miR-937-5p targets SOX17 to modulate breast cancer cell cycle and cell proliferation through the Wnt signaling pathway.

Abstract

Breast cancer stands as one of the most prevalent cancers affecting women globally and represents a primary cause of cancer-related deaths worldwide. Prior bioinformatics and experimental investigations have suggested that miR-937-5p might function as a proto-oncogene in the context of breast cancer; however, a more in-depth understanding of its specific impacts and underlying molecular mechanisms is still required.

Gene Set Enrichment Analysis of Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology terms indicated a potential involvement of miR-937-5p in cell cycle progression and DNA replication processes. Experimental findings demonstrated that inhibiting miR-937-5p significantly reduced the growth of breast carcinoma cells and induced an accumulation of cells in the S-phase of the cell cycle. Concurrently, the protein levels of the proliferation marker ki-67 and key cell cycle regulatory proteins including Cyclin A2, Cyclin B1, CDK1, and Cyclin D1 were also diminished upon miR-937-5p inhibition.

Further investigation revealed that miR-937-5p can directly interact with and negatively regulate the expression of SOX17. Notably, the overexpression of SOX17 also resulted in a significant decrease in the proliferation of breast carcinoma cells and induced S-phase cell cycle arrest. Additionally, SOX17 overexpression led to reduced protein levels of ki-67, β-catenin, c-Myc, Cyclin A2, Cyclin B1, Cyclin D1, and CDK1. Importantly, the effects observed upon miR-937-5p modulation were found to be reversed by the overexpression of SOX17.

Keywords: breast cancer; miR-937-5p; SOX17; the Wnt signaling pathway; cell cycle

Introduction

Breast cancer is recognized as one of the most frequently diagnosed cancers and represents the leading cause of cancer-related mortality among women. Standard treatment approaches for breast cancer often involve a combination of surgical removal of the tumor, radiation therapy, hormone therapy, chemotherapy, and molecularly targeted therapies. Despite these available treatments, there remains a critical need to identify more effective therapeutic targets, particularly for aggressive subtypes of breast cancer such as triple-negative breast cancer, to improve treatment outcomes.

MicroRNAs, which are small non-coding RNA molecules approximately 22 nucleotides in length, function as post-transcriptional regulators and are involved in a multitude of cellular processes. The typical biogenesis of miRNAs involves the processing of primary miRNA transcripts, which fold into hairpin structures known as pre-miRNAs. These pre-miRNAs are then cleaved by two different RNase enzymes to produce a double-stranded RNA molecule of about 22 nucleotides, which includes the mature miRNA. Mature miRNA sequences can originate from both the 5′ and 3′ arms of the pre-miRNA hairpin, and these are designated as the miRNA-5p and -3p species, respectively. Research suggests that both the 3p and 5p species derived from the same precursor miRNA often coexist and can both be functionally active.

Furthermore, miRNAs originating from the same pre-miRNA molecule can exhibit distinct target specificities and, consequently, perform different biological functions. miRNAs exert their regulatory effects by binding to the 3′ untranslated region of their target messenger RNAs, thereby influencing various cellular processes such as cell proliferation, differentiation, migration, invasion, programmed cell death, and numerous other biological pathways. A number of miRNAs, including miR-141, miR-122, miR-526b, and miR-655, have been shown to exhibit abnormal expression patterns in breast carcinoma and to function either as oncogenes, promoting cancer development, or as tumor suppressors, inhibiting cancer progression. It has been reported that miR-937-5p is significantly implicated in the advancement of several diseases, including pulmonary carcinoma and colon carcinoma. In the context of breast cancer, studies have indicated that miR-937-5p can promote the proliferation and colony formation of MCF-7 breast cancer cells. Preliminary bioinformatics analyses conducted on data from The Cancer Genome Atlas Breast Cancer database also suggested that higher expression levels of both precursor miR-937 and mature miR-937-5p were associated with poorer overall survival in breast cancer patients. Collectively, these findings suggest that miR-937-5p may play a role in promoting breast cancer development; however, a more detailed understanding of its specific effects and the underlying molecular mechanisms is still needed.

