MiRNA miR-148b-3p for targeted regulation of action of SLC7A11 in breast cancer as well as action mechanism and application of miRNA miR-148b-3p

Abstract

The invention provides miRNA miR-148b-3p for targeted regulation of the action of SLC7A11 in breast cancer as well as an action mechanism and application of the miRNA miR-148b-3p. It is found and proved for the first time that miR-148b-3p can inhibit proliferation and migration of breast cancer cells through targeted regulation of SLC7A11. Related action mechanisms are disclosed by researches such as construction of a ceRNA network, and potential targets and strategies are provided for breast cancer treatment. Meanwhile, it is found that the compound also has a regulation effect in other cancers, but the effect is weaker than that of the breast cancer, the specific regulation effect on the breast cancer is highlighted, and a solid theoretical foundation is laid for accurate treatment of the breast cancer, so that breast cancer treatment and research are more effectively carried out.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a miRNA miR-148b-3p that targets and regulates the role of SLC7A11 in breast cancer, its mechanism of action, and its applications. Background Technology

[0002] Cancer is one of the most serious threats to human health worldwide, and its occurrence and development involve a variety of complex molecular mechanisms. In recent years, research has found that solute carrier family 7 member 11 (SLC7A11) plays an important role in cancer.

[0003] SLC7A11 is a multi-pathway transmembrane protein that mediates the uptake of extracellular cystine and the exchange of glutamate, playing a crucial role in cell growth, proliferation, and metabolism. As a key cellular mechanism against ferroptosis, the SLC7A11-GSH system influences intracellular cystine and glutathione levels by regulating cystine metabolism pathways, thereby affecting cell death. Currently, increasing research indicates that SLC7A11 is overexpressed in various tumors, and its expression level is closely related to tumor cell proliferation, invasion, metastasis, and the tumor microenvironment. For example, in lung cancer, Has-mir-373 and Has-mir-372 can competitively bind to and upregulate SLC7A11 expression, regulating immune infiltration in lung adenocarcinoma; in melanoma, SLC7A11 increases intracellular GSH levels, confers resistance to BRAF inhibitors; and in bladder cancer, inhibiting SLC7A11 expression can reverse drug-resistant cells' resistance to cisplatin. However, there are few systematic studies on the role of SLC7A11 in pan-cancer, and the potential molecular mechanisms underlying changes in its levels during cancer formation are not fully understood.

[0004] In breast cancer, SLC7A11 expression is positively correlated with tumor immune cell infiltration, and high expression is significantly associated with poor prognosis, suggesting that it could serve as a biomarker for poor prognosis in breast cancer patients. However, how SLC7A11 can inhibit cancer, especially breast cancer, remains unclear.

[0005] Minimal RNAs (miRNAs) are a class of endogenous non-coding single-stranded RNA molecules that can bind to the 3'-untranslated region of target genes, negatively regulating their expression and cellular function. Previous studies have shown that miR-148a-3p promotes malignant behavior in breast cancer cells by downregulating DUSP1, but the miRNAs or mechanisms by which they target and regulate the SLC7A11 gene to inhibit breast cancer remain unclear.

[0006] Therefore, in order to gain a deeper understanding of the mechanism of action of SLC7A11 in cancer and to find effective methods to inhibit cancer, especially breast cancer, it is urgent to find a mechanism of action of SLC7A11 to inhibit cancer, as well as a novel miRNA that can target and regulate SLC7A11. By regulating the expression level of miRNA in vivo, the SLC7A11 gene can be targeted and regulated to achieve the goal of inhibiting breast cancer. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a miRNA, miR-148b-3p, that targets and regulates the role of SLC7A11 in breast cancer, along with its mechanism of action and applications. By constructing a ceRNA network to predict and screen miR-148b-3p, this invention is the first to discover and demonstrate that miR-148b-3p can inhibit the proliferation and migration of breast cancer cells by targeting and regulating SLC7A11. Furthermore, cell experiments verified that changes in relevant indicators after transfection with the miR-148b-3p inhibitor showed a good inhibitory effect on breast cancer cells. This reveals that the mechanism of action of SLC7A11 in breast cancer is related to the regulation of miR-148b-3p, providing a new perspective for a deeper understanding of the mechanisms of breast cancer development and progression. It also provides potential targets and strategies for breast cancer treatment. Additionally, the invention discovers the regulatory role and specificity of miR-148b-3p in other cancers, highlighting its relatively weak regulatory effect on breast cancer and providing a solid theoretical basis for precision treatment of breast cancer.

[0008] To achieve the above objectives, the present invention employs the following solution:

[0009] On one hand, the present invention provides the use of miRNA for preparing a formulation that regulates the proliferative activity or migration ability of tumor cells, wherein the miRNA is miR-148b-3p, and the nucleotide sequence of miR-148b-3p is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0010] To gain a deeper understanding of the mechanism of action of SLC7A11 in cancer and to find effective methods to inhibit cancer, especially breast cancer, this invention conducted a systematic pan-cancer analysis of SLC7A11 and a targeted study on breast cancer. This included analyzing the differential expression of SLC7A11 in normal and tumor samples, prognostic analysis, and gene analysis. The results showed that SLC7A11 expression in normal tissues is tissue-specific, with differences in the expression levels of its RNA and protein in normal tissues. Furthermore, its expression in different tumor tissues exhibited significant differential changes and was associated with poor prognosis in various cancers. However, the mechanism by which SLC7A11 regulates the biological behavior of breast cancer cells remains unclear.

[0011] Therefore, in some implementations, further exploration was conducted by predicting upstream binding miRNAs of SLC7A11 using multiple target gene prediction programs. Only predicted miRNAs that typically appeared in more than three of these programs were included in subsequent analyses. Long non-coding RNAs (lncRNAs) interacting with the screened miRNAs were predicted using the ENCORI and miRNet platforms. Based on the ceRNA hypothesis, a ceRNA network was constructed by performing negative correlation analysis between mRNA and miRNA, RNA expression analysis, and overall survival analysis in a BRCA cohort. Ultimately, 11 target miRNAs of the SLC7A11 gene were obtained. Given the negative correlation trend between miRNA expression and ceRNA expression, the most significantly correlated miRNA, miR-148b-3p, was screened from the matched miRNAs. The nucleotide sequence of miR-148b-3p contains a sense strand and an antisense strand, wherein the sense strand is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2.

[0012] Furthermore, in the tumor cells, the gene associated with their proliferation activity or migration ability is the SLC7A11 gene; by regulating the expression level of miR-148b-3p, the expression of the SLC7A11 gene can be regulated, thereby regulating the proliferation activity or migration ability of tumor cells.

[0013] Furthermore, by upregulating or downregulating the expression level of miR-148b-3p, the expression of the SLC7A11 gene can be inhibited or promoted, thereby reducing or increasing the proliferation activity and migration ability of tumor cells.

[0014] Furthermore, by adding miR-148b-3p analogues, the expression of SLC7A11 gene was inhibited, thereby suppressing the proliferation and migration of tumor cells; by adding miR-148b-3p inhibitors, the expression of SLC7A11 gene was increased, thereby enhancing the proliferation and migration of tumor cells.

[0015] Furthermore, the nucleotide sequence of the miR-148b-3p inhibitor is shown in SEQ ID NO.3.

[0016] In some embodiments, the sequence of the miR-148b-3p inhibitor is designed based on the sequence of SEQ ID NO.1, and the nucleotide sequence of the miR-148b-3p inhibitor is shown in SEQ ID NO.3.

[0017] Furthermore, miR-148b-3p analogs or miR-148b-3p inhibitors were added to tumor cells using transfection reagents.

[0018] In some embodiments, this invention identifies the SLC7A11 gene as the gene associated with proliferation or migration in tumor cells, and ultimately screens out miR-148b-3p as a regulator of its expression. Cellular experiments verify that altering miR-148b-3p expression levels can correspondingly inhibit or promote SLC7A11 gene expression, thereby regulating tumor cell proliferation and migration. Specifically: silencing miR-148b-3p significantly increases the mRNA and protein levels of SLC7A11, thereby enhancing the proliferation and migration rate of breast cancer cells; overexpressing miR-148b-3p significantly reduces the mRNA and protein levels of SLC7A11, thereby significantly inhibiting the proliferation and migration rate of breast cancer cells.

[0019] Furthermore, the tumor cells include any one or more combinations of breast cancer cells, colon cancer cells, or liver cancer cells.

[0020] Furthermore, the tumor cells include breast cancer cells.

