| PURPOSE Breast cancer has a high incidence and is considered to be one of the most common malignant cancers in women in the world.Although significant progress has been made in diagnosis and treatment,the long-term survival rate of breast cancer patients is still very low,posing a greater threat to women’s health.Breast cancer cells have the characteristics of immortal proliferation,accelerated growth,and abnormal energy metabolism.Aerobic glycolysis is one of the important signs of abnormal metabolic regulation,also known as the "Warburg effect".Hypoxia-inducible factor-1α(HIF-1α)is a key regulator of aerobic glycolysis of tumor cells.It participates in tumor cell energy metabolism and angiogenesis,thereby affecting cell proliferation and metastasis.Monocarboxylic acid transporter 4(MCT4)is an important factor for the transport of lactic acid output.Its expression is related to the high glycolysis of cells and the poor prognosis of tumors.However,the mechanism of HIF-1α/MCT4 in breast cancer aerobic glycolysis is still unclear.This study aims to explore the role and mechanism of HIF-1α/MCT4 in the metabolic pathways of aerobic glycolysis and cell proliferation in breast cancer MDA-MB-231 and MCF-7 cells.MATERIALS AND METHODS(1)Transiently transfected small interfering RNA(si RNA)was used to silence the expression of HIF-1α and MCT4 in MDA-MB-231 and MCF-7 cells,and the transfection efficiency of HIF-1α and MCT4 was detected by q RT-PCR,Screen out the appropriate interference sequence and concentration.(2)The effect of HIF-1α and MCT4 on aerobic glycolysis of MDA-MB-231 and MCF-7 cells was preliminarily determined by measuring glucose consumption and lactic acid accumulation after effective silences of HIF-1α and MCT4 expression in MDA-MB-231 and MCF-7 cells.(3)Effectively down-regulate the expression of HIF-1α and MCT4 in MDA-MB-231 and MCF-7,and detect the correlation of glycolysis pathway in cells by fluorescence quantitative PCR Enzyme m RNA expression level.(4)Divide MDA-MB-231 and MCF-7 cells into si RNA group,negative control group and untreated group.Incu Cyte S3 was used to detect the cell proliferation ability and analyze the effect of silencing HIF-1α and MCT4 expression on mammary glands.The effect of cancer cell proliferation.Using SPSS22.0 statistical software for statistical analysis,P<0.05 considered the difference to be statistically significant.RESULTS(1)Fluorescence quantitative PCR results showed that in MDA-MB-231 and MCF-7 cells,the m RNA expression level of the two target gene sequences specifically silenced HIF-1αdecreased significantly at a concentration of 100 n M.Similarly,the m RNA expression level of the two target gene sequences of specific silencing MCT4 decreased significantly at a concentration of 100 n M.Therefore,the sequence with better effect is selected for subsequent experiments.(2)After effectively silencing HIF-1α expression,the glucose consumption of MDA-MB-231 and MCF-7 cell si HIF-1α experimental group was(8.25883±0.73676)and(18.41963±1.52361)respectively,which were significantly lower than the negative control group(10.83626±0.90079),(27.13035±0.50939),the difference was statistically significant(P<0.05);the lactic acid production in the si HIF-1α experimental group was(1.85609±0.00337)and(3.21013±0.08225),which were significantly lower than negative The control group(3.3161±0.56836),(3.95045±0.06789),the difference was statistically significant(P<0.05);after effectively silencing the expression of MCT4,the glucose consumption of MDA-MB-231 cell si MCT4 experimental group was(9.37602±1.47343),Compared with the negative control group(9.08992±2.08014),there was no significant difference(P>0.05),while the glucose consumption of the MCF-7 cell si MCT4 experimental group was(18.79896±1.73912),which was significantly lower than the negative control group(27.13035±0.50939).The difference was statistically significant(P<0.05);the lactic acid production in the si MCT4 experimental group of MDA-MB-231 and MCF-7cells were(2.24794±0.03586)and(1.7281±0.15472),which were significantly lower than the negative control group(3.3161 ±0.56836),(3.95045±0.06789),the difference was statistically significant(P<0.05).It shows that inhibiting HIF-1α and MCT4 can reduce the glycolytic activity of breast cancer cells.(3)After the expression of HIF-1αwas effectively down-regulated,the m RNA expression levels of glycolysis-related enzymes GLTU1,LDHA and MCT4 in MDA-MB-231 and MCF-7 cells were significantly reduced,and the difference was statistically significant(P<0.05)).It shows that inhibiting HIF-1α can reduce the activity of glycolysis pathway-related enzymes;after down-regulating the expression of MCT4,in MDA-MB-231 and MCF-7 cells,glycolysis-related enzymes GLTU1 and LDHA m RNA expression levels were significantly decreased,and HIF-The expression level of 1α was also suppressed,and the difference was statistically significant(P<0.05).It indicates that MCT4 may be involved in the regulation of glycolytic enzyme activity,and inhibition of MCT4 expression may induce HIF-1α inactivation.(4)After the expression of HIF-1α was effectively down-regulated,the proliferation activity of MDA-MB-231 and MCF-7 cells was weakened(P<0.05);similarly,after the expression of MCT4 was effectively inhibited,MDA-MB-231 and MCF-7 cells The proliferation activity was also weakened(P<0.05).It shows that inhibiting the expression of HIF-1α and MCT4 can attenuate the proliferation activity of breast cancer cells.CONCLUSION(1)Down-regulating the expression of HIF-1α and MCT4 can reduce the glycolytic activity of breast cancer MDA-MB-231 and MCF-7 cells.(2)Down-regulating the expression of HIF-1α and MCT4 can attenuate the proliferation activity of breast cancer MDA-MB-231 and MCF-7 cells. |
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