In the current study, publicly available gene expression data from the Gene Expression Omnibus and The Cancer Genome Atlas, along with quantitative real-time polymerase chain reaction, were employed to further validate the expression patterns of miR-937-5p in breast carcinoma tissues and cell lines. Prior to investigating the specific impacts of miR-937-5p on the characteristics of breast carcinoma cells, Gene Set Enrichment Analysis of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways was performed on genes whose expression levels were correlated with miR-937 expression. This analysis aimed to identify the signaling pathways in which miR-937 might be involved. Based on the results of these enrichment analyses, subsequent investigations focused on elucidating the roles of miR-937-5p in the proliferation of breast cancer cells and the regulation of cell cycle components.

Regarding the downstream molecular mechanisms potentially involved, the Wnt/β-catenin signaling pathway is well-established for its role in regulating the cell cycle. Consequently, SOX17, a known antagonist of the Wnt/β-catenin pathway, garnered our interest. The expression levels of SOX17 were examined in tissue samples and cell lines, the predicted interaction between miR-937-5p and SOX17 was experimentally validated, and the effects of SOX17 on breast cancer cell proliferation and cell cycle regulators were also investigated. Finally, the dynamic interplay between miR-937-5p and SOX17 on breast carcinoma cell characteristics was examined to determine whether miR-937-5p exerts its effects through the modulation of SOX17.

Materials and methods Tissue sampling

Twelve pairs of breast cancer tissue samples and adjacent non-cancerous tissue samples were collected from patients who had been diagnosed with breast cancer and underwent surgical resection at the Second Xiangya Hospital. Experienced clinical pathologists conducted histological diagnosis and confirmation of the breast cancer samples. The collection of clinical samples was performed with the approval of the Ethics Committee of the Second Xiangya Hospital, and written informed consent was obtained from all participating patients.

Pathological analysis using Hematoxylin and eosin staining

Collected tissue specimens were fixed in a 10% neutral buffered formalin solution for a period of 24 hours. Following fixation, the tissues were embedded in paraffin wax and then sliced into thin sections with a thickness of 4 micrometers. These sections were subsequently processed for Hematoxylin and eosin staining. The pathological characteristics of both the breast cancer tissues and the adjacent non-cancerous tissues were then examined using a microscope.

Polymerase chain reaction-based analyses

The expression levels of both microRNAs and messenger RNAs were determined after extracting the total RNA from the designated tissue samples or cells. These extractions were performed following established protocols using an SYBR Green PCR Master Mix. The expression of RNU6B was used as an internal control for microRNA quantification, while the expression of GAPDH was used as an internal control for messenger RNA quantification. The resulting data were analyzed using the 2-ΔΔCT method to determine relative expression levels.

Normal and breast cancer cell lines

A human non-tumorigenic epithelial cell line, designated as MCF-10A, was obtained from the American Type Culture Collection and cultured using a specific Mammary Epithelial Cell Growth Medium Kit. This MCF-10A cell line served as a non-tumorigenic control in the experiments. Several breast cancer cell lines were also used in this study. The MCF-7 cell line was obtained from the American Type Culture Collection and cultured in Eagle’s Minimum Essential Medium. Another breast cancer cell line, MDA-MB-231, was also obtained from the American Type Culture Collection and cultured in Leibovitz’s L-15 Medium. The T47D breast cancer cell line was purchased from the American Type Culture Collection and cultured in RPMI-1640 medium. Finally, the BT-474 breast cancer cell line was purchased from the American Type Culture Collection and cultured in Hybri-Care Medium. All the aforementioned cell lines were maintained in their respective culture media supplemented with 10% fetal bovine serum and incubated at a temperature of 37 degrees Celsius in an atmosphere containing 5% carbon dioxide.

Cell transfection

The inhibition of miR-937-5p was achieved by introducing antagomir-937-5p into the target cells, with antagomir-NC serving as a control. Overexpression or silencing of SOX17 was accomplished by introducing a SOX17-overexpressing plasmid or small interfering RNA targeting SOX17 into the cells, respectively. Control groups received an empty vector or a non-targeting small interfering RNA. All transfection procedures were carried out using a specific transfection reagent.

