Effect of reversing methylation of SARI gene by 5-azacytidine on proliferation and invasion of human breast cancer cells

AIM: To observe whether reversing methylation of SARI (suppressor of activator protein-1 regulated by interferon) gene by 5-azacytidine (5-Aza) inhibits the proliferation and invasion of human breast cancer MDA-MB-231 cells. METHODS: MDA-MB-231 cells were treated with 5-Aza at 5 and 10 μmol/L. The methylation of SARI gene promoter was detected by methylation-specific PCR (MSP), and the mRNA expression of SARI was detected by RT-PCR. The protein expression of SARI was determined by Western blot. The cell growth was detected by MTT assay and colony formation assay. The cell invasion ability was detected by Transwell invasion assay. RESULTS: The results of MSP detection showed that the methylation levels of SARI promoter were significantly decreased after 5-Aza treatment (P<0.01). The results of RT-PCR and Western blot showed that the expression of SARI were significantly increased after 5-Aza treatment (P<0.05). The results of MTT and colony formation assays showed that the cell proliferation was significantly decreased after 5-Aza treatment (P<0.05). The results of Transwell invasion test showed that the invasive ability of the cells was significantly decreased after 5-Aza treatment (P<0.05). CONCLUSION: 5-Aza reverses the methylation status of SARI promoter in MDA-MB-231 cells, up-regulates the expression of SARI, and restores its ability to inhibit tumor cell growth and invasion.     

Methylation of TIMP-3 gene and their relationships with their clinical-pathological characteristics of colorectal cancers

AIM: To investigate whether methylation of the TIMP-3 gene is associated with clinical-pathological characteristics, recurrence and metastasis of the colorectal cancer. METHODS: Nest methylation specific PCR (nMSP) and RT-PCR techniques were used to detect methylation of TIMP-3 gene and its mRNA expression in the colorectal cancer specimen and adjacent non-cancerous tissues. RESULTS: The expression of TIMP-3 mRNA in tumor tissues was distinctly reduced (P<0.01). The expression of TIMP-3 mRNA in those without lymph node metastasis was higher than those with lymph node metastasis (P<0.01). The patients with Duke's C, D and lymph node metastasis were more to contain methylated TIMP-3 compared to those with Duke's A, B and no lymph node metastasis (P<0.05). Statistical differences in pathological characteristics such as tumor site, Duke's stage, histological differentiation and type between TIMP-3 methylation positive group and negative group were observed (P<0.05). CONCLUSION: Methylation of the TIMP-3 gene promoter usually occurs in the proximal site, infiltrating type, poor cellular differentiation, lymph node metastasis and advanced stage of colorectal cancers patients.     

3-MA combined with TSA inhibits growth of triple-negative breast cancer cells

AIM: To investigate the effect of 3-methyladenine (3-MA) combined with trichostatin A (TSA) on triple-negative breast cancer cells and the mechanism involved. METHODS: The viability of MDA-MB-231 cells was detected by MTT assay, the migration ability was determined by scratch assay, and the expression of autophagy-related proteins was detected by Western blot. RESULTS: TSA significantly inhibited the viability of MDA-MB-231 cells in a time-and dose-dependent manner. The results of scratch assay showed that TSA inhibited cell migration ability. Western blot data indicated that TSA resulted in a moderate increase in LC3-Ⅱ expression. Moreover, 3-MA inhibited cell autophagy induced by TSA, and combination of 3-MA and TSA further inhibited the viability of MDA-MB-231 cells. CONCLUSION: Combination of 3-MA and TSA may effectively inhibit the growth of triple-negative breast cancer cells.     

