| Background:Breast cancer is the most common malignancy in women.Triple-negative breast cancer(TNBC),which constitutes the most aggressive molecular subtype among breast tumors,is characterized by the lack of expression of estrogen receptors(ER)and progesterone receptors(PR)and the lack of overexpression of human epidermal growth factor 2(HER2).Due to the molecular and clinical features,it cannot benefit from endocrine therapy or molecular targeted therapy.TNBC has a higher rate of early recurrences,often with distant metastases,and is associated with a poorer prognosis compared to other breast cancer subtypes.Therefore,it is crucial to find effective targets for TNBC prevention and treatment.1α,25(OH)2D3,the active form of Vitamin D,plays important physiological roles in bone metabolism and is known to be associated with cancer.Studies have indicated that vitamin D had protective effects on TNBC,but little is known about the potential mechanism.In this study,we further examined the role of 1α,25(OH)2D3 in inhibiting glycolysis and metastasis of TNBC in vitro and in vivo.With the purpose of providing theoretical and experimental basis for the applications of vitamin D in TNBC prevention therapy and prevention.Methods:(1)Population analysis:a.Based on the bioinformatics method,the expression of Hypoxia Inducible Factor-1α(HIF-1α)、Glucose Transporter 1(GLUT 1)、Lactic dehydrogenase A(LDHA)were compared in TNBC and non-TNBC patients from The Cancer Genome Atlas(TCGA)database;The expression of HIF-lα,GLUT1,and LDHA were compared between carcinoma tissues and normal breast tissues;b.The microarray gene expression dataset(GSE58812)from the GEO website was used to examine the relationship between the expression of HIF-lα,GLUT1,and LDHA and the overall survival of TNBC patients.(2)Animal experiment:a.Establishment of tumor-bearing mice models:mouse TNBC cell line 4T1 cell suspension(1×105)was injected into the right axilla of BALB/c mice;Human TNBC cell line MDA-MB-231 cell suspension was mixed with MATRIGEL and was injected into the right axilla of BALB/c-nu nude mice to establish a nude mouse xenograft model.After 2 weeks of injection,TNBC xenografts were randomly divided into two groups,the control group(0.9%saline.N=5).the 1α,25(OH)2D3-treated group(1μg/kg,N=5).Saline or 1α,25(OH)2D3 was subcutaneous injected every two days.The body weight and the tumor size were measured every two days.At the end of the experiment,the tumor and other organs of the mice were obtained.b.(Hematoxylin-eosin staining,HE)method was used to observe the lung metastasis.c.The expression of Ki67,HIF-1α,Hexokinase 2(HK2),and Vimentin was measured by immunohistochemical method.d.The expression of glycolytic enzymes and EMT-related proteins of xenograft were detected by Western Blot assay.(3)Cell experiment:a.TNBC cells MDA-MB-231 and 4T1 were exposed to CoCl2 to mimic hypoxic conditions.b.Cell viability and proliferation were measured by the CCK8 method.c.The migration ability of cells was examined by Wound-healing assay.d.The invasion ability of cells was examined by Transwell assay respectively.e.Glycolytic enzymes and EMT-related molecules in 1α,25(OH)2D3-treated and control TNBC cells were examined by Quantitative real-time polymerase chain reaction(qRT-PCR)and Western Blot assay.f.Glucose uptake,lactate production,and ATP levels of TNBC cells were evaluated by commercial kits respectively.g.MDA-MB-231 cells with stable low expression of VDR were established by lentivirus transfection.Western blot assay was performed to determine the expression of HIF-1α;Glucose uptake and lactate production in TNBC cells were evaluated by commercial kits respectively;The migration and invasion ability of cells were detected respectively by Wound-healing assay and Transwell assay.h.The 4T1 cells with instantaneous low expression VDR were established by siRNA transfection.Transfected cells were treated with or without 1α,25(OH)2D3,and qRT-PCR assay was performed to determine the expression of HIF-1α and glycolysis-related enzymes before and after VDR knockdown.Results:1.Expression of HIF-1α and glycolysis-related molecules in human TNBC samples and the correlation with the overall survival of patients.According to bioinformatics analysis,the expressions of HIF-1α,GLUT1,and LDHA in TNBC samples were significantly higher than non-TNBC samples;in addition,the expression of HIF-1α,GLUT1,and LDHA was significantly higher in TNBC tumor tissues compared with normal tissues;Kaplan-Meier survival analysis demonstrated that higher expressions of HIF-1α,GLUT1,and LDHA were respectively associated with worse overall survival in TNBC patients.2.Effect of 1α,25(OH)2D3 on the TNBC xenograft model.(1)Effect of 1α,25(OH)2D3 on the growth of TNBC xenograft tumors.In TNBC xenograft models,there was no significant decrease in the weight and volume of the tumors compared with the control group.Immunohistochemical results showed that 1α,25(OH)2D3-treated tumors had lower expressions of Ki67 than controls.