Cell counting kit-8 assay for detecting cell viability

Following transfection, cells were seeded into 96-well plates at a density of 2 × 103 cells per well. Subsequently, the viability of the cells in each experimental group was assessed using a cell counting kit according to the provided instructions. The absorbance at 450 nm was measured for each group using a microplate reader.

Colony formation assay

Transfected MDA-MB-231 and BT-474 cells were cultured in 6-well plates at a density of 2 × 102 cells per well. After a period of two weeks, the formed colonies were fixed with methanol and then stained with a 0.1% crystal violet solution. The number of visible colonies was then quantified.

EdU assay for detecting DNA synthesis capacity

The capacity for DNA synthesis in the target cells was evaluated using a specific kit that detects the incorporation of EdU, an analog of the nucleoside thymidine, into newly synthesized DNA. Briefly, 1×104 cells were cultured in 24-well plates for 24 hours and then incubated with 10 μM EdU for 2 hours at 37°C with 5% CO2. Following incubation, the EdU solution was removed, and the cells were fixed with formaldehyde and permeabilized with Triton X-100. The cells were then incubated with a reaction cocktail for 30 minutes in the dark and subsequently stained with a DAPI solution. Under a fluorescence microscope, the cell nuclei stained with DAPI appeared blue, while the newly synthesized DNA incorporating EdU appeared green. The rate of EdU incorporation was calculated as the ratio of EdU-positive cells (green) to the total number of DAPI-positive cells (blue).

Flow cytometry assay for detecting cell cycle

Transfected or co-transfected cells were harvested, fixed in ethanol at −20°C for 2 hours, and then incubated with a propidium iodide staining reagent for 40 minutes in the dark. The distribution of cells across different phases of the cell cycle was then analyzed for all samples using a flow cytometry instrument.

Immunoblotting assay for detecting protein levels

Tissue samples and cells were lysed using a specific lysis buffer. Cytoplasmic and nuclear proteins were extracted using a dedicated kit following the manufacturer’s instructions. The protein concentration in each sample was determined using a bicinchoninic acid protein assay kit. Protein samples (50 μg) were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto nitrocellulose filter membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 for 2 hours at room temperature and subsequently incubated with primary antibodies against various proteins including ki-67, Cyclin A2, Cyclin B1, CDK1, Cyclin D1, SOX17, β-catenin, c-Myc, Histone H3, β-actin, and GAPDH. GAPDH was used as a control for total protein levels. Histone H3 and β-actin served as controls for nuclear and cytoplasmic proteins, respectively. Following incubation with the primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence substrates.

Immunohistochemistry staining

Immunohistochemistry staining was performed to examine the spatial distribution and protein levels of SOX17 in adjacent non-cancerous and breast cancer tissue samples. Embedded tissue samples were sectioned into 4-μm thick slices, deparaffinized in xylene, and rehydrated through a series of graded ethanol solutions in phosphate-buffered saline. Endogenous peroxidase activity was blocked using a 0.3% hydrogen peroxide solution in methanol. The tissue sections were then incubated overnight at 4°C with a primary antibody against SOX17, followed by incubation with a horseradish peroxidase-polymer-conjugated secondary antibody at 37°C for 30 minutes and staining with a diaminobenzidine staining kit. Cell nuclei were counterstained with hematoxylin. Image analysis software was used to quantify the integrated optical density of the stained sections by measuring the integrated optical density.

Dual-luciferase reporter assay for detecting β-catenin activity and miR-937-5p binding to SOX17

The dual-luciferase reporter assay was conducted according to previously described methods to validate the predicted binding of miR-937-5p to the 3′ untranslated region of SOX17. The 3′UTR of the SOX17 gene was amplified using polymerase chain reaction from the genomic DNA of the MDA-MB-231 cell line and inserted downstream of the Renilla luciferase gene in a specific reporter vector using restriction enzymes. To create a reporter plasmid with a mutated miR-937-5p binding site, the seed region of the predicted binding site in the SOX17 gene was altered. Subsequently, 293T cells were co-transfected with either an miR-937-5p mimic or inhibitor, along with the reporter vectors containing either the wild-type or mutant SOX17 3′UTR. The luciferase activity in each group was measured 48 hours after co-transfection using a dual-luciferase reporter assay system. The Renilla luciferase activity was normalized to the firefly luciferase activity, and the results were further normalized to cells transfected with an empty control vector. To investigate the regulation of β-catenin activity by SOX17 and miR-937-5p, MDA-MB-231 and BT-474 cells were co-transfected with a firefly luciferase reporter plasmid driven by β-catenin-responsive elements or a control reporter plasmid with mutated binding sites, along with a Renilla luciferase reporter plasmid for normalization. Cells were harvested 24 hours post-transfection, and firefly and Renilla luciferase activities were measured using a dual-luciferase assay kit. The activity of the β-catenin-responsive reporter was expressed as normalized relative light units relative to the Renilla control, and fold induction was determined by normalizing the activity of the β-catenin-responsive reporter to that of the control reporter.