Hypermethylation of SFRP genes in esophageal squamous cell cancer

AIM: To investigate the promoter methylation of secreted frizzled-related protein(SFRP) genes in esophageal squamous cell cancer(ESCC). METHODS: The methods of methylation-specific PCR(MS-PCR) and RT-PCR were applied to examine the CpG methylation of the SFRP promoter and the mRNA expression of SFRP genes,respectively, in 78 samples of ESCC and corresponding adjacent non-cancer tissues. The protein expression of β-catenin was determined by immunohistochemistry.RESULTS: In the ESCC tissues, the frequencies of promoter methylation in SFRP1 , SFRP2 , SFRP4 and SFRP5 genes were 65.4%(51/78), 69.2%(54/78), 62.8%(49/78) and 52.6%(41/78),respectively, significantly higher than those in the adjacent tissues(P<0.01). The hypermethylation of these genes had no correlation with clinical stage and pathological classification in ESCC tissues(P>0.05). The frequency of simultaneous methylation of the 4 genes was correlated with the clinical stage(P<0.05). The positive rates of mRNA expression of the 4 genes in ESCC tissues were 42.3%(33/78), 46.2%(36/78), 50.0%(39/78) and 39.7%(31/78), respectively lower than those in the adjacent tissues(P<0.01). The mRNA expression of SFRP genes and the ectopic expression of β-catenin were correlated with the methylation frequency of SFRP genes(P<0.01).CONCLUSION: Promoter methylation of SFRP1 , SFRP2 , SFRP4 and SFRP5 genes was a frequent event in ESCC, indicating a contribution to the pathogenesis of ESCC through aberrant canonical Wnt/β-catenin signaling pathway. Combination analysis of methylation status in SFRP genes may has definite value on estimating prognosis of ESCC.     

Promoter methylation status and demethylation-induced expression of SFRP genes in human acute leukemia cell lines

AIM: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) genes and acute leukemia (AL)，and to study the mechanism how 5-aza-2-deoxycytidine (5-Aza-CdR) reverses the hypermethylation of SFRP genes in human AL cell lines. METHODS：Methylation-specific PCR (MSP) was used to detect the methylation levels of SFRP1, SFRP2, SFRP4 and SFRP5 genes in different human AL cell lines (Molt-4, Jurkat, HL-60 and NB4). The methylation levels of these genes in Jurkat cell line before and after 5-Aza-CdR treatment were also analyzed by MSP. The expression of SFRP1, SFRP2, SFRP4 and SFRP5 mRNA was detected by real-time fluorescence quantitative RT-PCR. The mRNA levels of DNA methyltransferase (DNMT) 1, DNMT3A and DNMT3B were analyzed by semiquantitative RT-PCR. RESULTS：None of normal human blood or bone marrow mononuclear cells showed methylation of SFRP genes. Hypermethylation in the promoter regions of SFRP1, SFRP2 and SFRP5 genes was observed in all of the four AL cell lines. SFRP4 gene was totally methylated in NB4, Molt-4 and Jurkat cell lines but partially methylated in HL60 cell line. Treatment with 5-Aza-CdR for 72 h successfully reversed the hypermethylation of SFRP genes, and significantly increased the mRNA expression of SFRP. Moreover, the mRNA expression of DNMT1, DNMT3A and DNMT3B was down-regulated by 5-Aza-CdR in a concentration-dependent manner. CONCLUSION：Methylated SFRP genes may serve as potential independent biomarkers for early detection of AL. 5-Aza-CdR activates SFRP gene expression by demethylation of SFRP genes and down-regulation of DNMT1, DNMT3A and DNMT3B mRNA expression.     

Effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells

AIM: To observe the effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells. METHODS: Both hepatocellular carcinoma cells (H2M) and epidermoid carcinoma cells (A431) with methylation of TIMP-3 promoter gene were treated with 5-Aza-2'-deoxycytidine (5Aza-dc). Invasion ability and motility of the cells were detected by Transwell experiments. Expressions of TIMP-3 protein and mRNA were detected by Western blotting and RT-PCR, respectively. TIMP-3 gene promoter methylation was detected by methylation-specific PCR (MSP). RESULTS: ① Invasion ability and motility of H2M and A431 cells were declined after treatment with 5Aza-dc; ② After treatment with 5Aza-dc, the expression of TIMP-3 protein and mRNA were increased in H2M and A431 cells; ③ After treatment with 5Aza-dc, methylation of TIMP-3 promoter gene was not detectable in the cell lines. CONCLUSIONS: 5Aza-dc induces demethylation in TIMP-3 promoter gene, restores TIMP-3 gene and protein expression, and inhibits invasion ability and motility of the carcinoma cells.     