[0021] In some implementations, the regulatory role of miR-148b-3p in other cancers was further explored, contributing to a comprehensive understanding of its functional mechanisms in tumorigenesis and development. Experimental results showed that in the colon cancer cell line HT-29 and the liver cancer cell line HepG2, silencing miR-148b-3p expression could target and regulate SLC7A11, increasing SLC7A11 mRNA and protein levels. However, compared to breast cancer cells, the regulatory effect on its mRNA and protein levels was relatively weak. Similarly, in the colon cancer cell line HT-29 and the liver cancer cell line HepG2, transfection with a miR-148b-3p inhibitor enhanced cell proliferation and migration, but these effects were not as pronounced as in the breast cancer cell line. Therefore, it can be concluded that miR-148b-3p can also influence the biological behavior of tumor cells in other cancers (colon cancer, liver cancer, etc.) by targeting and regulating SLC7A11. However, compared to breast cancer, its regulatory effects on these cancers are relatively weak, including its influence on the regulation of SLC7A11 gene and protein expression, cell proliferation, and migration. This suggests that miR-148b-3p may have a relatively specific regulatory role in breast cancer, further highlighting its importance in breast cancer research.

[0022] On the other hand, the present invention provides the use of a miR-148b-3p inhibitor in the preparation of a formulation for regulating the proliferative activity or migration ability of tumor cells, wherein the nucleotide sequence of the miR-148b-3p inhibitor is shown in SEQ ID NO.3.

[0023] Furthermore, by adding a miR-148b-3p inhibitor, the expression of the SLC7A11 gene is enhanced, thereby increasing the proliferation and migration ability of tumor cells; the tumor cells include any one or more combinations of breast cancer cells, colon cancer cells, or liver cancer cells.

[0024] In another aspect, the present invention provides the use of miRNA in preparing a formulation that regulates ferroptosis in tumor cells, wherein the miRNA is miR-148b-3p, and the nucleotide sequence of miR-148b-3p is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0025] In some embodiments, this invention has explored the mechanism of action of SLC7A11 in breast cancer by constructing a ceRNA network, and found that miR-148b-3p may play a key role in breast cancer progression by targeting SLC7A11. Furthermore, silencing miR-148b-3p expression can increase the mRNA and protein levels of SLC7A11, thereby enhancing the proliferation and migration rate of breast cancer cells. However, tumor cell proliferation, migration, and survival are closely related to cell death mechanisms. Ferroplasmosis, as a novel cell death mechanism that has received widespread attention in recent years, may play an important role in the occurrence and development of tumors. SLC7A11 is known to play an important role in breast cancer and is associated with multiple biological processes; changes in its expression not only affect tumor cell proliferation and migration but may also influence cell death mechanisms, particularly ferroptosis. Meanwhile, miR-148b-3p, as a key factor regulating SLC7A11, may regulate SLC7A11 expression beyond cell proliferation and migration, potentially involving cell death mechanisms, especially ferroptosis.

[0026] Therefore, based on this, this invention further investigates the mechanism by which miR-148b-3p regulates ferroptosis in breast cancer cells by targeting SLC7A11. A deeper analysis of this mechanism is expected to reveal another important regulatory pathway of miR-148b-3p and SLC7A11 in breast cancer cells, further enriching our understanding of the functions of miR-148b-3p and SLC7A11, and providing new targets and strategies for breast cancer treatment.

[0027] Experimental results show that miR-148b-3p significantly affects the expression of ferroptosis-related genes by targeting and regulating SLC7A11, thereby influencing the ferroptosis process in breast cancer cells. Specifically, inhibiting miR-148b-3p increases GSH levels, decreases MDA content, and increases cell survival in tumor cells, indicating that miR-148b-3p can regulate breast cancer cell survival by modulating the ferroptosis-related gene SLC7A11.

[0028] Therefore, it can be concluded that miR-148b-3p can target and regulate SLC7A11. Increasing miR-148b-3p expression inhibits SLC7A11 expression, thereby promoting ferroptosis. Furthermore, this ferroptosis further inhibits the proliferation and migration of breast cancer cells. This discovery provides a new perspective for breast cancer treatment; targeting miR-148b-3p may become a novel therapeutic approach, inhibiting tumor growth by activating ferroptosis. It also provides a solid theoretical basis for further research on the biological behavior of breast cancer cells, offers crucial data support for research on the regulation of ferroptosis in breast cancer, and provides important new clues for future therapeutic targets.

[0029] Furthermore, in the tumor cells, the gene associated with ferroptosis is the SLC7A11 gene; by regulating the expression level of miR-148b-3p, the expression of the SLC7A11 gene can be regulated, thereby regulating ferroptosis in tumor cells.

[0030] Furthermore, by upregulating or downregulating miR-148b-3p expression, the expression of the SLC7A11 gene is inhibited or promoted, thereby promoting or inhibiting ferroptosis in tumor cells.

[0031] Furthermore, by adding miR-148b-3p analogues, the expression of the SLC7A11 gene was inhibited, promoting ferroptosis in tumor cells; by adding miR-148b-3p inhibitors, the expression of the SLC7A11 gene was increased, inhibiting ferroptosis in tumor cells.

[0032] Furthermore, the nucleotide sequence of the miR-148b-3p inhibitor is shown in SEQ ID NO.3.

[0033] Furthermore, miR-148b-3p analogs or miR-148b-3p inhibitors were added to tumor cells using transfection reagents.

[0034] Furthermore, the tumor cells include any one or more combinations of breast cancer cells, colon cancer cells, or liver cancer cells.

[0035] Furthermore, the tumor cells include breast cancer cells.

[0036] In another aspect, the present invention provides the use of a miR-148b-3p inhibitor in the preparation of an agent that regulates ferroptosis in tumor cells, wherein the nucleotide sequence of the miR-148b-3p inhibitor is shown in SEQ ID NO.3.

[0037] Furthermore, by adding a miR-148b-3p inhibitor, the expression of the SLC7A11 gene is increased, thereby inhibiting ferroptosis in tumor cells; the tumor cells include any one or more combinations of breast cancer cells, colon cancer cells, or liver cancer cells.

[0038] The beneficial effects of this invention are as follows:

[0039] 1. This invention provides a miRNA, miR-148b-3p, that targets and regulates the role of SLC7A11 in breast cancer, along with its mechanism of action and applications. It clarifies for the first time the regulatory effect of miR-148b-3p on breast cancer cells and experimentally verifies that miR-148b-3p can inhibit the proliferation and migration of breast cancer cells (BRCA) by targeting and regulating SLC7A11. In cell experiments, transfection with a miR-148b-3p inhibitor significantly increased the mRNA and protein levels of SLC7A11, while also significantly enhancing the proliferation activity and migration rate of breast cancer cells. Conversely, overexpression of miR-148b-3p inhibited breast cancer cell viability by 35.7% and breast cancer cell migration by 61.5%, indicating that miR-148b-3p has a good inhibitory effect on breast cancer cells.

[0040] 2. This invention, through a series of studies including the construction of ceRNA networks, reveals for the first time that the mechanism of action of SLC7A11 in breast cancer is related to the regulation of miR-148b-3p, providing a new perspective for a deeper understanding of the mechanisms of breast cancer development and progression. Furthermore, since miR-148b-3p can regulate SLC7A11 and thus affect the biological behavior of breast cancer cells, and SLC7A11 plays an important role in breast cancer (e.g., it is positively correlated with tumor immune cell infiltration, and high expression is significantly associated with poor prognosis), miR-148b-3p and its regulated SLC7A11 may become potential targets for breast cancer treatment, providing a theoretical basis for developing new breast cancer treatment strategies.

[0041] 3. This invention, through research on other cancers (such as colon cancer and liver cancer), found that miR-148b-3p can also influence the biological behavior of tumor cells in these cancers by targeting and regulating SLC7A11. However, compared with breast cancer, its regulatory effect on these cancers is relatively weak, including its influence on the regulation of SLC7A11 gene and protein expression, cell proliferation, and migration. This further highlights that miR-148b-3p may have a relatively specific regulatory role in breast cancer, providing a reference for precision treatment of different cancers. Attached Figure Description

[0042] Figure 1A represents the RNA-seq expression level of SLC7A11 in normal tissues.

[0043] Figure 1 B represents the protein expression level of SLC7A11 in normal tissue.

[0044] Figure 2 A is a box plot showing the mRNA expression levels of SLC7A11 in normal and cancerous human tissues, derived from data from the Cancer Genome Atlas (TCGA) database. Tumor tissues are represented by red dots and boxes, while normal tissues are represented by blue dots and boxes.

[0045] Figure 2 B represents a comprehensive analysis of SLC7A11 expression in tumor and healthy tissues using the TCGA and genotype-tissue expression (GTEx) datasets. The symbols “-”, “*”, “**”, and “***” indicate no significance, P<0.05, P<0.01, and P<0.001, respectively.