Statistical analysis

Data obtained from experiments performed in triplicate were analyzed using statistical software and are presented as the mean along with the standard deviation. The statistical significance of the observed results was determined using either an unpaired Student’s t-test or a one-way analysis of variance. A probability value of less than 0.05 was considered to be statistically significant.

Results

miR-937-5p expression in clinical specimens and cell lines

To ascertain the expression pattern of miR-937-5p in breast cancer, clinical tissue samples consisting of 12 pairs of adjacent normal control tissues and cancerous tissues were initially collected, and their histopathological features were examined using Hematoxylin and eosin staining. This staining revealed polymorphic nuclei with condensed chromatin, visible nucleoli, and nuclear division in the ducts of breast tissue. The expression levels of miR-937-5p were found to be significantly elevated in breast carcinoma tissues compared to the adjacent normal control tissue samples. Similarly, in breast carcinoma cell lines including MCF-7, T47D, MDA-MB-231, and BT-474, the expression of miR-937-5p was markedly increased when compared to MCF-10A, a non-tumorigenic human breast epithelial cell line. This upregulation was particularly pronounced in MDA-MB-231 and BT-474 cells. These findings indicate that miR-937-5p is abnormally upregulated in breast cancer, and the MDA-MB-231 and BT-474 cell lines were selected for subsequent experiments due to their higher levels of miR-937 expression.

Specific effects of miR-937-5p on breast cancer malignancy in vitro

To investigate the potential signaling pathways in which miR-937-5p might be involved, genes whose expression correlated with miR-937 expression in The Cancer Genome Atlas Breast Cancer dataset were identified using an online tool. These genes were then subjected to Gene Set Enrichment Analysis of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. The Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated a significant enrichment of miR-937-related genes in pathways associated with the cell cycle, ribosome biogenesis, and DNA replication. Similarly, the Gene Ontology analysis revealed a significant enrichment of miR-937-related genes in processes such as chromosome segregation, DNA replication, and telomere organization. Based on these findings, further experiments were conducted to examine the specific effects of miR-937-5p on breast cancer cell viability, DNA synthesis capacity, cell cycle distribution, and the expression of related protein markers.

Inhibition of miR-937-5p was achieved in MDA-MB-231 and BT-474 cells through transfection with antagomir-937-5p, with antagomir-NC serving as a negative control. The effective downregulation of miR-937-5p was confirmed by quantitative real-time polymerase chain reaction. In both MDA-MB-231 and BT-474 cell lines, the inhibition of miR-937-5p significantly reduced cell viability, DNA synthesis capacity, colony-forming ability, and induced an arrest in the S-phase of the cell cycle, which is the DNA replication phase. Consistent with these observations, the inhibition of miR-937-5p also led to a significant decrease in the protein levels of the proliferation marker ki-67 and key cell cycle regulators including Cyclin A2, Cyclin B1, CDK1, and Cyclin D1. These results suggest that the inhibition of miR-937-5p may suppress breast cancer cell proliferation by influencing cell cycle-related signaling pathways.

