RGS10 deficiency facilitates distant metastasis by inducing epithelial–mesenchymal transition in breast cancer

Distant metastasis is the major cause of death in patients with breast cancer. Epithelial–mesenchymal transition (EMT) contributes to breast cancer metastasis. Regulator of G protein-signaling (RGS) proteins modulates metastasis in various cancers. This study identified a novel role for RGS10 in EMT and metastasis in breast cancer. RGS10 protein levels were significantly lower in breast cancer tissues compared to normal breast tissues, and deficiency in RGS10 protein predicted a worse prognosis in patients with breast cancer. RGS10 protein levels were lower in the highly aggressive cell line MDA-MB-231 than in the poorly aggressive, less invasive cell lines MCF7 and SKBR3. Silencing RGS10 in SKBR3 cells enhanced EMT and caused SKBR3 cell migration and invasion. The ability of RGS10 to suppress EMT and metastasis in breast cancer was dependent on lipocalin-2 and MIR539-5p. These findings identify RGS10 as a tumor suppressor, prognostic biomarker, and potential therapeutic target for breast cancer.


Introduction
Breast cancer is the most common cancer among women worldwide.Globally, in 2020, an estimated 2.3 million new cases of breast cancer were diagnosed, and there were approximately 685,000 deaths from the disease (Sung et al., 2021).The majority of breast cancer mortality is due to distant metastasis, with 5-year survival estimated at 30% (American Cancer Society, 2023).There is a critical need to identify early breast cancer metastasis using prognostic biomarkers to ensure patients receive effective anticancer therapies in a timely manner (Miglietta et al., 2022).
Epithelial-mesenchymal transition (EMT) plays a critical role in tumor progression and metastatic invasion in breast cancer.EMT describes the process by which epithelial cells lose their epithelial characteristics and cell-cell contact and increase their invasive potential (Singh and Chakrabarti, 2019).

Expression and prognostic associations of RGS10 in breast cancer
To determine the role of RGS10 in breast cancer, we analyzed RGS10 mRNA levels in normal tissues (n = 31) from the Genotype-Tissue Expression dataset, which showed RGS10 mRNA levels were high in normal breast, blood, colon, and small intestine tissues and low in normal heart, liver, and pancreas tissues (Figure 1A).Next, we determined RGS10 mRNA levels in cell lines representing 21 human cancers from the Cancer Cell Line Encyclopedia database (Figure 1B).Finally, we applied RT-qPCR in freshly resected breast cancer tissues (n = 20) and matched adjacent normal breast tissues, and showed RGS10 mRNA levels were lower in breast cancer tissues compared to normal breast tissues (p=0.003; Figure 1C).The clinicopathological characteristics of these 20 patients are shown in Table 1.This pattern implied a downregulation of RGS10 expression in breast cancer tissues.
To investigate the biological role and clinical and prognostic significance of RGS10 in breast cancer tissues, we used survival analyses.In breast cancer samples from the Kaplan-Meier plotter database, high RGS10 mRNA level was associated with significantly improved DFS (p=0.0066; Figure 1D) and OS (p=0.027; Figure 1E).In surgically resected breast cancer tissues (n = 133), RGS10 protein expression level detected by immunohistochemistry (representative images shown in Figure 1F) was positively correlated with breast cancer subtype (p=0.043),distant metastasis (p=0.008), and survival status (p=0.024).There were no correlations with age, comorbid disease, histological grade, tumor size, number of positive axillary lymph nodes, number of pregnancies, number of births, age at first pregnancy, menopausal status, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor 2 (HER2) status, or Ki67 status   2).In these patients, a high RGS10 protein expression level was associated with a longer DFS (p=0.003, Figure 1G) and OS (p=0.022, Figure 1H).Clinicopathological characteristics associated with DFS and OS were identified with Cox regression analyses.On multivariate regression analysis, histological grade and RGS10 protein expression were independent predictors of DFS (Table 3).On The online version of this article includes the following source data for figure 1: Source data 1.Original files for Figure 1.     4).This suggests low RGS10 expression is associated with poor survival in patients with breast cancer.Taken together, these findings imply that RGS10 has a role in suppressing breast cancer and RGS10 may represent a potential prognostic biomarker in breast cancer.