Significance and alterations of p14ARF gene of CDKN2A locus in gastric cancer

AIM:To investigate the significance and changes of p14^ARF gene in gastric cancer.METHODS:The tumors and gastric tissues neighboring carcinoma from 48 patients with gastric cancer were studied. The homozygous deletions, mutations, methylation of the CpG islands, and mRNA expression of p14^ARF gene were assessed by PCR, PCR-SSCP, PCR based methylation assay, and RT-PCR.RESULTS:①The homozygous deletion rate of p14^ARF was 31.3% (15/48), and no homozygous deletions were examined in all the gastric tissues neighboring tumor. ②There were no point mutations of p14^ARF in 33 gastric cancers without homozygous deletion and in the matched gastric tissues adjacent to tumor. ③Methylation rate of the CpG islands of p14^ARF was significantly higher in gastric cancers(47.9%, 23/48) than that in gastric tissues neighboring cancer (4.2%, 2/48)(P＜0.01).④ No expression of p14^ARF mRNA was detected in 45.8%(22/48) of gastric cancers. Moreover, the negative rate (100%, 3/3) of p14^ARF mRNA of gastric cancers with the combined methylation of exons 1β and 2 was significantly higher than that (15%, 3/20) of the sole methylation of exon2(P＜0.05). CONCLUSION:p14^ARF gene is frequently inactivated by homozygous deletion and methylation of the 5' CpG islands in gastric cancer, which may play an important role in the development of gastric cancer.     

Rab1A knockdown inhibits malignant biological behaviors of breast carcinoma cells by inhibiting phosphorylation of AKT

AIM: To investigate the role of Rab1A gene in the malignant biological behaviors of breast carcinoma cells. METHODS: The expression levels of Rab1A in breast carcinoma tissues and normal adjacent tissues, and the basic expression level of Rab1A in different breast carcinoma cell lines were measured by Western blot. Small interfering RNA (siRNA) targeting Rab1A was designed, synthetized and transfected into the breast carcinoma MDA-MB-231 cells. After validation of efficiency of Rab1A gene expression knock-down, the malignant biological behaviors of the MDA-MB-231 cells were measured by CCK-8 assay, wound healing assay, Transwell assay and flow cytometry. The protein levels were determined by Western blot. RESULTS: Rab1A was expressed in normal breast tissue and cells at low level, and at high level in the cancer tissues and cancer cells (P<0.05). Compare with control group, after knock-down of Rab1A expression, the viability of MDA-MB-231 cells was significantly inhibited (P<005), the abilities of migration and invasion were reduced (P<0.05), the apoptosis was decreased (P<0.05), the percentage of G²/M phase was increased, the protein levels of p53, Bax, cleaved caspase-3 and PTEN were significantly increased (P<0.05), and the protein levels of Bcl-2, cyclin D1, cyclin B1, matrix metalloproteinase 2 (MMP2), p-AKT and mTOR were significantly decreased (P<0.05). CONCLUSION: Rab1A modulates the breast carcinoma cell viability, inhibits the migration and invasion abilities, induces G² arrest and effectively regulates the cell growth-, cell cycle-and apoptosis-related proteins. Knock-down of Rab1A expression inhibits the evolution and development of breast cancer by inhibiting the phosphorylation of AKT pathway, and Rab1A may function as a potential target in breast carcinoma treatment.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Full-length cDNA clone and protein expression of a solute carri er family 24 (sodium/potassium/calcium exchanger) member 6,SLC24A6