miR-937-5p inhibits SOX17 via targeting SOX17 3′UTR

The Wnt/β-catenin signaling pathway is known to promote cell cycle progression and consequently cell proliferation through the transcriptional upregulation of target genes, including c-Myc and cyclin D. To further elucidate the mechanism underlying the effect of miR-937-5p on the proliferation of breast carcinoma cells, a search for potential downstream targets of miR-937 was conducted using an online tool. Among the genes that showed a negative correlation with miR-937 expression based on The Cancer Genome Atlas Breast Cancer data, SOX17, an antagonist of canonical Wnt/β-catenin signaling, was identified. Analysis of expression data from a large cohort of pancreatic cancer tissue samples in The Cancer Genome Atlas database revealed a negative correlation between SOX17 and miR-937 expression, and higher SOX17 expression was associated with longer relapse-free survival. Similarly, both SOX17 protein and messenger RNA expression levels were found to be significantly lower in breast carcinoma specimens compared to adjacent normal control tissues. In breast tissue samples, a negative correlation between miR-937-5p and SOX17 messenger RNA expression was confirmed by Pearson’s correlation analysis. Immunohistochemistry staining also demonstrated an abnormal downregulation of SOX17 protein levels in breast cancer tissues. Quantitative analysis of the staining intensity showed that SOX17 protein levels were negatively correlated with miR-937 levels in the tissue samples. Therefore, further investigation focused on the regulation of SOX17 by miR-937-5p in breast carcinoma cells.

Overexpression or inhibition of miR-937-5p was achieved in MDA-MB-231 and BT-474 cell lines by transfecting them with agomir-937-5p or antagomir-937-5p, respectively, with appropriate negative controls. The efficiency of these transfections was confirmed by quantitative real-time polymerase chain reaction. In both MDA-MB-231 and BT-474 cell lines, overexpression of miR-937-5p led to a decrease in SOX17 protein levels, while inhibition of miR-937-5p resulted in an increase in SOX17 protein levels. To further validate the negative regulation of SOX17 by miR-937, a dual-luciferase reporter assay was performed. Reporter plasmids containing either the wild-type or a mutated 3′UTR of SOX17 were constructed and co-transfected into 293T cells along with either an miR-937-5p mimic or inhibitor. The results of the luciferase assay showed that overexpression of miR-937-5p decreased the luciferase activity of the reporter with the wild-type SOX17 3′UTR, whereas inhibition of miR-937-5p increased this activity. Importantly, neither overexpression nor inhibition of miR-937-5p affected the luciferase activity of the reporter with the mutated SOX17 3′UTR. These findings collectively demonstrate that miR-937-5p inhibits the expression of SOX17 by directly targeting its 3′UTR.

Specific effects of SOX17 on the malignant behaviors of breast cancer cells

Given the finding that miR-937-5p directly targets SOX17, the next step was to investigate the effects of SOX17 on breast cancer cell characteristics and Wnt signaling. Overexpression of SOX17 was achieved in MDA-MB-231 and BT-474 cells by transfecting a SOX17-overexpressing plasmid, with an empty vector serving as a negative control. The successful overexpression of SOX17 was confirmed by immunoblotting. In both MDA-MB-231 and BT-474 cell lines, SOX17 overexpression significantly inhibited cell viability, colony-forming ability, and DNA synthesis capacity, and induced cell cycle arrest in the S or G2/M phases. At the molecular level, SOX17 overexpression led to a significant decrease in the protein levels of the proliferation marker ki-67 and cell cycle regulators Cyclin A2, Cyclin B1, CDK1, c-myc, and Cyclin D1. Furthermore, SOX17 overexpression significantly reduced the levels of β-catenin in both the cytoplasm and the nucleus. Luciferase reporter assays using β-catenin-responsive reporters showed that SOX17 overexpression reduced the transcriptional activity of β-catenin. These data indicate that SOX17 overexpression inhibits Wnt signaling, induces cell cycle arrest, and suppresses breast cancer cell proliferation.