RGS10 silencing increases the proliferation and migration of breast cancer cells in vitro
To characterize RGS10 protein expression in the breast cancer cell lines MDA-MB-231, MCF7, and SKBR3, we conducted western blotting.MDA-MB-231 is a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer cell line (Bianchini et al., 2016).MCF7 is an adenocarcinoma cell line with estrogen, progesterone, and glucocorticoid receptors (Lee et al., 2015).The SKBR3 cell line overexpresses the HER2/c-erb-2 gene product (Merlin et al., 2002).RGS10 protein levels appeared lower in the highly aggressive cell line MDA-MB-231 compared to the poorly aggressive and less invasive cell lines MCF7 and SKBR3 (Figure 2A).This finding validates our previous observations suggesting that RGS10 acts as a tumor suppressor in breast cancer.
These findings show invasion and metastasis were enhanced in breast cancer cells lacking RGS10, suggesting an inhibitory effect of RGS10 in breast cancer metastasis.To explore the mechanisms by which RGS10 suppresses breast cancer invasion and metastasis, we analyzed potential downstream tumor metastasis-related genes by comparing the transcriptomes in RGS10-depleted (shRNA-RGS10-506, shRNA-RGS10-161) SKBR3 cells and SKBR3 cells transfected with shRNA-NC.Differential gene expression was visualized using a volcano plot, which revealed that 70 genes were significantly upregulated in RGS10-depleted SKBR3 cells (Figure 3A).Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) enrichment analysis of differentially expressed genes identified upregulated KEGG pathways, biological processes, cellular components, and molecular functions (Figure 4B-E).Upregulated KEGG pathways were associated with cytokine-cytokine receptor interactions and extracellular matrix-receptor interactions.Gene sets associated with high-and low-RGS10 mRNA expression were identified by gene set enrichment analysis using the Molecular Signatures Database (Liberzon et al., 2011).Gene sets associated with high-RGS10 mRNA expression included allograft rejection, apoptosis, interleukin (IL) 6/Janus kinase/ signal transducer and activator of transcription pathway (STAT) 3, IL2/STAT5 pathway, and inflammatory response (Table 5).Western blotting showed changes in biomarkers of EMT in RGS10-depleted SKBR3 cells compared to NC. Neutrophil-derived cytokine LCN2 and vimentin protein levels were higher and E-cadherin protein levels were lower in RGS10-depleted SKBR3 cells compared to NC (Figure 3F and G).These findings show that RGS10 deficiency promotes invasion and metastasis by activating the LCN2 pathway to induce EMT in breast cancer cells, supporting the potential of RGS10 as a prognostic biomarker in breast cancer.