AIM:To clone and express the gene of SLC24A6,which provide the basis to illuminate the relationship between the SLC24A6 and insulin release.METHODS:The gene expression of SLC24A6 was analyzed in the insu linoma and normal pancreatic tissues by RT-PCR.The full-length cDNA sequence wa s subcloned into pET32a vector,and induced expression and purified in ROSSET (D E3) strain.At the same time,the ORF of SLC24A6 was cloned into green fluoresce nce protein vector pEGFP-C3 to study the location of SLC24A6 in the mouse insuli noma β-TC3 cells.RESULTS:The mRNA expression of SLC24A6 in the human insulinoma tissue was significantly higher than that in normal pancreatic tissue.The fusio n protein of SLC24A6 was a 80 kD protein and was purified successfully by prokary otic vector in ROSSET strain.The localization of SLC24A6 in the mouse insulinom a β-TC3 cells was located in the membrane of the cells.CONCLUSION:SLC24A6 might be related with insulin release.The p rokaryotic expression of SLC24A6 provides the basis for the study on biological function and protein structure,and the location of SLC24A6 in the insulinoma ce ll will throw light on the relationship with insulin release.     

Effects of shRNA targeting COL1A1 gene on proliferation and apoptosis of human breast cancer cell line MDA-MB-231

AIM: To investigate the effects of shRNA-mediated collagen type I alpha 1 (COL1A1) gene silencing on the proliferation and apoptosis of human breast cancer cell line MDA-MB-231. METHODS: The specific recombinant vector pSilencer2.1-U6-COL1A1 was transiently transfected into human breast cancer cell line MDA-MB-231 with lipofectamine. RT-PCR and Western blotting were performed to detect the expression levels of COL1A1. MTT assay was employed to evaluate the effect of COL1A1 gene silencing on the cell proliferation. Flow cytometry was used to determine the apoptosis and cell cycle of transfected cells. The morphological characteristics of apoptosis were observed by Hoechst 33258 staining. RESULTS: Compared with mock group and scrambled group, the mRNA and protein levels of COL1A1 were reduced by pshRNA-COL1A1 transfection (P<0.05). The proliferation of MDA-MB-231 cells treated in shRNA-COL1A1 was significantly inhibited in a time-dependent way. The percentages of G₀/G₁ phase cells and early apoptotic rate were significantly higher in pshRNA-COL1A1 group than those in mock and scrambled group (P<0.05). The changes of apoptotic morphology such as cell shrinkage and nuclear condensation were also observed by staining with Hoechst 33258 under fluorescence microscope. CONCLUSION: Transfection of eukaryotic expression vector pshRNA-COL1A1 effectively inhibits the proliferation, induces apoptosis and arrests MDA-MB-231 cells in G0/G1 phase.     

Effect of DEC1 gene silencing on viability,invasion and migration of human breast cancer MDA-MB-231 cells under hypoxia

AIM: To investigate the effect of differentiated embryonic chondrocyte gene 1 (DEC1) expression silencing on viability, invasion and migration of human breast cancer MDA-MB-231 cells and its possible mechanism under hypoxia. METHODS: The expression of DEC1 was detected by RT-qPCR and Western blot in breast cancer MDA-MB-231 cells under normoxia and hypoxia. MDA-MB-231 cells were transfected with the siRNA targeting DEC1 and the protein levels of DEC1, Smad3 and phosphorylated Smad3 (p-Smad3) were examined under hypoxia. Subsequently, the changes in the viability, invasion and migration abilities of MDA-MB-231 cells were analyzed by CCK-8 assay, Transwell experiment and Scratch test, respectively. RESULTS: The expression of DEC1 in MDA-MB-231 cells under hypoxia was higher than that in the MDA-MB-231 cells under normoxia condition at both mRNA and protein levels (P<0.05). The viability, invasion and migration abilities of MDA-MB-231 cells in siRNA-DEC1 group were decreased significantly as compared with control group (P<0.01). Besides, the protein level of p-Smad3 in the MDA-MB-231 cells in siRNA-DEC1 group was lower than that in negative control group under hypoxia condition (P<0.05). CONCLUSION: Down-regulated DEC1 expression significantly decreases the viability, invasion and migration abilities of breast cancer MDA-MB-231 cells by blocking the TGF-β/Smad3 signaling pathway under hypoxia condition.     