Dynamic effects of miR-937-5p and SOX17 on breast cancer cell phenotype

Considering that miR-937-5p targets and inhibits SOX17 expression, the final step was to determine whether miR-937-5p exerts its effects on breast cancer cells through this mechanism. MDA-MB-231 and BT-474 cells were co-transfected with antagomir-937-5p and small interfering RNA targeting SOX17, and the protein levels of SOX17 were examined. The results showed that transfection with antagomir-937-5p increased SOX17 protein levels, while transfection with small interfering RNA targeting SOX17 decreased SOX17 protein levels. Importantly, the increase in SOX17 protein levels induced by antagomir-937-5p was significantly reversed by the co-transfection of small interfering RNA targeting SOX17. Regarding cellular functions, inhibition of miR-937-5p by antagomir-937-5p suppressed cell viability, colony-forming ability, and DNA synthesis ability in MDA-MB-231 and BT-474 cells, while silencing of SOX17 promoted these cellular functions. Notably, the inhibitory effects of antagomir-937-5p were significantly reversed by the silencing of SOX17. Consistent with these findings, antagomir-937-5p transfection induced S-phase cell cycle arrest, whereas small interfering RNA targeting SOX17 increased the percentage of cells in the G1 phase. Again, the effects of antagomir-937-5p on cell cycle distribution were significantly reversed by the silencing of SOX17. At the molecular level, inhibition of miR-937-5p decreased the protein levels of ki-67, c-Myc, Cyclin A2, Cyclin B1, CDK1, Cyclin D1, and both cytoplasmic and nuclear β-catenin, while silencing of SOX17 increased the levels of these proteins. The effects of antagomir-937-5p on these protein levels were significantly reversed by the silencing of SOX17. Furthermore, luciferase reporter assays showed that the activity of β-catenin was reduced by antagomir-937-5p transfection and increased by small interfering RNA targeting SOX17 in MDA-MB-231 and BT-474 cell lines. These collective data indicate that miR-937-5p affects breast cancer cell proliferation by targeting SOX17, and that SOX17 can counteract the effects of miR-937-5p.

Discussion

In this study, we observed an abnormal upregulation of miR-937-5p in breast cancer tissue samples and cell lines. Gene Set Enrichment Analysis of Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology terms suggested a potential link between miR-937 and cell cycle regulation and DNA replication. Experimental findings demonstrated that inhibiting miR-937-5p significantly reduced the proliferation of breast carcinoma cells and induced an arrest in the S-phase of the cell cycle. Concurrently, the protein levels of the proliferation marker ki-67 and key cell cycle regulators including Cyclin A2, Cyclin B1, CDK1, c-myc, and Cyclin D1 were also decreased upon miR-937-5p inhibition. Furthermore, we found that miR-937-5p can directly bind to and negatively regulate SOX17. Overexpression of SOX17 also significantly suppressed the proliferation of breast carcinoma cells, induced S-phase cell cycle arrest, and reduced the activity of β-catenin and the protein levels of ki-67, β-catenin, c-Myc, Cyclin A2, Cyclin B1, and CDK1. Importantly, the effects of miR-937-5p modulation were reversed by altering SOX17 expression.

Aberrant expression of microRNAs has been frequently observed in various tumor tissues compared to their non-cancerous counterparts. miR-937-5p has been reported to be abnormally expressed in lung carcinoma and breast carcinoma, where it can exhibit either tumor-suppressive or oncogenic properties depending on the context. In colon cancer tissues and cell lines, miR-937-5p levels were significantly elevated, and high levels were associated with reduced overall survival in patients. Similarly, analysis of The Cancer Genome Atlas data revealed an association between higher miR-937-5p expression and poorer survival rates in breast cancer. Consistent with these findings, our study also confirmed the correlation between higher miR-937-5p levels and poorer overall survival in breast carcinoma patients using datasets from The Cancer Genome Atlas Breast Cancer database. We observed that miR-937-5p expression was significantly upregulated in breast tissue samples and cell lines compared to adjacent non-cancerous tissue samples and cell lines, respectively. Collectively, these findings support the notion that miR-937-5p acts as a proto-oncogene during the progression of breast cancer.

It has been reported that miR-937-5p is involved in various aspects of cancer pathogenesis, including the proliferation, invasion, and migration of cancer cells. The role of mature miR-937, encompassing both miR-937-5p and miR-937-3p, in breast cancer appears complex, possibly due to differences in the sequences and expression levels of these two mature forms, as well as the heterogeneity of breast cancer subtypes, such as estrogen receptor-positive, triple-negative, or human epidermal growth factor receptor 2-positive. In breast carcinoma, inhibition of miR-937-3p was shown to attenuate the proliferation, migration, and invasion of estrogen receptor-positive MCF-7 breast cancer cells by targeting CCRL2.

