The Expression And Mechanism Studies Of BCAT1 In Breast Cancer

Background and objectiveBreast cancer is a malignant tumor and resulted from the canceration of mammary gland epithelial cells.It is one of the malignant tumors that seriously threating the health of women in the world.In developed countries and regions,the morbidity and mortality of breast cancer are among the top of the female malignant tumors.In recent years,treatments for breast cancer have a perpetual progress,and molecular targeted therapy and comprehensive treatment combined by traditional Chinese medicine have become the mains pattern for the treatment of breast cancer.The continuous improvement of treatment on breast cancer has a good prognosis.In china,the incidence of breast cancer has become the first female malignant tumor and has become the top killer of threat to the health of women.Therefore,prevention and treatment on breast cancer is of great significance.Recently,the studies of metabolic reprogramming in human breast cancer have achieved some progress.For instance,pyruvate dehydrogenase kinase-1(PDK1)regulates hypoxia-inducible factor-la(HIF-1?)to trigger metabolic reprogramming and dictates metastatic potential in breast cancer.Branched-chain amino acids(BCAAs),including leucine,isoleucine,and valine,are important nutrient signals that have direct and indirect effects.Plasma BCAAs level is associated with the incident of cardiovascular diseases.In addition,the high serum level of BCAAs has highly significant associations with future diabetes in human,which may due to the effects of BCAAs on insulin resistance.Defects in BCAAs catabolism also causes diseases.For instance,impaired amino acid transport at the blood-brain barrier decreases normal levels of brain BCAAs and induces the metabolic defect in BCAAs.In addition,the BCAA catabolic defect is a metabolic hallmark of failing heart.BCAAs catabolic defect imposes a significant contribution to heart failure.BCAAs also participates in the development of cancer.Elevated plasma levels of BCAAs in humans are associated with a greater than two-fold increased risk of future pancreatic cancer diagnosis,which was confirmed in rodent models.Branched-chain amino acid transaminase 1(BCAT1)is the enzyme that initiates the catabolism of BCAAs.In human gliomas carrying wild-type isocitrate dehydrogenase 1,BCAT1 promotes cell proliferation through amino acid catabolism.In non-small cell lung carcinoma(NSCLC),tumors incorporate free BCAAs into tissue protein and use BCAAs as a nitrogen source.Loss of BCAT1 and BCAT2 impairs NSCLC tumor formation.However,the role of BCAAs metabolism in human breast cancer remains unknown.In this paper,we aimed to explore the potential role of BACC metabolism in human breast cancer.The whole dissertation can be divided into four parts.In the first part,we collected twenty human patients with breast cancer and ten healthy donors from 2010?2015 at Shandong Provincial Hospital Affiliated to Shandong University.We performed LC-MS and RT-PCR to explore the expression of BACC and key enzymes in serum and tissues of breast cancer patients.In the second part,we used shRNA and siRNA transfection technique to disturb the expression of BCAT1 in breast cancer cell lines MCF-7 and T47D.The influence of BCAT1 after transfection on the proliferation and cloning formation was observed.In the third part,we used RT-PCR.spectrophotometry and mitoSOX staining to determine the expression of regulators of mitochondrial biogenesis,the activity of citrate synthase,cellular ATP level,mitochondrial ROS and antioxidants to explore the influence of BCAT1 on mitochondrial biogenesis and function.In the fourth part,we explored the mechanism about the effect of BCAT1 on the proliferation and cloning formation of breast cancer cells by determining the expression of mTOR,mitochondrial DNA content and mitochondrial ROS.Part IExpression of BACC and core enzyme in breast cancerObjective:1.To explore the level of BACC in serum and tissues of breast cancer patients.2.To explore the expression of core enzymes involved in BCAAs metabolism.Methods:1.Two hundred cases of breast cancer patients and one hundred of healthy volunteers were collected from 2010 to 2015 at the Provincial Hospital Affiliated to Shandong University.The plasma,tumor tissues and the adjacent tissues were collected from breast cancer patients and then are kept at-80?.2.Liquid chromatography-mass spectrometry(LC-MS)was used to determine the profile of plasma level of amino acids in breast cancer patients and normal people.3.The expression of leucine,isoleucine,and valine in plasma of breast patients and the healthy volunteers,and in tumor tissues and the adjacent tissues of breast cancer patients.4.RT-PCR was used to determine the expression of BCAT1 in breast cancer patients and healthy volunteers.Results:1.Compared with the normal,the expression of leucine,isoleucine,and valine in breast cancer patients was significantly upregulated and the difference is statistically significant(p<0.05).2.The plasma levels of leucine,isoleucine,and valine in breast cancer patients was significantly higher than the normal(p<0.05).3.The expression of leucine,isoleucine,and valine in tumor tissues was higher than the adjacent tissues of breast cancer patients(p<0.05).4.The expression levels of BCAT1 in tumor tissues was higher than the adjacent tissues of breast cancer patients(p<0.05).Conclusions:In conclusion,the expression of BACC and BCAT1 was increased markedly in breast cancer patients(p<0.05),which indicated BCAAs