[0046] Figure 3 To analyze SLC7A11 protein levels in primary tumors and normal tissues using UALCAN;

[0047] Figure 4 A is a forest plot used for pan-cancer analysis of SLC7A11 with overall survival (OS), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI). *P<0.05, **P<0.01, ***P<0.001.

[0048] Figure 4 B is a Venn diagram showing significant differences in prognosis among OS, DSS, DFI, and PFI for high SLC7A11 expression.

[0049] Figure 4 C represents the receiver operating characteristic (ROC) curve for pan-cancer diagnosis.

[0050] Figure 5 A represents a pan-cancer analysis of genomic variations in SLC7A11 from the Cancer Genome Atlas (TCGA) database, including mutation, amplification, and deep deletion analysis.

[0051] Figure 5 B represents the distribution of SLC7A11 gene mutations in pan-cancer.

[0052] Figure 5 C represents SLC7A11 single nucleotide variants (SNVs), including missense, truncation, and splice site mutations.

[0053] Figure 5D is a lollipop plot used to visualize the correlation between SLC7A11 levels and tumor mutational burden (TMB), microsatellite instability (MSI), and homologous recombination deficiency (HRD);

[0054] Figure 6 Kaplan-Meier curves generated using the TIDE tool were used to assess the prognostic association between SLC7A11 copy number variants (CNVs) and survival. Red line: SLC7A11 with copy number variants; Blue line: SLC7A11 without copy number variants.

[0055] Figure 7 A shows the association between SLC7A11 and four mismatch repair (MMR) genes in different cancer types using a heatmap.

[0056] Figure 7 B uses a lollipop plot to show the correlation between SLC7A11 levels and mRNA si stemness, where the size of the dots is proportional to the sample size, and the color represents the p-value.

[0057] Figure 7 C shows the correlation between pan-oncology RNA regulation and SLC7A11 levels in the form of a heatmap. *P<0.05, **P<0.01, ***P<0.001.

[0058] Figure 8 Kaplan-Meier curves were used to analyze the prognostic association between SLC7A11 methylation and survival. Red line: methylated SLC7A11; Blue line: unmethylated SLC7A11.

[0059] Figure 9 The correlation between SLC7A11 expression and 60 immune checkpoint pathway genes in different cancers was investigated.

[0060] Figure 10 A represents the correlation coefficient between SLC7A11 expression and six types of tumor-infiltrating immune cells (TIIC) (B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells) in different tumor patients.

[0061] Figure 10 B represents the three types of tumors that showed the most significant correlation with SLC7A11 expression in terms of stromal score, immune score, and estimate score.

[0062] Figure 11 The association between SLC7A11 expression and tumor purity.

[0063] Figure 12A represents the relationship between SLC7A11 expression and the number of immunosuppressive cells in breast cancer (BRCA).

[0064] Figure 12 B represents the relationship between SLC7A11 and tumor immune infiltration in breast cancer (BRCA).

[0065] Figure 12 C represents the correlation between SLC7A11 expression levels and gene markers of exhausted T cells in breast cancer (BRCA).

[0066] Figure 12 D represents the relationship between different expression levels of SLC7A11 in breast cancer (BRCA) and prognosis.

[0067] Figure 12 E represents the relationship between anti-PD-L1 therapy and prognosis in breast cancer (BRCA).

[0068] Figure 13 miRNAs that interact with SLC7A11 in breast cancer A (BRCA).

[0069] Figure 13 Real-time quantitative polymerase chain reaction (RT-PCR) data showed that the miR-148b-3p inhibitor significantly reduced miR-148b-3p mRNA expression. U6 was used as an internal control for miR-148b-3p expression levels.

[0070] Figure 13 C represents real-time quantitative polymerase chain reaction (RT-PCR) data, indicating that the miR-148b-3p inhibitor significantly reduced miR-148b-3p expression. U6 was used as an internal control for miR-148b-3p expression levels.

[0071] Figure 13 Data from real-time quantitative polymerase chain reaction (RT-PCR) showed that the miR-148b-3p inhibitor significantly reduced SLC7A11 mRNA expression. β-actin was used as an internal control for SLC7A11 expression levels.

[0072] Figure 13 E represents the use of anti-SLC7A11 antibody to verify the expression of SLC7A11 by Western blotting.

[0073] Figure 13 F represents the transfection of MDA-MB-231 cells with either the miR-148b-3p inhibitor or the control. Cell proliferation was assessed using a CCK-8 assay. MDA-MB-231 cells transfected with the miR-148b-3p inhibitor were collected after 48 hours.

[0074] Figure 13 G represents the migration of MDA-MB-231 cells as detected using a Transwell assay. MDA-MB-231 cells transfected with the miR-148b-3p inhibitor were collected after 48 hours.

[0075] Figure 14A is a heatmap showing the correlation between SLC7A11 and ferroptosis biomarkers.

[0076] Figure 14B The graph shows the correlation between miR-148b-3p and ferroptosis biomarkers.

[0077] Figure 14C is a chord diagram showing the association between SLC7A11 and ferroptosis marker genes.

[0078] Figure 14D Heatmap showing the differential expression of ferroptosis markers in the SLC7A11 high-expression and low-expression groups in breast cancer.

[0079] Figure 14E shows the change in MDA levels after transfection with the miR-148b-3p inhibitor (under RSL3-induced ferroptosis).

[0080] Figure 14F shows the changes in GSH levels in cells after transfection with the miR-148b-3p inhibitor (under RSL3-induced ferroptosis).

[0081] Figure 14G This image shows the change in viability of MDA-MB-231 cells after transfection with the miR-148b-3p inhibitor (under RSL3-induced ferroptosis).

[0082] The data above represent the mean ± standard deviation of three biological replicates. *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0083] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0084] Example 1: The role and validation of SLC7A11 in pan-cancer and breast cancer

[0085] SLC7A11, a member of the solute transporter family, is a multi-pathway transmembrane protein that mediates the uptake of extracellular cystine and the exchange of glutamate. Cystine is reduced to cysteine, the rate-limiting precursor for glutathione synthesis, a process crucial in cell growth, proliferation, and metabolism. As an amino acid transporter, the SLC7A11-GSH system is a key cellular mechanism for combating ferroptosis. Inhibition of SLC7A11 expression disrupts the cystine metabolic pathway, leading to decreased intracellular cystine levels and depletion of glutathione biosynthesis, indirectly inhibiting glutathione peroxidase 4 activity, resulting in lipid peroxide accumulation and ultimately inducing ferroptosis. SLC7A11 also plays an important role in a newly discovered form of cell death—disulfide death. Furthermore, an increasing number of studies use various SLC7A11 modulators to regulate its expression or activity to influence ferroptosis, thereby affecting the tumor microenvironment, immune escape, tumor progression, chemotherapy resistance, and targeted cancer therapy. Numerous studies have shown that SLC7A11, a core subunit of SystemXc, is overexpressed in various tumors, and its expression level is closely related to tumor cell proliferation, invasion, metastasis, and the tumor microenvironment. However, there is still very little systematic research on the role of SLC7A11 in pan-cancer.

[0086] Therefore, in this embodiment, a systematic pan-cancer analysis is performed to determine the full picture of SLC7A11 in various cancers, and potential cancer treatment strategies are formulated through a competitive endogenous RNA (ceRNA) network targeting SLC7A11. Simultaneously, the specific role of SLC7A11 in breast cancer (BRCA) is verified.

[0087] The specific procedures and corresponding experimental results are as follows (the statistical analysis in this study was automatically calculated by the above online database. All experimental data are expressed as the mean ± standard deviation of three independent biological experiments. GraphPadPrism 8.0 was used for statistical analysis of the experimental data. Student's t-test was used to compare differences. A p-value <0.05 or a log-rank p <0.05 was considered statistically significant):

[0088] I. Differential expression of SLC7A11 in normal and tumor samples

[0089] 1. Experimental Procedure

[0090] (1) Differential analysis of SLC7A11 mRNA expression

[0091] SLC7A11 mRNA expression data from normal human tissues (including large tissue blocks and single tissues) were downloaded from the GTEx database (https: / / gtexportal.org / home / ). To compare the expression levels of SLC7A11 in normal tissues and pan-cancer tissues from the TCGA database, the inventors used the TIMER 2.0 online tool (http: / / timer.comp-genomics.org / ) and performed statistical analysis and graphical visualization of normalized gene expression data of SLC7A11 in cancerous and normal tissues using data from the TCGA, GTEx, and UCSC Xena databases. Furthermore, normalized pan-cancer datasets TCGA, TARGET, and GTEx (PANCAN, N=19131) were downloaded from the UCSC database (https: / / xenabrowser.net / ). SLC7A11 was extracted.