Another study indicated that miR-937-5p promotes cell viability, downregulates the proportion of cells in the G1 phase of the cell cycle, and increases colony formation ability in MCF-7 cells by targeting APAF1. Inhibition of miR-937-5p in that study induced G1 phase arrest. In contrast, our present study found that inhibition of miR-937-5p induced S phase arrest in triple-negative MDA-MB-231 and human epidermal growth factor receptor 2-positive BT-474 cells. Furthermore, the protein levels of the proliferation marker ki-67 and cell cycle regulators Cyclin A2, Cyclin B1, Cyclin D1, and CDK1 were all decreased upon miR-937-5p inhibition. These findings suggest that miR-937-5p influences the breast cancer cell cycle, thereby promoting breast cancer cell proliferation. Moreover, we also observed that miR-937-5p promotes the proliferation of the non-tumorigenic breast cell line MCF-10A, indicating that miR-937-5p may enhance cell proliferation in both tumorigenic and non-tumorigenic breast cells.

MicroRNAs typically function by binding to their target messenger RNAs through partial or complete complementary base pairing, leading to gene silencing via inhibition of translation, messenger RNA degradation, and/or messenger RNA cleavage. In this study, both bioinformatics analysis and experimental validation identified SOX17 as a direct downstream target gene of miR-937-5p. SOX17 belongs to the SOX gene family, which was initially identified due to its significant sequence similarity to the high mobility group box of SRY, the sex-determining region Y gene.

Accumulating evidence has highlighted the critical role of SOX17 in suppressing canonical Wnt signaling, effectively acting as an antagonist of this pathway. SOX17 can compete with TCF transcription factors and redirect β-catenin to different target genes, and disruption of the β-catenin interaction motif in SOX17 abolishes its antagonistic effect on Wnt signaling. In embryonic stem cells, SOX17-mediated inactivation of the canonical Wnt pathway is crucial for the specification of cardiac mesoderm. In cholangiocarcinoma cells, SOX17 inhibited migration, anchorage-independent growth, and Wnt/β-catenin-dependent proliferation. In cervical cancer, studies using luciferase reporter assays and Western blotting demonstrated that SOX17 inhibited the activity of the Wnt/β-catenin signaling pathway, thereby restraining proliferation and tumor formation. It has also been reported that mouse SOX17 can degrade β-catenin and TCF/LEF in a glycogen synthase kinase 3 beta-independent manner in the human colorectal cancer cell line SW480, leading to the inactivation of the canonical Wnt signaling pathway. The multifaceted roles of Wnt signaling in regulating cell behaviors are well-established, with a significant involvement in cell cycle mechanisms. Consistent with these previous studies, overexpression of SOX17 in breast carcinoma cells in our study suppressed cell viability and DNA synthesis capacity and induced cell cycle arrest in the S and G2/M phases. Notably, SOX17 overexpression decreased the protein levels of ki-67, Wnt signaling factors β-catenin and c-Myc, and major cell cycle activators Cyclin A2, Cyclin B1, Cyclin D1, and CDK1, indicating that SOX17 inhibits Wnt signaling, thereby affecting cell cycle distribution in breast carcinoma cells. Furthermore, we also observed that knockdown of SOX17 promotes the proliferation of the non-tumorigenic breast cell line MCF-10A, suggesting that SOX17 depletion may enhance cell cycle progression in both tumorigenic and non-tumorigenic breast cells.

Finally, to validate the hypothesis that miR-937-5p exerts its oncogenic functions in breast cancer cells by targeting SOX17, we examined the dynamic effects of miR-937-5p inhibition and SOX17 silencing on breast cancer cells. As anticipated, silencing of SOX17 exhibited oncogenic effects by promoting cell viability and DNA synthesis ability and alleviating cell cycle arrest. Concurrently, SOX17 silencing increased the protein levels of ki-67, β-catenin, c-Myc, Cyclin A2, Cyclin B1, and CDK1. Importantly, silencing of SOX17 reversed the effects of miR-937-5p inhibition, indicating that miR-937-5p modulates breast cancer cell proliferation and cell cycle distribution by targeting SOX17. Taken together, our findings demonstrate a miR-937-5p/SOX17 axis that regulates breast cancer cell cycle and cell proliferation through the Wnt inhibitor signaling pathway.