MicroRNA-200a affects malignant biological behaviors of breast cancer cells by inhibiting epithelial-mesenchymal transition

AIM: To investigate the effect of microRNA-200a (miR-200a) on the malignant biological beha-viors of breast cancer cells and its regulatory mechanism. METHODS: The expression of miR-200a in human breast can-cer cell lines MDA-MB-231, MDA-MB-468 and MCF-7, and normal human mammary epithelial cell line MCF-10A was detected by RT-qPCR. CCK-8 assay was used to detect the viability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. Flow cytometry method and Transwell assay were used to detect the apoptosis and invasive ability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. The expression of SIP1, E-cadherin, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was determined by RT-qPCR and Western blot. RESULTS: Compared with MCF-10A cells, the lowest expression of miR-200a was observed in the MDA-MB-231 cells (P<0.05). Over-expression of miR-200a attenuated the viability of MDA-MB-231 cells (P<0.05), increased apoptosis (P<0.05) and decreased the invasion ability (P<0.05). The expression of SIP1, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was also significantly down-regulated, while the mRNA and protein expression of E-cadherin was significantly increased (P<0.05). Transfection with miR-200a inhibitor reversed the above results. CONCLUSION: Up-regulation of miR-200a inhibits the viability and invasion ability of MDA-MB-231 cells and promotes the apoptosis of MDA-MB-231 cells. miR-200a may regulate the biological behaviors of breast cancer by inhibiting epithelial-mesenchymal transition.     

Linarin inhibits migration and invasion abilities of human breast cancer MDA-MB-231 cells through IKK/NF-κB signaling pathway

AIM: To investigate the effect of linarin (LIN) on the migration and invasion abilities of human breast cancer MDA-MB-231 cells and its underlying mechanism. METHODS: MCF-7, MDA-MB-231 and MCF-10A cells were cultured in vitro and treated with LIN at 5, 10, 20, 40, 80 and 160 μmol/L for 24 h, and the cell proliferation was measured by CCK-8 assay and colony formation assay. The protein levels of Snail, E-cadherin, matrix metalloproteinase-9 (MMP-9), IκBα, p-IKKα/β and p-p65 were determined by Western blot. RESULTS: LIN remarkably reduced the viability of MDA-MB-231 cells in a dose-dependent manner (P<0.05), and the IC₅₀ was 55.89 μmol/L for 24 h. LIN decreased the colony formation rate of MDA-MB-231 cells at the concentration of 20 μmol/L (P<0.05). After exposed to LIN at 5 μmol/L and 10 μmol/L for 24 h, the migration and invasion abilities of the MDA-MB-231 cells were significantly reduced (P<0.05), the protein expression levels of E-cadherin and IκBα were up-regulated (P<0.05), the protein expression levels of Snail and MMP-9 were down-regulated (P<0.05), and the phosphorylation levels of IKKα/β and p65 were decreased (P<0.05) in comparison with the control group. Meanwhile, IKK-16 (IKKα/β inhibitor) and PDTC (NF-κB inhibitor) also down-regulated the protein expression levels of Snail and MMP-9 (P<0.05), and up-regulated the protein expression level of E-cadherin (P<0.05). CONCLUSION: LIN down-regulates the protein expression levels of Snail and MMP-9, and up-regulates the protein expression level of E-cadherin most likely through inhibiting IKK/NF-κB signaling pathway, and ultimately lead to decreases in the migration and invasion abilities of MDA-MB-231 cells.     

PCDH10 inhibits proliferation of breast cancer MDA-MB-231 cells by NF-κB/cyclin D1 signaling pathway