[0092] Expression data for SLC7A11 (ENSG00000151012) were collected. The expression data of the SLC7A11 gene in each sample were categorized into normal solid tissue, primary solid tumor, primary tumor, normal tissue, primary blood-derived cancer (bone marrow), primary blood-derived cancer (peripheral blood), and primary tumor. The data were standardized by a log2(x+0.001) transformation. Cancer types with fewer than three samples were excluded, resulting in expression data for 34 cancer types. Differential expression analysis of SLC7A11 was performed using the R package "limma". A p-value <0.05 was considered statistically significant.

[0093] (2) Differential analysis of SLC7A11 protein expression

[0094] The protein levels of SLC7A11 in various cancer types (including breast cancer (BRCA), colorectal cancer (COAD), glioblastoma (GBM), head and neck squamous cell carcinoma (HNSCC), clear cell renal cell carcinoma (KIRC), liver cancer (LIHC), lung adenocarcinoma (LUAD), and ovarian cancer (OV)) were analyzed using data from the Clinical Proteomics Tumor Analysis Consortium (CPTAC). Furthermore, the differential expression of SLC7A11 protein in lung squamous cell carcinoma, head and neck squamous cell carcinoma, and glioblastoma was investigated by comparing tumor tissues with adjacent normal tissues using the UALCAN cancer database (http: / / ualcan.path.uab.edu).

[0095] 2. Experimental Results:

[0096] The expression levels of SLC7A11 ribonucleic acid (RNA) and protein differ in normal tissues. According to genotype-tissue expression (GTEx) data, SLC7A11 mRNA levels vary among normal tissues. SLC7A11 protein expression is significantly higher in the frontal cortex and spinal cord of the nervous system, as well as in the heart and lung tissues. Figure 1 ).

[0097] To investigate SLC7A11 mRNA levels between cancerous and normal tissues, the inventors downloaded SLC7A11 mRNA expression data from the Cancer Genome Atlas (TCGA) and GTEx databases. The results showed differences in SLC7A11 expression across 34 cancer types. The data indicated that, except for bladder cancer (BLCA) and acute lymphoblastic leukemia (ALL), SLC7A11 mRNA levels were significantly upregulated in 88.24% (30 / 34) of tumors and significantly downregulated in 5.88% (2 / 34) of tumors. Figure 2 Protein expression data from the UALCAN database showed that SLC7A11 protein levels were upregulated in head and neck squamous cell carcinoma and lung squamous cell carcinoma, while significant downregulation was observed in glioblastoma multiforme. Figure 3 ).

[0098] 3. Experimental Conclusions

[0099] Therefore, it can be seen that the expression of SLC7A11 in normal tissues is tissue-specific, and the expression levels of its RNA and protein differ in normal tissues; at the same time, the expression of SLC7A11 in different tumor tissues also shows significant differences.

[0100] II. Prognostic Analysis of SLC7A11

[0101] 1. Experimental Procedure

[0102] The inventors obtained SLC7A11 expression data from the UCSC database and integrated consolidated survival data from TCGA covering 44 cancer types included in UCSC. They also evaluated four key outcome measures: overall survival (OS), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI), aiming to comprehensively explore the prognostic impact, predictive value, and potential diagnostic applications of SLC7A11 mRNA levels in each cancer type.

[0103] For survival analysis, a Cox proportional hazards regression model was built using the Coxph function to analyze the relationship between gene expression and prognosis in each tumor type. The log-rank test was used to statistically assess the prognostic significance. Furthermore, the Kaplan-Meier Plotter (http: / / kmplot.com / analysis / ) was used to investigate the association between SLC7A11 expression and overall survival (OS) and disease-free survival (DFS) in various tumor types. Kaplan-Meier curves generated using the TIDE tool were used to assess the prognostic association between SLC7A11 methylation and survival. Based on data from the TCGA database, ROC curves were used to assess the potential value of SLC7A11 in cancer diagnosis; an AUC > 0.5 was considered to have high diagnostic value.

[0104] 2. Experimental Results

[0105] Based on the TCGA and GTEx pan-cancer cohorts, SLC7A11 was evaluated as a prognostic predictor of overall survival (OS), disease-specific survival (DSS), progression-free interval (PFI), and disease-free interval (DFI). Univariate Cox regression analysis was performed. Figure 4 A) showed that high SLC7A11 expression was significantly associated with poor overall survival (OS) across 14 tumor types (HR > 1, p < 0.05). Further evaluation of the effects of SLC7A11 expression levels on disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI) revealed that high SLC7A11 expression was significantly associated with poor DSS in 12 cancer types (HR > 1, p < 0.05), with poor DFI in 4 cancer types, and with poor PFI in 11 cancer types (HR > 1, p < 0.05). The above results indicate that elevated SLC7A11 expression is generally associated with poorer prognosis in six cancer types, including KIPAN, chromophobe renal cell carcinoma (KIRP), adrenocortical carcinoma (ACC), hepatocellular carcinoma (LIHC), clear cell renal cell carcinoma (KICH), and breast cancer (BRCA). Figure 4 B). Furthermore, the potential diagnostic value of SLC7A11 in cancer was evaluated using receiver operating characteristic (ROC) curve analysis. The ROC curves indicate that SLC7A11 could serve as a diagnostic biomarker for certain cancers, such as KICH, KIRP, and esophageal squamous cell carcinoma (ESCA). Figure 4 C).

[0106] 3. Experimental Conclusions

[0107] Therefore, it can be seen that high expression of SLC7A11 is associated with poor prognosis in various cancers, indicating that SLC7A11 may be a potential risk factor for poor prognosis in these cancers. It also suggests that SLC7A11 may be a potential risk factor for poor prognosis in the aforementioned cancers and has certain application prospects in the early diagnosis of different cancers. The detection of its expression level is helpful for cancer screening and diagnosis.

[0108] III. Genetic Analysis of SLC7A11 in Cancer

[0109] 1. Experimental Procedure

[0110] The inventors used the cBioPortal platform (https: / / www.cbioportal.org / ) and the UALCAN portal (http: / / ualcan.path.uab.edu / index.html / ) to examine the location, type, and mutation distribution of SLC7A11 gene variants. The SangerBox website was used to investigate the relationship between SLC7A11 expression and microsatellite instability (MSI), tumor mutational burden (TMB), and homologous recombination deficiency (HRD) in different cancers within the TCGA cohort. Pearson rank correlation tests were performed to generate partial correlation coefficients (cor) and p-values.

[0111] 2. Experimental Results

[0112] (1) Genomic analysis and clinical significance of SLC7A11 in cancer

[0113] Genomic strategies provide a powerful tool for cancer analysis. When TIDE analysis was performed based on SLC7A11 copy number variation (CNV) levels, patients with lower SLC7A11 CNV levels in clear cell renal cell carcinoma (KIRC) had a better prognosis, while the opposite was true in head and neck squamous cell carcinoma (HNSC). Figure 5 A, Figure 6 To assess the potential genomic alterations of SLC7A11 in specific cancers, the inventors examined its mutational status across various tumors. A total of 83 missense mutations, 13 truncation mutations, and 1 splice mutation were found in SLC7A11. Figure 5C). Frameshift deletions of SLC7A11 are primarily detected in gastric adenocarcinoma (STAD), gastroesophageal junction adenocarcinoma (STES), low-grade glioma (LGG), and glioblastoma-low-grade glioma (GBMLGG), while nonsense mutations are observed only in urinary system cancers (UCS). Missense mutations are the predominant type and are widely distributed across various tumor types. Endometrial cancer (UCEC), colon cancer (COAD), and colon cancer-rectal adenocarcinoma (COADREAD) have relatively high mutation frequencies. Figure 5 B). Microsatellite instability (MSI) status and tumor mutational burden (TMB) are currently considered promising predictive biomarkers for immunotherapy efficacy, while homologous recombination deficiency (HRD) status is a key indicator of treatment options and prognosis for various cancers. This study experimentally evaluated the association between SLC7A11 and TMB, MSI, and HRD. A positive correlation was observed between SLC7A11 and TMB in adrenocortical carcinoma (ACC) and thymoma (THYM), and between SLC7A11 and MSI in colorectal cancer (COAD). Except for a positive correlation observed in chromophobe renal cell carcinoma (KIRP) and acute myeloid leukemia (LAML), no significant correlation was found between SLC7A11 and HRD. Figure 5 D). The above data indicate an association between SLC7A11 and genomic instability.

[0114] (2) SLC7A11 levels are closely related to DNA mismatch repair (MMR), tumor stemness, and methylation.

