064]

[0065] 2.3 Diagnostic value of 5 candidate miRNAs

[0066] To further validate the potential of the five selected miRNAs as serum biomarkers for BC screening, we expanded the sample size. Seventy-five BC patients and fifty benign controls were included in the study. Relative serum levels of the five miRNAs were detected using RT-qPCR.

[0067] In our study, Figure 2 Differential expression and ROC curves of five candidate miRNAs in section 2.3. mir-10b-5p in BC patients ( Figure 2 A), mir-133a-3p ( Figure 2 B), mir-155-3p ( Figure 2 C) and mir-195-5p Figure 2 The expression levels of E) were significantly lower than those of the benign control, and mir-195-3p ( Figure 2 D) The expression level in BC was higher than that in the benign control.

[0068] To evaluate the diagnostic value of these five miRNAs, we performed ROC curve analysis. The area under the ROC curve (AUC) for each miRNA is shown below. Figure 2 As shown in FJ. Then, the optimal cutoff value, specificity, and sensitivity for diagnosing BC using the Youden index were calculated (Table 4). The ROC curves show that mir-10b-5p, mir-133a-3p, mir-195-5p, mir-195-3p, and mir-155-3p exhibited good diagnostic potential for BC, with AUCs of 0.85, 0.84, 0.87, 0.62, and 0.83, respectively.

[0069] Table 4. Optimal cutoff values, specificity, and sensitivity of five miRNAs for diagnosing BC.

[0070]

[0071] 2.4 Construction of the Optimal Diagnostic Model

[0072] During the experimental phase, we found that mir-10b-5p, mir-133a-3p, mir-195-5p, mir-195-3p, and mir-155-3p had good diagnostic capabilities for BC. Therefore, we investigated whether combinations of several miRNAs had higher diagnostic value for BC and determined the optimal combination for BC diagnosis from all possible miRNA combinations (Table 5).

[0073] Table 5. Specificity and sensitivity of all miRNA combination models in Table 5.

[0074]

[0075] The study found that, except for models 15, 17, and 21, almost all constructed models had extremely high specificity (>0.90), suggesting that all models could accurately identify the occurrence of breast cancer. Furthermore, the study found that models 4, 19, and 24 also had high diagnostic value (AUC>0.90), possibly because they collectively included miR-10b-5p and miR-195-5p, the best-diagnostic single miRNAs.

[0076] Model 25 consists of 5 miRNAs and exhibits extremely high diagnostic capability, with an AUC of 0.948 (95% CI: 0.914–0.981; specificity = 100%, sensitivity = 79%) as shown by the ROC curve. The formula for this model is: Logit(P) = 0.757 + (-46.427 * miR-10b-5p) + (29.354 * miR-133a-3p) + (-20.170 * miR155-3p) + (3.144 * miR-195-3p) + (17.138 * miR-195-5p). Figure 3 The ROC curve for model 25, composed of a combination of five miRNAs, shows that this result is superior to that of any single miRNA. Figure 3 As shown.

[0077] 2.5 Bioinformatics analysis of five miRNAs

[0078] The miRNAWalk2.0 assay was used to explore and visualize the potential target genes of mir-10b-5p, mir-133a-3p, mir-155-3p, mir-195-5p, and mir-195-3p. Figure 4 In addition, the potential target genes of the above 5 miRNAs were included in KEGG pathway enrichment analysis and GO function analysis to further analyze their potential functions. Figure 4The target gene prediction, GO functional annotation, and KEGG pathway enrichment analysis of five miRNAs were performed using miRWalk3.0.

[0079] These findings indicate that the target genes of mir-10b-5p, mir-133a-3p, mir-195-5p, mir-195-3p, and mir-155-3p are involved in key pathways and processes related to cancer development and progression. GO analysis showed that these miRNAs are mainly involved in biological processes such as WNT-mediated cell-cell signaling, the WNT signaling pathway, and mitotic cell cycle phase transitions. Figure 4 F), and also mainly participates in cell junctions, transcriptional regulatory complexes and transferase complexes, transferring cellular components such as phosphate groups. Figure 4 In addition, our study also found that they are involved in molecular functions such as DNA binding to transcription factor binding and protein serine / threonine kinase activity. Figure 4 H). KEGG analysis results indicate that the above five miRNAs mainly exert their effects through mechanisms such as mediating microRNAs, the PI3K-Akt signaling pathway, and cellular senescence in cancer. Figure 4 I). The enrichment of these target genes in the above pathways reflects their potential role in regulating key cellular functions related to tumorigenesis.

[0080] Summarize

[0081] Breast cancer (BC) is one of the most common types of malignant tumors. In recent years, the incidence and mortality rates of BC have been increasing annually, and the age of onset is trending younger. BC often does not present obvious symptoms in its early stages, making it difficult to detect during routine physical examinations and distinguish it from benign breast diseases. However, as the disease progresses, the tumor may become malignant and metastasize, leading to a poor prognosis. Therefore, early diagnosis of BC is challenging. The emergence of biomarkers has aided in the clinical diagnosis of BC. Currently, some serum biomarkers have been found to be related to the pathogenesis of BC and have been clinically validated. In recent years, some non-coding RNAs have also been studied to assist in the diagnosis of BC. miRNAs are short fragments of non-coding ribonucleic acid (ncRNAs) present in bodily fluids. Due to their extremely high tissue specificity and stability, they have been increasingly used to differentiate normal tissues from tumor subtypes. Previous studies have shown that plasma miRNAs can serve as biomarkers for detecting BC.