MIR539-5p regulates the migration, invasion, proliferation, and EMT of breast cancer cells
To study the upstream regulatory mechanism of RGS10 in breast cancer, we used the StarBase database to predict miRNAs that could potentially bind to RGS10.The luciferase reporter assay identified MIR539-5p as a miRNA that targets RGS10 in breast cancer cells (Figure 3G and H).
To predict the potential effects of MIR539-5p on breast cancer cells, we transfected SKBR3 and MDA-MB-231 cells with a MIR539-5p mimic to represent MIR539-5p overexpression, a MIR539-5p inhibitor, or appropriate NCs (Figure 4A).RT-qPCR and western blotting validated transfection efficiency and showed that RGS10 mRNA and protein levels were significantly decreased in SKBR3 cells overexpressing MIR539-5p compared to SKBR3 cells transfected with miRNA-NC or the wild type (Figure 4B and C).CCK-8 analysis, and colony formation and transwell migration/invasion assays showed SKBR3 cell proliferation, colony formation, migration, and invasion were significantly increased in SKBR3 cells overexpressing MIR539-5p compared to SKBR3 cells transfected with miRNA-NC or the wild type (Figure 4D-G).In contrast, RGS10 protein levels were significantly increased (Figure 5A-C) and MDA-MB-231 cell proliferation, colony formation, migration, and invasion were significantly decreased in MDA-MB-231 cells transfected with MIR539-5p inhibitor compared to anti-miRNA-NC or the wild type (Figure 5D-G).
Based on these findings, for the first time, we propose a MIR539-5p/RGS10/LCN2 regulatory axis in breast cancer.Consistent with this, western blotting and immunofluorescence assays showed decreased E-cadherin protein expression and increased LCN2, vimentin, and snail protein expression in SKBR3 cells overexpressing MIR539-5p compared to SKBR3 cells transfected with miRNA-NC or the wild type (Figure 4H and I).These effects were reversed in MDA-MB-231 cells transfected with a MIR539-5p inhibitor compared to anti-miRNA-NC or the wild type (Figure 5H and I).
These findings identify MIR539-5p as a critical factor in breast cancer metastasis by regulating RGS10/LCN2 expression.

RGS10 inhibits breast cancer growth by targeting LCN2 in vivo
To investigate tumorigenicity, we subcutaneously transplanted SKBR3 cells transfected with shRNA-RGS10-506 or shRNA-NC into nude mice.The growth of tumors derived from RGS10-depleted SKBR3 cells was significantly increased compared to NC, manifested by larger tumor size compared to NC (Figure 6A-C).Immunohistochemistry showed LCN2, snail, and vimentin protein expression were increased and E-cadherin protein expression was decreased in tumor tissues derived from RGS10depleted SKBR3 cells compared to NC (Figure 6D).These in vivo data confirm the previous in vitro observations that RGS10 deficiency promotes invasion and metastasis by activating the LCN2 pathway to induce EMT in breast cancer cells, and demonstrate that RGS10 has utility as a prognostic biomarker in breast cancer.