[0115] DNA mismatch repair (MMR) mechanisms are highly complex and aim to ensure genomic stability and integrity by detecting and correcting abnormal sequences and structures in chromosomes. Tumor cells frequently utilize MMR pathways to evade treatment and acquire self-renewal capabilities similar to stem cells. Therefore, this study investigated the potential association between SLC7A11 expression levels and MMR-related genes (such as PMS2, MSH6, MSH2, and EPCAM) and tumor stemness. In various cancers, including chromophobe renal cell carcinoma (KIRP), testicular germ cell tumor (TGCT), low-grade glioma (LGG), colorectal cancer (COAD), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), pancreatic cancer (PAAD), bladder cancer (BLCA), and pheochromocytoma and paraganglioma (PCPG), SLC7A11 expression is significantly negatively correlated with most MMR genes. Figure 7 A).

[0116] Furthermore, we calculated the correlation between the RNAss index and SLC7A11. We observed significant correlations across 16 tumor types, with a significant positive correlation in 12 types (including lung adenocarcinoma (LUAD), acute myeloid leukemia (LAML), breast cancer (BRCA), esophageal squamous cell carcinoma (ESCA), gastroesophageal junction adenocarcinoma (STES), renal chromophobe carcinoma (KIRP), KIPAN, gastric adenocarcinoma (STAD), prostate cancer (PRAD), lung squamous cell carcinoma (LUSC), bladder cancer (BLCA), and adrenocortical carcinoma (ACC)), and a significant negative correlation in 4 tumor types (including glioblastoma (GBM), glioblastoma-low-grade glioma (GBMLGG), low-grade glioma (LGG), and pheochromocytoma with paraganglioma (PCPG)). Figure 7 B). Epigenetic modifications play a crucial role in cancer development and progression, and have become a major research focus. Among these, methylation plays a key role in SLC7A11-mediated ferroptosis. We further analyzed the relationship between SLC7A11 and the expression of 44 marker genes involved in epigenetic modifications (such as m1A (10 genes), m5C (13 genes), and m6A (21 genes)). Figure 7 C). The results showed that SLC7A11 expression was positively correlated with m1A, m5C, and m6A-related genes in most tumors, particularly in cancers such as liver cancer (LIHC), ovarian cancer (OV), prostate cancer (PRAD), thyroid cancer (THCA), adrenocortical carcinoma (ACC), clear cell renal cell carcinoma (KIRC), and breast cancer (BRCA). Furthermore, in cancers such as cutaneous melanoma (SKCM), diffuse large B-cell lymphoma (DLBC), and glioblastoma-low-grade glioma (GBMLGG), the observation of SLC7A11 promoter methylation significantly improved patient survival. Figure 8 The above analysis reveals the important role of SLC7A11 in DNA methylation and mRNA modification in various cancer types.

[0117] 3. Experimental Conclusions

[0118] Therefore, it can be seen that SLC7A11 is associated with genomic instability and its mutation status varies in different cancers. At the same time, SLC7A11 expression is closely related to DNA mismatch repair, tumor stemness and methylation and has cancer type specificity.

[0119] IV. Correlation analysis of SLC7A11 expression with immunomodulatory genes and immune infiltration

[0120] 1. Experimental Procedure

[0121] The correlation between SLC7A11 expression levels and the levels of immune cell infiltration or immune checkpoint expression in pan-cancer was analyzed using TIMER (https: / / cistrome.shinyapps.io / timer / ). A p-value <0.05 was considered statistically significant. The association between SLC7A11 and chemokines, chemokine receptors, and major histocompatibility complex (MHC) molecules was analyzed using the TISIDB tool (cis.hku.hk / TISIDB / index.php). Furthermore, using the SangerBox website, the inventors explored the relationship between SLC7A11 expression and stromal scores, immune scores, estimate scores, and tumor-infiltrating immune cells (TIICs) in various cancers. These immune cells included B cells, CD4+ T memory cells, CD8+ T cells, NK cells, monocytes, macrophages, and neutrophils.

[0122] 2. Experimental Results

[0123] Given the crucial role of immune checkpoint gene expression in determining the efficacy of immunotherapy, this study investigated the potential correlation between SLC7A11 expression and 60 immune checkpoint pathway genes across various cancers. SLC7A11 showed a significant association with pan-cancer immunosuppressive and stimulatory genes, and most immune checkpoint pathway genes were positively correlated. Figure 9 ).

[0124] Simultaneously, using the TIMER database, the relationship between SLC7A11 expression levels and tumor-infiltrating immune cell (TIIC) infiltration levels in pan-cancer was assessed. The inventors evaluated and presented, in heatmap format, the correlation coefficients of six TIIC types (B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells) in each patient across different tumors. Figure 10 A). The most significant positive correlation between immune cell infiltration and SLC7A11 was found in clear cell renal cell carcinoma (KIRC), while the strongest negative correlation was observed in head and neck squamous cell carcinoma (HNSC). Significant changes in dendritic cell (DC) infiltration were observed in various malignancies, including clear cell renal cell carcinoma (KIRC), glioblastoma-low-grade glioma (GBMLGG), low-grade glioma (LGG), KIPAN, hepatocellular carcinoma (LIHC), pheochromocytoma and paraganglioma (PCPG), head and neck squamous cell carcinoma (HNSC), endometrial cancer (UCEC), breast cancer (BRCA), and lung squamous cell carcinoma (LUSC)).

[0125] In addition, the inventors also studied the relationship between SLC7A11 expression and tumor purity. Figure 11In testicular germ cell tumors (TGCT), gastroesophageal junction adenocarcinoma (STED), lung squamous cell carcinoma (LUSC), esophageal squamous cell carcinoma (ESCA), lung adenocarcinoma (LUAD), breast cancer (BRCA), and gastric adenocarcinoma (STAD), SLC7A11 expression was significantly positively correlated with tumor purity, while a significant negative correlation was observed in renal chromophobe cell carcinoma (KIRP) and renal clear cell carcinoma (KIRC). Using the ESTIMATE algorithm based on TCGA data, stromal scores, immune scores, and evaluation scores were calculated for relevant tumor samples, and the correlation between SLC7A11 expression and these scores was assessed. The strongest correlation between SLC7A11 and stromal scores was observed in gastroesophageal junction adenocarcinoma (STES), CGA-KIPAN, and pheochromocytoma and paraganglioma (PCPG). The strongest correlation between SLC7A11 and immune scores was found in head and neck squamous cell carcinoma (HNSC), thyroid carcinoma (THCA), and lung adenocarcinoma (LUAD). The highest correlation between SLC7A11 and assessment scores was found in gastroesophageal junction adenocarcinoma (STES), lung adenocarcinoma (LUAD), and thyroid carcinoma (THCA). Figure 10 B).

[0126] The above results indicate a close relationship between SLC7A11 expression, tumor purity, and TIIC invasion levels.

[0127] 3. Experimental Conclusions

[0128] Therefore, it can be seen that SLC7A11 plays an important role in immune regulation, and its effect on immune cell infiltration varies depending on the tumor type; at the same time, there is a close relationship between SLC7A11 expression and tumor purity and immune-related scores, and this relationship is tumor type specific.

[0129] V. Correlation analysis of SLC7A11 expression with immune characteristics and prognosis of breast cancer (BRCA)

[0130] In this embodiment, to further explore the mechanism of action of SLC7A11 in breast cancer and its relationship with immune characteristics and prognosis, the various correlations between SLC7A11 and tumor-infiltrating immune cells in breast cancer were also investigated. The TIMER2 database was used to study the correlation between SLC7A11 expression and the abundance of common immunosuppressive cells in breast cancer. Specifically, as follows... Figure 12As shown in Figure A, SLC7A11 expression was significantly positively correlated with the levels of CD8+ T cells (r = 0.07, p < 0.05), neutrophils (r = 0.21, p < 0.001), macrophages (r = 0.13, p < 0.001), and dendritic cells (r = 0.13, p < 0.001). This confirms that SLC7A11 expression is positively correlated with tumor immune cell infiltration in breast cancer (BRCA).

[0131] Furthermore, using the ImmuCellAI and xCell databases, the inventors further analyzed the relationship between SLC7A11 and tumor-infiltrating immune cells in breast cancer (BRCA). The study found that SLC7A11 was significantly negatively correlated with activated NK cells, plasma cells, follicular helper T cells, CD8+ T cells, regulatory T cells (Tregs), naive B cells, and resting mast cells. Conversely, it was significantly positively correlated with resting NK cells, activated dendritic cells, M1 macrophages, M0 macrophages, neutrophils, M2 macrophages, activated CD4+ memory T cells, and resting mast cells. Specific results are shown in Table 1.

