Potential value of MicroRNA-21 as a biomarker for predicting the prognosis of patients with breast cancer

Abstract Background: The latest global cancer data from 2020 shows that breast cancer has replaced lung cancer as the number one cancer in the world. Searching for new biomarkers of breast cancer has important clinical significance for early diagnosis, prediction of prognosis, and targeted therapy. MicroRNA-21 (miRNA-21) can be used as a new molecular marker for early diagnosis, prognosis, and treatment of tumors. However, the expression of miRNA-21 in breast cancer and its prognosis are not clear. Therefore, this study conducted a meta-analysis to further clarify the relationship between the expression of miRNA-21 in breast cancer and prognosis. At the same time, we carried out bioinformatics analysis to further analyze the possible molecular mechanism of miRNA-21, so as to provide potential clinical indicators for the diagnosis, treatment, and prognosis of patients. Methods: PubMed, Medline, Embase, Web of Science, Wanfang, Chinese Biomedical Literature Database, Chinese National Knowledge Infrastructure, and other databases were used to retrieve the published relevant literatures. Include the eligible research, extract the survival data hazard ratios and 95% confidence intervals and other information. STATA16.0 software was used for meta-analysis. Download the miRNA data of breast cancer through the Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database. The data extracted for independent sample t test and ROC curve was drawn. OncomiR plotted the survival curve of miRNA-21 on the prognosis of breast cancer. The target genes of miRNA-21 were predicted, and the Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were analyzed. STRING database and Cytoscape construct protein-protein interaction (PPI) network to obtain Hub gene. The correlation between the expression level of Hub gene in breast cancer and the abundance of immune cell infiltration was analyzed by TIMER database and verified by Kaplan–Meien plotter database. Results: The results of this meta-analysis will be submitted to a peer-reviewed journal for publication. Conclusion: In this study, meta-analysis and bioinformatics analysis were used to further explore the prognosis, mechanism, and related pathways of miRNA-21 in breast cancer. Ethics and dissemination: The private information from individuals will not be published. This systematic review also should not damage participants’ rights. Ethical approval is not available. The results may be published in a peer-reviewed journal or disseminated in relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/R32A9.


Introduction
Breast cancer is one of the most common cancers in women around the world, which usually occurs in breast ducts and breast lobules, accounting for 22.90% of female cancers. [1] At present, the diagnosis and treatment of breast cancer not only burden the physiology and psychology of patients, but also adversely affect the family and society. [2] Therefore, in-depth understanding of the molecular pathogenesis of breast cancer, looking for biomarkers that are more sensitive to breast cancer, early detection, and treatment are of great significance to improve the overall survival time of patients with breast cancer. [3] Micro-RNA (miRNA) is a kind of small molecular singlestranded non-coding RNA with a length of about 18-25 nucleotides. [4,5] It can bind to the 3-non-coding region of the target gene RNA and function through RNA-mediated silencing complexes to prevent protein translation and expression or inhibit biological processes such as homologous mRNA degradation, thus exerting the function of tumor suppressor genes or tumor promoting genes. [6] It has been found that miRNA plays an important role in apoptosis, proliferation, metabolism, and differentiation. [7][8][9] This is closely related to the occurrence, proliferation, metastasis, diagnosis, treatment, and prognosis of the tumor. [10][11][12] As the most frequently studied cancer-related miRNA, in clinic, the abnormal expression of MiRNA-21 is related to a variety of malignant tumors such as liver cancer, thyroid cancer, colorectal cancer, and so on. [4,[13][14][15] Studies have shown that in breast cancer patients, the up-regulated expression of miRNA-21 is closely related to tumor cell proliferation, invasion, and drug resistance. [16,17] At present, the lack of sample size in the study of miRNA-21 expression and prognosis in patients with breast cancer leads to inconsistent results. [18][19][20][21][22][23] The purpose of this study is to explore the prognostic significance of miRNA-21 expression in patients with breast cancer by meta-analysis, and to provide accurate medical basis for the clinical application of miRNA-21. In addition, we carried out bioinformatics studies to clarify the expression and significance of miRA-21, to explore its possible mechanism, and to provide experimental basis for the study of molecular markers of breast cancer.