metabolism was activated in human breast cancer tissues.Part ?Interference on the expression BCAT1 influences the growth and cloning formation of breast cancer cellsObjective:1.To explore the influence of BCAT1 overexpression on the proliferation and cloning formation in breast cancer.2.To explore the influence of interference on BCAT1 expression on the proliferation and cloning formation in breast cancer.Methods:1.Purchased transfection plasmid was transfected to breast cancer cells and blank plasmid was taken as the control.We constructed BCAT1 silent group(shBCAT1),control group(shCtrl),BCAT1 overexpression group(siCtrl)and control group(Ctrl).Western blotting was used to determine the transfection result.2.CCK-8 assay was used to determine the effect of BCAT1 overexpression and knockdown on the proliferation of breast cancer cells.3.Cloning formation assay was used to determine the influence of BCAT1 overexpression and knockdown on cloning formation of' MCF-7 and T47D cells.Results:1.Results of western blotting showed the protein expression level of BCAT1 in MCF-7and T47D cell lines in shBCAT1 group was effectively decreased compared with control group(P<0.05)after transfection for 72h.However.the protein expression level of BCAT1 in MCF-7and T47D cell lines in BCAT1 group was effectively increased(P<0.05)2.Results of CCK-8 showed the proliferation capacity of MCF-7and T47D cell lines began to decrease dramatically at 24h compared with shCtrl group(P<0.05).Results of cloning formation assay showed the number of cloning cells in shBCAT1 group reduced significantly compared with shCtrl group(P<0.05).3.Results of CCK-8 showed the proliferation capacity of breast cancer cells in BCAT1 group began to increased dramatically at 24h compared with Ctrl group(P<0.05).Results of cloning formation assay showed the number of cloning cells in BCATI group increased significantly compared with Ctrl group(P<0.05).Conclusions:1.Lentivirus and plasmid transfection could effectively upregulate or downregulate the expression of BCAT1.2.BCAT1 overexpression could promote the proliferation and cloning formation of breast cancer cells,while BCAT1 knockdown could inhibit the proliferation and cloning formation of breast cancer cells.Part ?BCAT1 regulates mitochondrial biogenesis and functionObjective:1.To explore the role of BCAT1 on mitochondrial biogenesis.2.To explore the role of BCAT1 on mitochondrial function.Methods:1.To explore the role of BCAT1 on mitochondrial biogenesis and function by determining mitochondrial DNA content in breast cancer cell lines.2.RT-PCR was used to determine the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC1?),nuclear respiratory factor 1(NRF-1),mitochondrial transcription factor A(TFAM).and ?-F1-ATPase in Ctrl,BCAT1,shCtrl and shBCAT1 group.3.Spectrophotometry was used to determine the activity of citrate synthase in shCtrl and shBCAT1 group.4.mitoSOX staining was used to determine mitochondrial ROS in breast cancer cell lines MCF-7/T47D in shCtrl and shBCAT1 group.5.RT-PCR was used to determine the mRNA levels of antioxidants(SOD1,SOD2,catalase,and Gpxl)in shCtrl and shBCAT1 group.Results:1.BCAT1 knockdown decreased mitochondrial DNA content,while BCAT1 overexpression increased mitochondrial DNA content in MCF-7and T47D cell lines2.BCAT1 knockdown decreased the mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC1?),nuclear respiratory factor 1(NRF-1),mitochondrial transcription factor A(TFAM),and P-F1-ATPase,while BCAT1 overexpression has the opposing results.3.BCAT1 knockdown decreased the activity of citrate synthase,reduced cellular ATP level,repressed the mRNA level of antioxidants(SOD1,SOD2,catalase,and Gpx1),and induced mitochondrial ROS.Conclusions:1.BCAT1 could affect mitochondrial biogenesis by increasing mitochondrial number and enhancing regulators.2.BCAT1 could affect mitochondrial function by increasing the activity of citrate synthase,cellular ATP level,mitochondrial ROS,antioxidants(SOD1,SOD2,catalase,and Gpx1)and mitochondrial ROS.Part IVInfluence of BCAT1 on mTOR in breast cancer cellsObjective:1.To explore the influence of BCAT1 on phosphorylation of AMPK and mTOR.2.To explore the role of mTOR affected by BCAT1 on the proliferation and cloning formation of breast cancer cells.Methods:1.Western blotting assay was used to determine the protein expression of AMPK,SIRT1,mTOR and S6K1 in shCtrl and shBCAT1 group.We also used western blotting assay to determine the protein expression of ?-mTOR.mTOR.?-S6K1and S6K1 in shCtrl,shBCAT1,Ctrl and BCAT1 group.2.Rapamycin was used to inhibit the expression of mTOR in breast cancer cell lines.RT-PCR was used to determine the mRNA levels of PGC1?,NRF-1,Tfam,?-F1-ATPase.SOD1,SOD2,catalase,and Gpx1.3.Mitochondria DNA content,mitochondria ROS.number of cloning cells and situation of cell proliferation in breast cancer cell lines in Ctrl+Vechic.BCAT1+Vechicle,Ctrl+Rapamycin and BCAT1+Rapamycin group was determined.Results:1.BCAT1 knockdown had no influence on the expression of AMPK.SIRT1,mTOR and S6K1.2.Rapamycin could inhibit mTOR signaling,while BCAT1 overexpression had no influence on PGC1?,NRF-1,Tfam,?-F1-ATPase.SOD1,SOD2,catalase,and Gpx1,.3.Inhibition on mTOR could prevent the effect of BCAT1 on mitochondrial biogenesis and mitochondrial ROS.3.Inhibition on mTOR could weaken the promotion of BCAT1 on the proliferation and cloning formation of breast cancer cells.Conclusions:1.BCAT1 regulated mitochondrial biogenesis by mTOR signaling.2.mTOR contributed to the effect of BCAT1 on breast cancer cell growth.