[0132] Table 1. Correlation between SLC7A11 and immune-related cells in breast cancer (BRCA)

[0133]

[0134] Furthermore, this study also demonstrated a positive correlation between SLC7A11 expression levels in breast cancer (BRCA) and gene markers of exhausted T cells, including CD274 (PD-L1, r = 0.27, p < 0.001), CTLA4 (r = 0.1, p < 0.001), HAVCR2 (TIM-3, r = 0.213, p < 0.001), LAG3 (r = 0.097, p = 0.001), and TOX (r = 0.066, p = 0.029). Figure 12 B). The correlation between SLC7A11 and immune checkpoint inhibitory genes (including PD1, PD-L1, CTLA-4, and LAG-3) was further validated using the TIMER 2.0 database, among which PD-L1 (CD274) showed a significant positive correlation with SLC7A11. Figure 9 , Figure 12 C).

[0135] Further analysis of prognostic differences associated with SLC7A11 in breast cancer (BRCA) revealed that high expression of SLC7A11 was significantly associated with poor prognosis. Figure 12 D; HR = 1.63, p = 3.6e -5Furthermore, patients with high SLC7A11 expression who receive anti-PD-L1 therapy have a better prognosis. Figure 12 E; HR = 0.48, p = 1.5e -6 ).

[0136] Therefore, based on the role of SLC7A11 in prognosis and its correlation with immune infiltration and immune regulatory genes, SLC7A11 can serve as a biomarker for poor prognosis in breast cancer (BRCA) patients.

[0137] In summary, SLC7A11 expression in normal tissues exhibits tissue specificity, with differences in RNA and protein expression levels. Simultaneously, its expression in different tumor tissues also shows significant differences, with mRNA levels significantly upregulated in most tumors. High SLC7A11 expression is associated with poor prognosis in various cancers, potentially representing a risk factor for these diseases. It holds promise for early diagnosis in different cancers, and detecting its expression level can aid in cancer screening and diagnosis. SLC7A11 is associated with genomic instability, and its mutation patterns vary across different cancers. Simultaneous expression is closely related to DNA mismatch repair, tumor stemness, and methylation, exhibiting cancer type specificity. It plays a crucial role in immune regulation, and its impact on immune cell infiltration varies depending on tumor type. Simultaneous expression is closely related to tumor purity and immune-related scores, and this relationship is tumor type specific.

[0138] In breast cancer, SLC7A11 expression is positively correlated with tumor immune cell infiltration and exhibits specific correlations with various immune cells. Its expression level is positively correlated with gene markers of exhausted T cells and immune checkpoint inhibitor genes. High SLC7A11 expression is significantly associated with poor prognosis, and patients with high SLC7A11 expression who receive anti-PD-L1 therapy have better prognoses. Therefore, SLC7A11 can serve as a biomarker for poor prognosis in breast cancer (BRCA) patients and may regulate the immune microenvironment and disease progression in breast cancer by influencing immune cell infiltration and related gene expression.

[0139] Example 2: Investigating the mechanism of action of SLC7A11 in breast cancer

[0140] In Example 1, although a series of analyses clearly demonstrated that SLC7A11 expression in breast cancer was positively correlated with tumor immune cell infiltration, and that high expression was significantly associated with poor prognosis, the mechanism by which SLC7A11 regulates the biological behavior of breast cancer cells remained unclear. Therefore, in this example, the inventors constructed a ceRNA network to explore its mechanism of action.

[0141] I. Constructing a ceRNA network to predict miRNAs targeting SLC7A11

[0142] Upstream binding miRNAs of SLC7A11 were predicted using multiple target gene prediction programs, including PITA, RNA22, miRmap, microT, miRanda, PicTar, and TargetScan. Only predicted miRNAs that typically appeared in more than three of these programs were included in subsequent analyses. These predicted miRNAs were considered candidate miRNAs for SLC7A11. Long non-coding RNAs (lncRNAs) interacting with the screened miRNAs were predicted using the ENCORI platform (https: / / rna.sysu.edu.cn / encori / index.php) and the miRNet platform (https: / / www.mirnet.ca / ). Based on the ceRNA hypothesis, a ceRNA network was constructed by performing negative correlation analysis between mRNA and miRNA, RNA expression analysis, and overall survival analysis in the BRCA cohort.

[0143] Furthermore, the regulatory network of SLC7A11 in breast cancer (BRCA) was explored. First, using the ENCORI database, RNA22, DIANA-micro, miRWalk, miRcode, and TargetScan were used to identify miRNAs targeting SLC7A11. MiRNAs predicted from three or more databases were selected, resulting in 37 miRNAs. In addition, the experimentally validated miRTarbase database contained 102 SLC7A11-targeting miRNAs, and 11 miRNAs were identified as overlapping between the two datasets. Figure 13 A). Given the negative correlation between miRNA expression and ceRNA (mRNA, lncRNA, etc.) expression, the most significantly correlated miRNA was selected from the matched miRNAs: hsa-mir-148b-3p. Figure 13 B). Here, hsa-mir-148b-3p is identical to miR-148b-3p. The nucleotide sequence of miR-148b-3p contains a sense strand and an antisense strand, wherein the sense strand is shown in SEQ ID NO.1 and the antisense strand is shown in SEQ ID NO.2. Based on the sequence in SEQ ID NO.1, the sequence of the miR-148b-3p inhibitor was designed, and the nucleotide sequence of the miR-148b-3p inhibitor is shown in SEQ ID NO.3 (synthesized by Suzhou Jima Gene Co., Ltd.).

[0144] Therefore, the inventors initially predicted that miR-148b-3p may play a key role in the progression of breast cancer (BRCA) by targeting SLC7A11.

[0145] II. Cellular Experiment Validation: miR-148b-3p can inhibit the proliferation and migration of breast cancer cells (BRCA) by targeting and regulating SLC7A11.

[0146] To verify the above hypothesis, the following steps are performed:

[0147] 1. Cell culture and antibody preparation

[0148] MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM medium containing 10% fetal bovine serum (Glbco; Thermo Fisher Scientific) at 37°C in a humidified incubator containing 5% CO2. Mycoplasma testing using a mycoplasma detection kit (Biotool, USA) was negative. The following antibodies were used: SLC7A11 (A2413, Abclonal) and GAPDH (MB374, Millipore).

[0149] 2. CCK-8 Experiment

[0150] MDA-MB-231 cell proliferation was measured using the CCK-8 assay kit (C0042, Beyotime) according to the manufacturer's protocol. 2000 cells, with or without miR-148b-3p inhibitor, were seeded into each well of a 96-well plate and incubated for 1, 2, 3, and 4 days. At the specified time points, 10 μL of CCK-8 reagent was added to each well, and the plates were then incubated at 37°C with 5% CO2 for 1 hour. Absorbance was analyzed at 450 nm using a microplate reader (Bio-Rad Laboratories, USA), with cell-free wells serving as blank controls. Cell proliferation is expressed as absorbance. At least six control wells were included at each time point in all experiments. All experiments were performed in triplicate.

[0151] 3. Transwell experiment

[0152] The migration ability of MDA-MB-231 cells with and without miR-148b-3p inhibitor was assessed using a Transwell assay. 5000 cells / well were seeded into 24-well Transwell chambers (ECM508; Merck Millipore) and incubated at 37°C with 5% CO2 for 48 h. Migrating cells were stained with a cell staining reagent for 20 min and extracted with extraction buffer to measure the optical density at 560 nm. Images were captured using a Leica DM5000 microscope. Data are presented as mean ± standard deviation of three independent biological experiments.

[0153] 4. Results Analysis and Conclusions

[0154] (1) Silencing miR-148b-3p expression can target and regulate SLC7A11, increasing the mRNA and protein levels of SLC7A11.

[0155] MDA-MB-231 cells were collected 48 hours after transfection with the miR-148b-3p inhibitor.

[0156] Real-time quantitative PCR results showed that miR-148b-3p inhibitors significantly reduced miR-148b-3p expression. Figure 13 C). Silencing miR-148b-3p expression significantly increased the mRNA and protein levels of SLC7A11. Figure 13 D、 Figure 13 E) This indicates that miR-148b-3p can target and regulate SLC7A11, and by inhibiting the expression of miR-148b-3p, the mRNA and protein levels of SLC7A11 can be significantly increased.

[0157] (2) Silencing miR-148b-3p expression significantly increased the mRNA and protein levels of SLC7A11, thereby enhancing the proliferation and migration rate of breast cancer cells.

[0158] Cell viability at different proliferation stages was determined using the CCK-8 assay. The results showed that miR-148b-3p knockdown significantly enhanced cell viability compared to the miR-148b-3p inhibitor-negative control (NC) cells. Figure 13 F). Furthermore, cell migration rates measured using Transwell assays revealed that cells transfected with the miR-148b-3p inhibitor had significantly higher migration rates compared to the miR-148b-3p inhibitor-negative control (NC) cells. Figure 13 G).