(2)Effect of 1α,25(OH)2D3 on the lung metastasis of TNBC xenograft tumors.In 4T1 xenograft models,there was a significant reduction in the numbers of lung metastatic lesions in 1α,25(OH)2D3-treated tumors compared with controls.Western blot assay results showed that 1α,25(OH)2D3 decreased the expression of N-cadherin,Vimentin,and Non-p-β-catenin,and increased the expression of E-cadherin in xenograft tumors.While 1α,25(OH)2D3 decreased the expression of N-cadherin,Non-p-β-catenin,and increased the expression of E-cadherin in MDA-MB-231 xenograft.(3)Effect of 1α,25(OH)2D3 on the glycolysis of TNBC xenograft tumors.Western blot assay results showed that compared with the control group,the expression of HK2,and GLUT 1was decreased in the 1α,25(OH)2D3 group in MDA-MB-231 xenograft.While the expression of HK2,GLUT1,MCT1,and LDHA was decreased in the 1α,25(OH)2D3-treated 4T1 xenograft.In parallel,immunohistochemical results showed that 1α,25(OH)2D3-treated tumors had lower expressions of HK2 than controls.Meanwhile,lactate concentrations in TNBC xenograft were decreased in the 1α,25(OH)2D3-treated compared with the control group.(4)Immunohistochemistry and Western blot assays results showed that 1α,25(OH)2D3 significantly decreased the expression of HIF-1α in TNBC xenograft compared with the control group.3.Effect of 1α,25(OH)2D3 on TNBC cells.(1)Effect of 1α,25(OH)2D3 on the ability of proliferation,migration and invasion in TNBC cells.The CCK8 assay showed that 1α,25(OH)2D3 inhibited the viability of TNBC cells in a timeand dose-dependent manner.Compared to the control group,1α,25(OH)2D3 inhibited the migration ability of MDA-MB-231 and 4T1 cells.Transwell assay showed that overexpression of HIF-1α by CoCl2 enhanced the invasion ability of TNBC cells.And compared to the control group,1α,25(OH)2D3 inhibited the invasion ability of MDA-MB-231 and 4T1 cells.(2)Effect of 1α,25(OH)2D3 on the glycolysis of TNBC cells.The results showed that 1.0 μM of 1α,25(OH)2D3 significantly inhibited the glucose uptake,lactate production,and ATP production of TNBC cells.Western Blot assay results showed that 1.0 μM of 1α,25(OH)2D3 significantly reduced the expression of GLUT1 and LDHA compared with the control group in MDA-MB-231 cells,while 1.0 μM of 1α,25(OH)2D3 significantly reduced the expression of HK2,PKM2 and LDHA compared with the control group in 4T1 cells.qRT-PCR assay showed that compared with the control group,1.0 μM of 1α,15(OH)2D3 can significantly reduce the relative expression of PKM2,GLUT1,LDHA when HIF-1α was overexpressed in MDA-MB-231 cells,while 1.0 μM of 1α,25(OH)2D3 can significantly reduce the relative expression of HK2,PKM2,GLUT1 and LDHA when HIF-1α was overexpressed in 4T1 cells.4.VDR was involved in the effect of 1α,25(OH)2D3 on glycolysis and metastasis ability in HIF-1α overexpressed TNBC cells.(1)VDR was involved in the effect of 1α,25(OH)2D3 on the expression of HIF-1α in HIF-1α overexpressed TNBC cells.Western blot assay showed that compared to the Sh-NC group,knockdown of VDR reversed the inhibitory effect of 1α,25(OH)2D3 on the expression of HIF-1α in MDA-MB-231 cells.qRT-PCR assay results showed that compared to the Si-NC group,VDR knockdown attenuated the inhibitory effect of 1α,25(OH)2D3 on the mRNA expression of HIF-1α in 4T1 cells.(2)VDR was involved in the effect of 1α,25(OH)2D3 on metastasis ability in HIF-1α overexpressed TNBC cells.Western blot assay showed that compared to the Sh-NC group,VDR knockdown attenuated the effect of 1α,25(OH)2D3 on the expression of E-cadherin and vimentin in MDA-MB-231 cells.Meanwhile,VDR knockdown attenuated the inhibitory effect of 1α,25(OH)2D3 on the ability of migration and invasion in MDA-MB-231 cells.(3)VDR was involved in the effect of 1α,25(OH)2D3 on glycolysis in HIF-1α overexpressed TNBC cells.Compared to the Sh-NC group,VDR knockdown attenuated the inhibitory effect of 1α,25(OH)2D3 on glucose uptake,lactate production and the expression of GLUT1 in MDA-MB-231 cells.qRT-PCR assay results showed that compared to the Si-NC group,VDR knockdown attenuated the inhibitory effect of 1α,25(OH)2D3 on the mRNA expression of HK2,PKM2,and LDHA in 4T1 cells.Conclusion:1.Compared with non-TNBCs,the expression of HIF-1α,GLUT1,and LDHA was higher in TNBC tissues.Compared with normal breast tissues,the expression of HIF-1α,GLUT1,and LDHA was higher in TNBC tissues.Higher expression of HIF-1α,GLUT1,and LDHA was associated with poor overall survival in TNBC patients.2.1α,25(OH)2D3 significantly reduced lung metastasis of TNBC xenograft by inhibiting the expression of HIF-1α,glycolysis,and EMT-related genes.3.1α,25(OH)2D3 inhibited glycolysis and metastasis of TNBC cells by HIF-1α via VDR. |
PDF Full Text Request