Discussion
Distant metastasis is a main cause of death in patients with breast cancer.Identification of prognostic biomarkers for early distant metastasis may inform clinical decision-making and improve patient outcomes.To the best of our knowledge, this is the first study to characterize the role of RGS10 as a tumor suppressor and biomarker of EMT in breast cancer.RGS10 was expressed at lower levels in breast cancer tissues than in adjacent normal breast tissues.RGS10 expression was associated with molecular subtypes of breast cancer, distant metastasis, and survival status.Patients with high compared to low RGS10 mRNA expression in breast cancer tissues had improved DFS and OS.RGS10 protein levels were lower in the highly aggressive breast cancer cell line MDA-MB-231 compared to the poorly aggressive and less invasive breast cancer cell lines MCF7 and SKBR3.RGS10 reduced breast cancer cell proliferation, colony formation, invasion, and migration by inhibiting EMT via a novel mechanism dependent on LCN2 and MIR539-5p.
EMT is involved in normal development and morphogenic processes, including embryogenesis and tissue regeneration.Pathological EMT promotes invasion and metastasis in tumors through intracellular signaling, transcription factors, miRNAs, and epigenetic and posttranslational regulators (Huang et al., 2022;Lamouille et al., 2014).Signaling pathways such as TGF-β, Wnt, Notch, and phosphoinositide 3-kinase/AKT contribute to EMT, often with cross-talk at various levels and feedback activation/repression mechanisms (Deshmukh et al., 2021;Huang et al., 2022).Transcription factors such as snail, slug, ZEBl/ZEB2, and Twist1/Twist2 induce EMT by acting on the E-box sequence of the CDH1 promoter.Noncoding miRNAs regulate EMT by selectively targeting mRNAs of cell receptors, signaling pathways, the cell cycle, or cell adhesion (Górecki and Rak, 2021;Huang et al., 2022;Li et al., 2019).Epigenetic modifications, including DNA methylation and histone modifications, alter the expression of EMT transcription factors involved in the molecular pathways of metabolism, transcription, differentiation, and apoptosis (Górecki and Rak, 2021).
The present study identifies RGS10 as an important mediator of EMT in breast cancer.Previous studies have demonstrated links between several RGS proteins and various cancers, with RGS proteins acting as tumor initiators or suppressors depending on the RGS protein and type of cancer (Li et al., 2023).RGS10 is the smallest protein of the RGS D/12 subfamily, which functions as GAPs for the Gi family Gα subunits (Almutairi et al., 2020).RGS10 has been linked to a poor prognosis in patients with laryngeal cancer (Yin et al., 2013), liver cancer (Wen et al., 2015), and childhood acute myeloid leukemia (Chaudhury et al., 2018).RGS10 may represent a biomarker of clinical staging for ovarian cancer and is one of five signature genes involved in the occurrence and development of ovarian cancer.This five-gene signature (RGS11, RGS10, RGS13, RGS4, and RGS3) is overexpressed in ovarian cancer and involved in extracellular matrix-receptor interaction, the TGF-β signaling pathway, the Wnt signaling pathway, and the chemokine signaling pathway.These pathways mediate the proliferation, migration, and invasion of ovarian cancer cells; in particular, TGF-β signaling plays an important role in EMT in ovarian cancer (Hu et al., 2021).The novel action of RGS10 in EMT in breast cancer appears to be dependent on LNC2, also known as neutrophil gelatinase-associated lipocalin, siderocalin, uterocalin, and oncogene 24p3.LNC2 is a secreted glycoprotein of the adipokine superfamily.LCN2 expression levels are particularly high in breast, pancreatic, ovarian, colorectal, thyroid, and bile duct cancer tissues and tumor cell lines.Previous studies show LCN2 can promote tumorigenesis by increasing invasion, metastasis, and proliferation while decreasing apoptosis, possibly because LCN2 can facilitate iron intake to cancer cells and form a heterodimer with matrix metalloproteinase-9 (Santiago-Sánchez et al., 2020).In breast cancer, LCN2 can promote progression by inducing EMT through the estrogen receptor alpha/Slug axis (Morales-Valencia et al., 2022;Yang et al., 2009).
The regulation of EMT in breast cancer by RGS10 may rely on upstream regulation by MIR539-5p.miRNAs play a critical role in the cellular processes of breast cancer, including EMT (Zhang et al., 2013;Zhao et al., 2017).Previous reports indicate that MIR539 expression is downregulated in breast cancer tissues and cell lines (Guo et al., 2018), and MIR539 acts as a tumor suppressor by targeting epidermal growth factor receptor (Guo et al., 2018), specificity protein 1 (Cai et al., 2020), or laminin subunit alpha 4 (Cai et al., 2020) expression.In patients with breast cancer, decreased expression of MIR539 was significantly associated with lymph node metastasis.In breast cancer cells, overexpression of MIR539 inhibited the proliferation and promoted apoptosis of breast cancer cells, suppressed EMT, and sensitized cells to cisplatin treatment (Cai et al., 2020).Further studies are required to fully elucidate miRNA regulation of the gene expression networks that are essential to EMT in breast cancer.
Inflammation promotes EMT in tumors, and EMT induces the production of proinflammatory factors by cancer cells (Suarez-Carmona et al., 2017).The present study showed that RGS10 expression in SKBR3 cells was associated with the inflammatory response.Previous reports show that RGS10 regulates cellular physiology and fundamental signaling pathways in microglia, macrophages, and T-lymphocytes.In microglia and ovarian cancer cells, RGS10 regulates inflammatory signaling by a G protein-independent mechanism, linking RGS10 to the inflammatory signaling mediators tumor necrosis factor-alpha (TNFα) and cyclooxygenase-2 (Alqinyah et al., 2018).In macrophages, RGS10 regulates activation and polarization by suppressing the production of the inflammatory cytokines TNFα and IL6 (Dean and Hooks, 2020).These findings suggest a potential role for RGS10 in the development of an inflamed and immunosuppressive tumor microenvironment, which may have important implications for the progression in breast cancer (Suarez-Carmona et al., 2017).Our study had some limitations.First, this was a retrospective study with a small sample size.Thus, the conclusions should be confirmed in meta-analyses or large randomized controlled trials.Second, the mechanism by which the MIR539-5p/RGS10/LCN2 axis may be related to outcomes in patients with breast cancer remains to be elucidated.Biochemical characterization of the molecular mechanisms of RGS10 in breast cancer should provide additional insight into the potential of RGS10 as a biomarker of EMT, metastasis, and prognosis in breast cancer and the role of RGS10 as a therapeutic target.
In conclusion, the results of this study show that RGS10 expression is related to survival outcomes in patients with breast cancer.RGS10 protein levels were lower in the highly aggressive breast cancer cell line MDA-MB-231 compared to the poorly aggressive and less invasive breast cancer cell lines MCF7 and SKBR3.Silencing RGS10 expression effectively increased the proliferation, colony formation, invasion, and migration ability of SKBR3 cells.The MIR539-5p/RGS10/LCN2 pathway was identified as an important regulatory axis of EMT in breast cancer.These data demonstrate that RGS10 may play a tumor suppressor role and be considered a biomarker of EMT and prognosis in breast cancer.et al., 2019).The Kaplan-Meier plotter (http://kmplot.com)was used to assess the relevance of RGS10 mRNA expression for disease-free survival (DFS) and overall survival (OS) in patients with breast cancer (Győrffy, 2021).