[0159] Therefore, the above results indicate that miR-148b-3p inhibits the proliferation and migration of breast cancer cells by targeting and regulating SLC7A11, revealing that the mechanism of action of SLC7A11 in breast cancer is related to the regulation of miR-148b-3p. Furthermore, after 48 hours, overexpression of miR-148b-3p resulted in a 35.7% inhibition rate of breast cancer cell viability and a 61.5% inhibition rate of breast cancer cell migration, demonstrating a good inhibitory effect on breast cancer cells.

[0160] Example 3: Study on the mechanism by which miR-148b-3p regulates ferroptosis in breast cancer cells by targeting SLC7A11.

[0161] In Example 2, this invention explored the mechanism of action of SLC7A11 in breast cancer by constructing a ceRNA network and found that miR-148b-3p may play a key role in breast cancer progression by targeting SLC7A11. Furthermore, silencing miR-148b-3p expression can increase the mRNA and protein levels of SLC7A11, thereby enhancing the proliferation and migration rate of breast cancer cells. However, tumor cell proliferation, migration, and survival are closely related to cell death mechanisms. Ferroplasmosis, as a novel cell death mechanism that has received widespread attention in recent years, may play an important role in the occurrence and development of tumors. SLC7A11 is known to play an important role in breast cancer and is associated with multiple biological processes; changes in its expression not only affect tumor cell proliferation and migration but may also influence cell death mechanisms, particularly ferroptosis. Meanwhile, miR-148b-3p, as a key factor regulating SLC7A11, may regulate SLC7A11 expression beyond cell proliferation and migration, potentially involving cell death mechanisms, especially ferroptosis.

[0162] Therefore, based on this, this embodiment further investigates the mechanism by which miR-148b-3p regulates ferroptosis in breast cancer cells by targeting SLC7A11. A deeper analysis of this mechanism is expected to reveal another important regulatory pathway of miR-148b-3p and SLC7A11 in breast cancer cells, further enriching our understanding of the functions of miR-148b-3p and SLC7A11, and providing new targets and strategies for breast cancer treatment.

[0163] I. To illustrate the potential regulatory role of SLC7A11 in ferroptosis through correlation analysis.

[0164] To investigate the potential regulatory role of SLC7A11 in ferroptosis, a correlation heatmap between SLC7A11 and ferroptosis biomarkers was constructed, as shown in Figure 14A. Figure 14A displays the correlation heatmap between SLC7A11 and a series of ferroptosis biomarkers. The intensity of the color indicates the strength of the correlation, as explained by the color bar in the upper right corner. Genes with positive correlations (red) and negative correlations (blue) are shown in the figure, with significance indicated (**p<0.01). The figure shows that SLC7A11 is positively correlated with several ferroptosis-related genes, particularly CS, EMC2, and FANCD2. Furthermore, some genes showed negative correlations, such as GPX4 and ACSL4, which play important roles in ferroptosis regulation. These results suggest that SLC7A11 may play an important regulatory role in ferroptosis through its interaction with these ferroptosis-related genes.

[0165] II. Correlation analysis to illustrate the potential regulatory role of miR-148b-3p in ferroptosis

[0166] Meanwhile, to illustrate the potential regulatory role of miR-148b-3p in ferroptosis, this embodiment constructed a correlation analysis between miR-148b-3p and different ferroptosis biomarkers, and the specific results are as follows: Figure 14B As shown. Figure 14B The correlations between miR-148b-3p and different ferroptosis biomarkers were demonstrated. The most significant negative correlations were observed between miR-148b-3p and genes such as SLC7A11 and CARS1 (p<0.001), suggesting that miR-148b-3p may promote ferroptosis by inhibiting these genes. For biomarkers such as CS and EMC2, miR-148b-3p showed significant negative correlations, further supporting its potential role in ferroptosis regulation. This suggests that miR-148b-3p may influence the ferroptosis process through interactions with genes associated with these ferroptosis biomarkers (such as SLC7A11 and CARS1).

[0167] III. Illustrating the Complex Regulatory Network of SLC7A11 in Ferric Death by Constructing Correlation String Diagrams

[0168] To illustrate the complex regulatory network of SLC7A11 in ferroptosis, this embodiment also constructed association chord diagrams between SLC7A11 and different ferroptosis biomarkers, as shown in Figure 14C. Figure 14C shows the association chord diagrams between SLC7A11 and different ferroptosis biomarkers. Red lines represent positive correlations, and blue lines represent negative correlations. It can be seen that SLC7A11 has complex positive and negative correlations with multiple ferroptosis-related genes, particularly negative correlations with GPX4 and CARS1, while showing positive correlations with some other genes such as CS and FANCD2. This complex network relationship demonstrates the key regulatory role of SLC7A11 in ferroptosis, potentially achieving its function by influencing multiple molecular pathways. This further emphasizes the importance and complexity of SLC7A11 in ferroptosis regulation.

[0169] IV. Demonstrating the Regulatory Role of SLC7A11 in Ferric Death Biomarkers through Differential Expression Analysis

[0170] Furthermore, to illustrate the regulatory role of SLC7A11 in ferroptosis biomarkers, this embodiment also included a differential expression analysis of ferroptosis biomarkers between the SLC7A11 high-expression group and the low-expression group in breast cancer. Specific results are as follows: Figure 14D As shown. Figure 14DA heatmap showing the differential expression of ferroptosis biomarkers between SLC7A11 high-expression and low-expression groups in breast cancer is presented. The populations of upregulated and downregulated genes are significantly different (labeled "UP" and "DOWN"). For example, genes such as ALOX15, SLC1A5, and CARS1 are significantly upregulated in the SLC7A11 high-expression group, while genes such as ACSL4 and GPX4 are upregulated in the low-expression group, indicating that SLC7A11 expression may affect the ferroptosis status of cancer cells by regulating the expression of these ferroptosis-related genes. This provides further evidence supporting the role of SLC7A11 in the regulation of ferroptosis.

[0171] V. Verification Experiment on the Effect of miR-148b-3p on RSL3-Induced Ferric Death by Modulating SLC7A11

[0172] Meanwhile, to illustrate the effect of miR-148b-3p on RSL3-induced ferroptosis by regulating SLC7A11, a functional verification experiment was also conducted in this embodiment to demonstrate the effect of miR-148b-3p on RSL3-induced ferroptosis by regulating SLC7A11. The specific results are shown in Figure 14E-. Figure 14G As shown in the figure. Among them, RSL3 is a commonly used inducer in the field of ferroptosis research.

[0173] Figure 14E shows the changes in malondialdehyde (MDA) levels, a product of lipid peroxidation. After transfection with the miR-148b-3p inhibitor, the MDA level in the inhibitor + RSL3 group was significantly lower than that in the Control + RSL3 group, indicating that miR-148b-3p inhibition weakens RSL3-induced lipid peroxidation, thereby inhibiting ferroptosis. Previous studies have also shown that miR-148b-3p exerts its effects by targeting SLC7A11, which plays a crucial role in ferroptosis regulation and has complex correlations with multiple ferroptosis-related genes. When miR-148b-3p inhibits SLC7A11, it may alter SLC7A11's regulation of these related genes, enhancing its role in promoting lipid peroxidation, thus leading to increased MDA levels and promoting ferroptosis.

[0174] Figure 14F shows the changes in cellular glutathione (GSH) levels after transfection with a miR-148b-3p inhibitor. The results show that miR-148b-3p inhibition significantly increased cellular GSH levels, suggesting that miR-148b-3p may mediate ferroptosis by regulating GSH metabolism. SLC7A11 is also associated with GSH metabolism-related genes, showing positive or negative correlations with some genes affecting GSH synthesis or transport. Inhibition of SLC7A11 by miR-148b-3p may have interfered with SLC7A11's regulation of these genes, thereby affecting GSH metabolism and ultimately leading to decreased GSH levels and promoted ferroptosis.

[0175] Figure 14G This study demonstrates changes in cell viability. Transfection with a miR-148b-3p inhibitor significantly increased the viability of MDA-MB-231 cells, indicating that miR-148b-3p inhibition simultaneously suppresses ferroptosis and improves cell survival. SLC7A11 is associated with genes related to cell survival and may influence cell viability by regulating genes affecting cell proliferation, apoptosis, or metabolism. When miR-148b-3p inhibits SLC7A11, it alters SLC7A11's regulation of these related genes, promoting ferroptosis and thus reducing cell viability.

[0176] Therefore, it can be concluded that miR-148b-3p significantly affects the expression of ferroptosis-related genes by targeting and regulating SLC7A11, thereby influencing the ferroptosis process in breast cancer cells. Specifically, inhibiting miR-148b-3p increases GSH levels in tumor cells, decreases MDA content, and increases cell survival, indicating that miR-148b-3p can regulate the survival of breast cancer cells by modulating the ferroptosis-related gene SLC7A11.