[0082] This study first selected eight miRNAs with diagnostic potential in benign prostatic hyperplasia (BC) through extensive literature review. The diagnostic value of these eight miRNAs in BC was then assessed by analyzing serum samples from 15 benign controls and 15 BC patients. The results confirmed that the expression levels of mir-10b-5p, mir-133a-3p, mir-195-5p, and mir-155-3p were significantly lower than in healthy controls, while the expression level of mir-195-3p in BC was higher than in healthy controls. These differences in expression suggest that these miRNAs may be involved in the development and progression of BC.

[0083] Regarding diagnostic potential, ROC curve analysis showed that all five miRNAs exhibited significant diagnostic capabilities, indicating their potential as non-invasive biomarkers for early detection and diagnosis of breast cancer. We subsequently expanded the sample size to 75 breast cancer patients and 50 benign control patients for further validation. Based on these five high-quality miRNAs, we successfully established an optimal diagnostic panel with high sensitivity and specificity. Previous studies have explored the individual diagnostic efficacy of these five miRNAs, but no study has yet incorporated all five miRNAs together into a diagnostic tool. Our study found that the combined panel's diagnostic efficacy is superior to that of each individual miRNA. Therefore, combining these five miRNAs into a single diagnostic panel could be considered as a novel biomarker for diagnosing breast cancer.

[0084] mir-195-3p and mir-195-5p both belong to the miR-195 family and are involved in the occurrence and development of various tumors. Studies have shown that serum miR-195 levels in the early-stage BC group were more than twice that of healthy controls, suggesting that miR-195 may serve as a biomarker for the early diagnosis of BC.

[0085] In addition, the study found that both complementary mature miRNAs of miR-195 have good diagnostic value. Among the five single miRNAs studied, mir-195-5p showed the best diagnostic ability, exhibiting excellent sensitivity and specificity (65% and 100%, respectively). In the constructed diagnostic panel, mir-195-3p was the only miRNA overexpressed in breast cancer. Previous studies have reported the relationship between mir-195-3p and breast cancer. However, the pathogenesis of mir-195-3p in breast cancer has not yet been investigated. mir-195-5p is considered a potential novel prognostic marker for rectal cancer. In cervical cancer, mir-195-5p expression was lower than in the control group, potentially inhibiting the malignant progression of cervical cancer by targeting YAP1. These studies provide evidence that miR-195 may serve as a biological marker for the diagnosis of BC.

[0086] Previous studies have reported that the miR-10b family is involved in the development and metastasis of various tumors. It has been reported that serum miR-10b-5p expression levels are significantly elevated in patients with early-stage hepatocellular carcinoma (HCC), and miR-10b-5p has high diagnostic value (ROC=0.934). Studies have found a significant correlation between miR-10b-5p expression and molecular subtypes of early-stage invasive ductal carcinoma. Further research has shown that high miR-10b-5p expression is a protective factor for patients with invasive ductal carcinoma of the breast, reducing recurrence and mortality, and may become a potential therapeutic target for these patients. These studies suggest that miR-10b-5p could serve as a novel biomarker for the diagnosis and prognosis of breast cancer patients.

[0087] mir-133a-3p, also an important regulator of HCC, has been found to be a potential non-invasive biomarker for the diagnosis of HCC patients (AUC=0.67). mir-133a-3p has a strong ability to distinguish between breast cancer and benign breast diseases (ACU=0.84).

[0088] miR-155, a miRNA that plays a core role in cancer regulation, has been found to be highly correlated with gynecological tumors, digestive system tumors, and hematologic malignancies. Increasing evidence suggests that its 3p chain plays a role in these areas. Studies have found that miR-155-3p expression levels can serve as a biomarker for early diagnosis of cancer with high sensitivity, specificity, and accuracy.

[0089] Several advantages of using miRNA-based biomarkers include their stability in serum samples, non-invasive detection, and potential for early diagnosis. This study screened five miRNAs highly correlated with breast cancer (BC) using clinical samples to create a diagnostic model with higher efficacy, overcoming the limitations of single miRNA diagnosis, better distinguishing BC patients from those with benign breast diseases, and providing the possibility of early BC diagnosis using combined models. Currently, some researchers have also found that certain miRNAs can serve as potential diagnostic biomarkers for BC patients. Our study incorporated five miRNAs with individual diagnostic efficacy. The development of a diagnostic model composed of mir-10b-5p, mir-133a-3p, mir-195-5p, mir-195-3p, and mir-155-3p as BC diagnostic biomarkers can facilitate timely intervention and treatment decisions and improve patient management. However, further validation studies in larger cohorts are needed to confirm the diagnostic accuracy and clinical efficacy of these miRNAs.

[0090] in conclusion

[0091] In this study, we preliminarily identified serum miRNA markers associated with breast cancer (BC) and identified five serum miRNAs that could be used for breast cancer screening and early diagnosis. Furthermore, we successfully constructed a diagnostic panel with excellent diagnostic performance, sensitivity, and specificity using these five miRNAs, providing a new possibility for the early diagnosis of BC.

[0092] This invention specification provides a detailed description of specific embodiments. Those skilled in the art should recognize that the above embodiments are exemplary and should not be construed as limiting the invention. For those skilled in the art, various improvements and modifications can be made to the invention without departing from its principles, and the resulting technical solutions also fall within the scope of protection of the claims of this invention.

Claims

1. The application of reagents for detecting serum biomarker expression levels in the preparation of products for diagnosing breast cancer, characterized in that: The biomarkers consist of miR-195-3p, miR-10b-5p, miR-133a-3p, miR-195-5p, and miR-155-3p.

Images