Clinical specimens
This study included an additional 153 patients with histologically confirmed invasive ductal breast carcinoma who received treatment at the Shengjing Hospital of China Medical University from April 2006 to April 2008.Patients had (1) undergone surgery with no distant metastases at the time of the operation; (2) received standard adjuvant therapy after surgery; (3) ≥10 axillary lymph nodes dissected and examined pathologically after surgery (Coates et al., 2015;Gradishar et al., 2021;Wolff et al., 2013); and (4) ≥10 years of follow-up as outpatients or by telephone interviews.Twenty paired breast cancer and normal adjacent tissues were obtained from patients undergoing surgical resection at Shengjing Hospital of China Medical University to evaluate the status of RGS10.Clinical specimens from 133 patients were used to assess the effect of RGS10 on prognosis.The clinicopathological characteristics of these 153 patients are shown in Tables 1 and 2.
The study was approved by the Institutional Ethics Committee of Shengjing Hospital of China Medical University (Permit Number: 2024PS171K) and complied with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines of the National Medical Products Administration of China.Informed consent was obtained from all the participants.

Breast cancer cells and culture
Human breast cancer cell lines SKBR3, MCF-7, and MDA-MB-231 were obtained from American Type Culture Collection (ATCC, Manassas, VA).The identity of cell lines had been authenticated by using STR profiling and with no contamination in the mycoplasma test.SKBR3 cells were cultured in McCoy's 5A (modified) medium supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 .MCF7 cells were cultured in DMEM high glucose (Invitrogen) medium supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 .MDA-MB-231 cells were cultured in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 37°C without CO 2 .

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated from breast cancer tissues and breast cancer cell lines using Triquick reagent (Solarbio Life Science, Beijing, China) and reverse-transcribed using a cDNA synthesis kit (Takara Bio, at 37°C for 24 hr, cells that had migrated to the lower surface through the membrane were fixed with 4% paraformaldehyde for 10 min.Fixed cells were stained with crystal violet.
Detection of cell migration was similar to cell invasion, except the upper chamber of the Transwell chamber was not precoated with Matrigel.