[0177] In summary, miR-148b-3p can target and regulate SLC7A11. Increasing miR-148b-3p expression inhibits SLC7A11 expression, thereby promoting ferroptosis. This ferroptosis further suppresses the proliferation and migration of breast cancer cells. This discovery provides a new perspective for breast cancer treatment; targeting miR-148b-3p may become a novel therapeutic approach, inhibiting tumor growth by activating ferroptosis. It also provides a solid theoretical basis for further research on the biological behavior of breast cancer cells, offers crucial data support for research on the regulation of ferroptosis in breast cancer, and provides important new clues for future therapeutic targets.

[0178] Example 4: Study on the regulatory role of miR-148b-3p in other cancers

[0179] In Example 1, it was clearly demonstrated that SLC7A11 exhibits differential expression in various cancers and is closely related to processes such as tumor occurrence, development, prognosis, and immune regulation. Meanwhile, Example 2 verified that miR-148b-3p can inhibit breast cancer cell proliferation and migration by targeting and regulating SLC7A11. Building on this, further exploration of the regulatory role of miR-148b-3p in other cancers will contribute to a comprehensive understanding of its functional mechanisms in tumorigenesis and development. Furthermore, by comparing the regulatory effects of miR-148b-3p on SLC7A11 in breast cancer and other cancers, as well as its impact on tumor cell proliferation, migration, and other biological behaviors, it is further illustrated that miR-148b-3p's regulatory effect on SLC7A11 is most significant in breast cancer.

[0180] I. Experimental Materials and Methods

[0181] 1. Cell line selection

[0182] Colorectal cancer cell line (HT-29) and liver cancer cell line (HepG2) were selected. Previous studies have shown that certain biological characteristics of these cell lines (such as cell proliferation) are positively correlated with the mRNA and protein expression of SLC7A11.

[0183] 2. Cell Culture and Processing

[0184] The cell lines described above were cultured in DMEM medium containing 10% fetal bovine serum in a humidified incubator at 37°C with 5% CO2. CCK-8 and Transwell assays were performed, and real-time quantitative PCR was used to detect the expression of miR-148b-3p, the mRNA and protein levels of SLC7A11, and the proliferation and migration rates of colon and liver cancer cells (the specific experimental procedures were the same as those for breast cancer cells in Example 2).

[0185] II. Results Analysis and Conclusions

[0186] (1) Silencing miR-148b-3p expression can target and regulate SLC7A11, increasing the mRNA and protein levels of SLC7A11, but the increase is relatively weak.

[0187] Real-time quantitative PCR results showed that in the colon cancer cell line HT-29 and the liver cancer cell line HepG2, miR-148b-3p inhibitors reduced miR-148b-3p expression. Silencing miR-148b-3p expression increased the mRNA and protein levels of SLC7A11 compared to the negative control (NC) group, but the increase was less than that in breast cancer cell lines. This indicates that although miR-148b-3p can also target and regulate SLC7A11 in the colon cancer cell line HT-29 and the liver cancer cell line HepG2, increasing the mRNA and protein levels of SLC7A11 by inhibiting miR-148b-3p expression, the regulatory effect on its mRNA and protein levels is relatively weaker compared to that in breast cancer cells.

[0188] (2) Silencing miR-148b-3p expression significantly increased the mRNA and protein levels of SLC7A11, thereby enhancing the proliferation and migration rate of colon cancer cells and liver cancer cells, but the enhancement effect was relatively weak.

[0189] In the colon cancer cell line HT-29, transfection with a miR-148b-3p inhibitor and subsequent Transwell assays revealed an increase in cell migration compared to the negative control (NC) group, but the increase was less significant than in breast cancer cell lines, approximately 15% of the increase observed in breast cancer cell lines. Similarly, CCK-8 assays showed that transfection with the miR-148b-3p inhibitor enhanced cell proliferation in the HT-29 colon cancer cell line compared to the NC group, but the enhancement was also less pronounced, approximately 10% of the increase observed in breast cancer cell lines.

[0190] In addition, the HepG2 liver cancer cell line also showed a similar trend of proliferation and migration changes, namely, the cell proliferation activity and migration were enhanced after transfection with miR-148b-3p inhibitor, but not as obvious as those of breast cancer cell lines.

[0191] Therefore, it can be concluded that miR-148b-3p can also influence the biological behavior of tumor cells in other cancers (such as colon cancer and liver cancer) by targeting and regulating SLC7A11. However, compared with breast cancer, its regulatory effect on these cancers is relatively weak, including its influence on the regulation of SLC7A11 gene and protein expression, cell proliferation, and migration. This suggests that miR-148b-3p may have a relatively specific regulatory role in breast cancer, further highlighting its importance in breast cancer research.

[0192] In this embodiment, a comparative study of miR-148b-3p-regulated ferroptosis was also conducted on different tumor cell types. The results showed that the inhibitory effect of miR-148b-3p on cell viability varied significantly among different tumor cell types. Specifically, in breast cancer cells, the inhibition rate reached approximately 73%, significantly higher than that in colon cancer cells and liver cancer cells. This indicates that the effect of miR-148b-3p-regulated ferroptosis on the viability of different tumor cells exhibits significant heterogeneity, which may be related to the intrinsic biological characteristics of different tumor cells and their differences in sensitivity to ferroptosis.

[0193] Furthermore, based on the above, it can be inferred that miR-148b-3p may have a relatively specific regulatory effect on breast cancer, a phenomenon that may be closely related to miR-148b-3p's regulation of ferroptosis. In breast cancer cells, miR-148b-3p's effect of inhibiting cell activity by regulating ferroptosis is most significant, resulting in a stronger impact on the cell proliferation, migration, and other biological behaviors of breast cancer cells under miR-148b-3p regulation. This difference in ferroptosis regulation may stem from differences between breast cancer cells and colon cancer cells and liver cancer cells in metabolic pathways, gene expression profiles, and cell membrane lipid composition. These differences further affect the activity of ferroptosis-related signaling pathways and the degree of cellular response to miR-148b-3p regulation.

[0194] In summary, we can conclude that miR-148b-3p can influence the biological behavior of tumor cells in various cancers, including colon cancer, liver cancer, and breast cancer, by targeting and regulating SLC7A11. However, its regulatory effect is more pronounced in breast cancer, particularly in regulating the activity of ferroptosis-inhibiting cells. This specificity suggests that the specific responses of different tumor types to miR-148b-3p should be fully considered in the diagnosis and treatment of tumors. This provides a theoretical basis for developing miR-148b-3p-based breast cancer-specific therapeutic strategies and also offers a reference example for studying similar specific regulatory mechanisms in other cancers.

[0195] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The use of a miRNA in the preparation of a formulation that regulates the proliferative activity or migration ability of tumor cells, characterized in that, The miRNA is miR-148b-3p, and the nucleotide sequence of miR-148b-3p is shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The use as described in claim 1, characterized in that, In the tumor cells, the gene associated with their proliferation activity or migration ability is the SLC7A11 gene; by regulating the expression level of miR-148b-3p, the expression of the SLC7A11 gene can be regulated, thereby regulating the proliferation activity or migration ability of tumor cells.

3. The use as described in claim 2, characterized in that, By upregulating or downregulating miR-148b-3p expression, the expression of the SLC7A11 gene can be inhibited or promoted, thereby reducing or increasing the proliferation and migration of tumor cells.

4. The use as described in claim 3, characterized in that, By adding miR-148b-3p analogues, the expression of the SLC7A11 gene is inhibited, thereby suppressing the proliferation and migration of tumor cells; by adding miR-148b-3p inhibitors, the expression of the SLC7A11 gene is increased, thereby enhancing the proliferation and migration of tumor cells.

5. The use as described in claim 4, characterized in that, The nucleotide sequence of the miR-148b-3p inhibitor is shown in SEQ ID NO.

3.

6. The use as described in claim 5, characterized in that, miR-148b-3p analogs or miR-148b-3p inhibitors are introduced into tumor cells using transfection reagents.

7. The use as described in claim 6, characterized in that, The tumor cells include any one or more combinations of breast cancer cells, colon cancer cells, or liver cancer cells.

8. The use as described in claim 7, characterized in that, The tumor cells include breast cancer cells.

9. The use of miR-148b-3p inhibitors in the preparation of agents that regulate the proliferative activity or migration ability of tumor cells, characterized in that, The nucleotide sequence of the miR-148b-3p inhibitor is shown in SEQ ID NO.

3.

10. The use as described in claim 9, characterized in that, By adding a miR-148b-3p inhibitor, the expression of the SLC7A11 gene is increased, thereby enhancing the proliferation and migration of tumor cells; the tumor cells include any one or more combinations of breast cancer cells, colon cancer cells, or liver cancer cells.

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