AIM To investigate the inhibitory effect of protocadherin 10 （PCDH10） on proliferation of human breast cancer cells and its possible mechanism. METHODS RT-PCR was used to measure the mRNA expression levels of PCDH10 in 4 human breast cancer cell lines and normal mammary epithelial MCF-10A cells. The breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed by recombinant lentivirus （pLV-PCDH10） infection, and blank control （blank） group and negative control （pLV-NC） group were also set up. The cell proliferation ability was detected using methyl thiazolyl tetrazolium （MTT） and colony formation experiments. The cell cycle was detected by flow cytometry. Western blot was used to determine the expression of cyclin D1, cyclin-dependent kinase 4 （CDK4）, nucear factor-κB p65 subunit （NF-κB p65） and NF-κB inhibitor α （IκBα）. RESULTS The mRNA expression levels of PCDH10 in 4 breast cancer cell lines were lower than that in normal mammary epithelial MCF-10A cells （P<0.05）. A breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed successfully. Compared with negative control group, PCDH10 overexpression significantly inhibited cell proliferation, induced cell cycle arrest at G₁ phase, down-regulated the expression of cyclin D1 and CDK4, and decreased phosphorylation of NF-κB p65 and IκBα（P<0.05）. CONCLUSION PCDH10 inhibits the proliferation and blocks cell cycle progression of breast cancer cells by targeting NF-κB/cyclin D1 signaling pathway.     

T0901317 induces apoptosis of human breast cancer MDA-MB-231 cells by up-regulating LXRα expression

AIM: To investigate the pro-apoptotic effect of T0901317, an artificial agonist of liver X receptor α (LXRα), on human breast cancer MDA-MB-231 cells and its mechanism. METHODS: MDA-MB-231 cells were treated with different concentrations (0, 10, 20 and 40 μmol/L) of T0901317 for different time (0, 12, 24 and 48 h). The cell apoptosis was determined by Annexin V/propidium iodide staining and Hoechst 33342 staining. The expression of apoptosis-related proteins, such as Bcl-2, caspase 3 and cleaved caspase-3, and LXRα was determined by Western blot. The mRNA expression of Bcl-2 and LXRα was analyzed by RT-qPCR. RESULTS: T0901317 induced the cell apoptosis in a dose-and time- dependent manner. The expression of cleaved caspase-3 and LXRα was up-regulated, but Bcl-2 was down-regulated by T0901317. The mRNA expression of Bcl-2 was down-regulated, while LXRα was up-regulated by T0901317.CONCLUSION: T0901317 up-regulates LXRα expression and induces the apoptosis of MDA-MB-231 cells.     

miR-125a-5p suppresses epithelial-mesenchymal transition via GSK-3β/Snail signaling pathway in breast cancer cells

AIM: To investigate the function of microRNA-125a-5p (miR-125a-5p) on epithelial-mesenchymal transition (EMT) of breast cancer cells via GSK-3β/Snail signaling pathway.METHODS: The expression of miR-125a-5p in normal breast epithelial cells and breast cancer cells, as well as the transfection efficiency of miR-125a-5p plasmid in MDA-MB-231 cells was detected by RT-qPCR. The chemotaxis ability and invasion ability were detected by chemotaxis assay and Transwell invasion assay. The changes of EMT-related markers, the protein level of phosphorylated glycogen synthase kinase-3β (p-GSK-3β) and the nuclear translocation of Snail were determined by Western blot. RESULTS: The expression of miR-125a-5p in the breast cancer cells was significantly lower than that in the normal breast epithelial cells. The expression of miR-125a-5p was significantly higher in MDA-MB-231/miR-125a-5p cells than that in MDA-MB-231/NC cells. The ability of epithelial growth factor (EGF) at 10 μg/L to induce chemotaxis of MDA-MB-231 cells was the strongest. Compared with MDA-MB-231/NC group, stimulation of EGF decreased the invasion ability of MDA-MB-231/miR-125a-5p cells, and resulted in the increase in E-cadherin expression, while significantly decreased the protein levels of vimentin and p-GSK-3β. Meanwhile, the nuclear localization of Snail was significantly inhibited. The invasion capacity of MDA-MB-231/miR-125a-5p+GAB2 cells was significantly enhanced compared with MDA-MB-231/miR-125a-5p+Con cells, the expression of E-cadherin was decreased, and the protein levels of vimentin and p-GSK-3β were significantly increased, while the nuclear localization of Snail was promoted. CONCLUSION: miR-125a-5p suppresses EMT via GSK-3β/Snail signaling pathway, thus inhibiting the invasion ability of breast cancer cells.