Dual-luciferase reporter assay
The StarBase database was used to predict potential binding sites between RGS10 mRNA and hsa-MIR539-5p.Sequences containing the 3'-untranslated region fragments of RGS10 and the mutated binding site of hsa-MIR539-5p or wild type were designed and constructed by GeneChem Biotechnology (Shanghai, China).The MIR539-5p mimic or NC was cotransfected with the wild-type or mutant plasmid using Lipofectamine 3000 (Invitrogen).Luciferase intensity was recorded 48 hr after transfection using the dual-luciferase reporter assay system (Promega, Madison, WI).

Molecular interaction networks
The Search Tool for the Retrieval of Interacting Genes/Proteins database (https://string-db.org/)was used to identify proteins that interact with RGS10 and conduct a protein-protein network interaction analysis (Szklarczyk et al., 2015).Subsequently, the Database for Annotation, Visualization, and Integrated Discovery (https://david.ncifcrf.gov)was used to perform GO and KEGG pathway-enrichment analysis.

In vivo animal experiments
Nine BALB/C female nude mice (6 weeks old; 19-22 g) (Beijing Huafukang Biotechnology Company) were randomly assigned into three groups (n = 5 each).Mice were maintained in a specific-pathogenfree environment at 28°C and 50% humidity.

Figure 1 .
Figure 1.The expression and prognostic associations of RGS10 in breast cancer.(A) RGS10 mRNA levels in 31 normal human tissues.Data were derived from the Genotype-Tissue Expression database.(B) RGS10 mRNA levels in cell lines representing 21 human cancers.Data were derived from the Cancer Cell Line Encyclopedia database.(C) qRT-PCR showing RGS10 mRNA levels in freshly resected breast cancer tissues (n = 20) and matched adjacent normal breast tissues.**p<0.01,Student's t -test.(D, E) Survival analyses showing disease-free survival (DFS) (D) and overall survival (OS) (E) in patients with breast cancer stratified by high versus low RGS10 mRNA levels.Data were derived from the Kaplan-Meier plotter database.(F) Representative images showing immunohistochemical staining of RGS10 protein expression in breast cancer tissues or normal tissues (n = 133) (magnification: ×200 and ×400).(G, H) Kaplan-Meier analysis showing DFS (G) and OS (H) in patients with breast cancer stratified by presence versus absence of RGS10 protein in breast cancer tissues (n = 133).

Figure 1
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Figure 2 .
Figure 2. RGS10 silencing increases the proliferation and migration of breast cancer cells in vitro.(A) Western blotting showing RGS10 protein levels in molecularly distinct breast cancer cell lines.The original files of the full raw unedited blots are provided in Figure 2-source data 1.The uncropped gels with the relevant bands labeled are provided in Figure 2-source data 2. The statistical data is provided in Figure 2-source data 3. (B) Western blotting showing RGS10 protein levels in SKBR3 cells transfected with three independent shRNA constructs, shRNA-RGS10-161, shRNA-RGS10-321, and shRNA-RGS10-506, and shRNA-NC.The original files of the full raw unedited blots are provided in Figure 2-source data 4.The uncropped gels with the relevant bands labeled are provided in Figure 2-source data 4.The statistical data is provided in Figure 2-source data 6.(C) CCK-8 assay showing the viability of SKBR3 cells transfected with shRNA-RGS10-161, shRNA-RGS10-506, or shRNA-NC.**p <0.01, one-way ANOVA.(D-F) Colony formation (D) and transwell migration/invasion (E, F) assays in SKBR3 cells transfected with shRNA-RGS10-161, shRNA-RGS10-506, or shRNA-NC.***p<0.001,one-way ANOVA.The online version of this article includes the following source data for figure 2: Source data 1.Original files for the gels in Figure 2A.Source data 2. Uncropped gels with the relevant bands labeled in Figure 2A.Source data 3. Statistical data for Figure 2A.Source data 4. Original files for the gels in Figure 2B.Source data 5. Uncropped gels with the relevant bands labeled in Figure 2B.Source data 6.Statistical data for Figure 2B.Source data 7. Original files for Figure 2C.Source data 8. Original files for Figure 2D.