Study registration
The protocol of the systematic review has been registered on Open Science Framework. The registration number is DOI 10.17605/OSF.IO/R32A9. This meta-analysis protocol is based on the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols (PRISMA-P) Statement Guidelines. [24]

Data sources and search strategy
Comprehensive search of PubMed, Medline, Embase, Web of Science, Wanfang, Chinese Biomedical Literature Database, Chinese National Knowledge Infrastructure databases. The retrieval time is from the establishment to April 2021. Languages are limited to English and Chinese. Literature traceability, manual retrieval, and other methods are used to search the relevant literature. The search strategy for PubMed is shown in Table 1. According to the characteristics of each database, the retrieval strategy can be changed slightly.

Inclusion criteria for study selection
(1) Literature inclusion criteria: the study on breast cancer diagnosed by pathology; the expression level of miRNA-21 was detected in the study; the results showed the relationship between the expression level of miRNA-21 and the survival and prognosis of patients. (2) Literature exclusion criteria: review, meta-analysis, conference summary, etc; repeated published articles or experimental data; articles that cannot be extracted from HR.   [25] to extract effective information according to the data in the article.

Assessment of quality in included studies
Literature quality was evaluated based on the Newcastle-Ottawa Quality Assessment Scale (NOS). [26] The total score is 9 points, and NOS score ≥7 is classified as a high-quality study. [27] 2.6. Measures of prognosis OS and DFS will be taken as prognostic outcomes. The results will be expressed as HRs, with 95% CIs.

Management of missing data
If there exists insufficient or missing data in the literature, we would only analyze the currently available data and discuss its potential value.

Statistical analysis
STATA 16.0 (STATA Corporation, College Station, TX) was used for this meta-analysis. HR and its 95% CIs were used to evaluate the relationship between miRNA-21 expression and clinical prognosis in patients with breast cancer. The chi-squared test and I 2 values were carried out to assess the heterogeneity among the pooled analysis. If P < .1 or I 2 > 50% indicates that there is significant heterogeneity between studies, the random effect model is used to deal with the survival data, otherwise the fixed effect model would be used.
2.9. Additional analysis 2.9.1. Subgroup analysis. We will conduct a subgroup analysis based on cutoff value of miRNA-21 expression, ethnicity, and survival data sources.
2.9.2. Sensitivity analysis. The sensitivity analysis was carried out by using the method of excluding the study one by one.

Publication bias.
If no <10 articles are included, we will use funnel chart to test publication bias. [28,29]

Ethics
Our research data were derived from published literatures, because there were no patient recruitment and personal information collection. Therefore, ethical approval was not needed.

Discussion
As a kind of miRNA, the expression level of MiRNA-21 was first reported to be related to cancer in 2005. [30] In recent years, studies have shown that the abnormal expression of miRNA-21 is closely related to the occurrence and development of many kinds of malignant tumors. [15] MiRNA-21 is an oncogene, which not only plays a role in the proliferation of breast cancer, but also relates to tumor invasion, metastasis, and apoptosis. It reduces the expression of tumor suppressor genes to promote the occurrence and development of tumors. At present, many target genes of miRNA-21 have been found, including programmed death protein 4, tropomyosin 1, homologous lost phosphatase tensin on chromosome 10, Fas ligand, tissue inhibitor of metalloproteinase-3, tumor-associated protein 63, and so on. [10,[31][32][33] MiRNA-21 participates in a series of life activities of tumor by regulating these target genes.
MiRNA-21 is a serotype marker, which can be released into the blood from tissue cells when breast cancer occurs, and rises in the early stage of breast cancer. It can sensitively and specifically reflect the occurrence and development of breast cancer. In this study, meta-analysis was used to further analyze the relationship between the expression of miRNA-21 and the prognosis of patients with breast cancer, so as to determine that miRNA-21 can be used as a potential clinical indicator. By means of bioinformatics, we can obtain the regulatory targets and signal pathways of miRNA-21. In addition, by analyzing the correlation between Hub gene expression and immune infiltrating cells, we can provide experimental basis for how miRNA-21 target genes Ding et al. Medicine (2021)