Figure 2
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Source data 9 .
Original files for Figure 2E.Source data 10.Original files for Figure 2F.

Figure 4 .
Figure 4. MIR539-5p regulates the migration, invasion, proliferation, and epithelial-mesenchymal transition (EMT) of breast cancer cells.(A) qPCR showing the transfection efficiency of the MIR539-5p mimic.***p<0.001,one-way ANOVA.(B, C) qRT-PCR and western blotting showing RGS10 mRNA and protein levels in SKBR3 cells transfected with the MIR539-5p mimic, negative control (NC), or wild type (WT).***p<0.001,one-way ANOVA.The original files of the full raw unedited blots are provided in Figure 4-source data 1.The uncropped gels with the relevant bands labeled are provided in Figure 4-source data 2. The statistical data is provided in Figure 4-source data 3. (D) CCK-8 assay showing the viability of SKBR3 cells transfected with the MIR539-5p mimic, NC, or WT.***p<0.001,one-way ANOVA.(E-G) Colony formation (E) and transwell migration/invasion (F, G) assays in SKBR3 cells transfected with the MIR539-5p mimic, NC, or WT.*p<0.05,**p<0.01,Student's t-test.(H) Western blotting showing protein levels of LCN2 and biomarkers of EMT in SKBR3 cells transfected with the MIR539-5p mimic or NC.The original files of the full raw unedited blots are Figure 4 continued on next page provided in Figure 4-source data 4.The uncropped gels with the relevant bands labeled are provided in Figure 4-source data 5.The statistical data is provided in Figure 4-source data 6.(I) Immunofluorescence staining showing E-cadherin, vimentin, and snail protein expression in SKBR3 cells transfected with the MIR539-5p mimic or NC.Scale bar: 50 µm.

Figure 5 .
Figure 5. MIR539-5p inhibitor suppresses breast cancer cell proliferation and invasion.(A) qPCR showing transfection efficiency of the MIR539-5p inhibitor after 48 hr.***p<0.001,one-way ANOVA.(B, C) qRT-PCR and western blotting showing RGS10 mRNA and protein levels in MDA-MB-231 cells transfected with the MIR539-5p inhibitor, negative control (NC), or wild type (WT).***p<0.001,one-way ANOVA.The original files of the full raw unedited blots are provided in Figure 5-source data 1.The uncropped gels with the relevant bands labeled are provided in Figure 5-source data Figure 5 continued on next page

Figure 6 .
Figure 6.RGS10 inhibits breast cancer growth by targeting LCN2 in vivo.(A) Size of tumors derived from RGS10-depleted SKBR3 cells, negative control (NC), and wild type (WT).(B) Volume of tumors derived from RGS10-depleted SKBR3 cells, NC, and WT.***p<0.001,one-way ANOVA.(C) Hematoxylin and eosin staining of tumors derived from RGS10-depleted SKBR3 cells and NC.(D) Immunohistochemical staining showing LCN2, E-cadherin, snail, and vimentin protein expression in tumors derived from RGS10-depleted SKBR3 cells and NC.The online version of this article includes the following source data for figure 6: Source data 1.Original files for the gels in Figure 6A.Source data 2. Uncropped gels with the relevant bands labeled in Figure 6B.Source data 3. Statistical data for Figure 6C.Source data 4. Original files for the gels in Figure 6D.

Table 2 .
Correlations between RGS10 expression and clinicopathological characteristics.

Table 3 .
Univariate and multivariate Cox regression analyses of clinicopathological risk factors for disease-free survival (DFS).

Table 4 .
Univariate and multivariate Cox regression analyses of clinicopathological risk factors for overall survival.