The Expression And Function Of BCAT1 In Melanoma

BackgroundMelanoma is the most malignant tumor in skin cancer,with a mortality rate of about 80%.The survival rate of patients with melanoma after traditional treatment can reach 95% within 5 years,but the survival rate of patients with metastatic melanoma is only about 10% within 5 years.The main reason for inducing the occurrence of melanoma is the excessive irradiation of ultraviolet rays,which makes the irradiated parts cancerous.Studies have found that malignant melanoma can occur in pigment cells in various parts of the body,most of them occur in the skin,and a few occur in the eyes,ears,meninges and other parts.Because melanoma is prone to metastasis,traditional treatment is not optimistic.Up to now,the number of melanoma cases in the world is 2 to 3 million cases and is still increasing.At the same time,its case fatality rate is also increasing.It is the tumor type with the fastest increase in the number of cases every year.Therefore,melanoma has become one of the important diseases that endanger human health.Branched-chain amino acids(BCAAs)include leucine,isoleucine,and valine.Branched-chain amino acid transaminase(BCAT)is the first step to catalyze the decomposition of branched-chain amino acids(BCAT)Of enzymes.BCAT includes two types: BCAT1 and BCAT2.BCAT1 is located in the cytoplasm and BCAT2 is located in the mitochondria.The two isozymes transfer the α-amino group of BCAAs to α-ketoglutarate to produce glutamate and corresponding branches Keto acid.BCAT1 is associated with the prognosis of various cancers,Studies have shown that BCAT1 is highly expressed in a variety of tumor types,such as liver cancer,non-small cell lung cancer,ovarian cancer and breast cancer,but is low in pancreatic ductal adenocarcinoma,so the expression level of BCAT1 varies depending on the tumor type.BCAT1 participates in tumor metabolic reprogramming,and promotes the proliferation,migration and invasion of tumor cells,thereby promoting tumor development.Therefore,BCAT1 gene is also called "longevity gene".However,there is no research on the expression and function of BCAT1 in melanoma.We will first explore the differential expression of BCAT1 in melanoma tumors and normal tissuesand further study the function of BCAT1 in melanoma cells,which may become a new molecular target for melanoma treatment.ObjectiveExplore the differential expression of BCAT1 in melanoma tissues and normal tissues,and explore the effect of reducing BCAT1 expression on cell proliferation,migration,and metabolism by constructing stable BCAT1 knockdown cell lines to provide a theoretical basis for the treatment of melanoma.Methods1.The differential expression of BCAT1 in melanoma tissues and normal tissues was determined by immunohistochemical test to detect tissue staining intensity and positive cell staining rate.2.Construction of lentivirus and transfection of B16 cells,selection of three stable transfected cell lines of B16-BCAT1 sh NC,B16-BCAT1sh1,B16-BCAT1sh2 by puromycin.3.Explore the effect of reducing BCAT1 expression on the proliferation,cycle,migration and other functions of melanoma B16 cells.(1)The effect of reducing BCAT1 expression on the proliferation of B16 cells by real-time label-free cell function instrument(RTCA).(2)The effect of reducing the expression of BCAT1 on the cloning ability of B16 cells was detected by cloning experiment.(3)The effect of reducing BCAT1 expression on B16 cell cycle by PI staining cell cycle test.(4)Explore the effect of reducing the expression of BCAT1 on the migration ability of B16 cell line through scratch experiments.4.The effect of knocking down BCAT1 on glycolysis and mitochondrial respiration of B16 cells was detected by XF seahorse energy metabolism experiment.(1)Glycolysis stress test to detect the effect of knocking down BCAT1 on the rate of extracellular acidification of glycolysis in B16 cells.(2)The mitochondrial stress test detects the effect of knocking down BCAT1 on the oxygen consumption of mitochondrial aerobic respiration of B16 cells.5.Targeted-energy metabolite detection explores the effect of knocking down BCAT1 on metabolites during glycolysis,TCA cycle and oxidative phosphorylation.6.To investigate the expression of EMT pathway markers and the expression of key enzymes of mitochondrial function in B16-BCAT1 sh NC and B16-BCAT1sh2 cells by Western blot.Results1.The results of immunohistochemical experiments showed that BCAT1 expression in malignant melanoma tissues was higher than normal tissues.2.Western blot detection of BCAT1 protein expression levels in 5 cell lines including B16,A2508,A375,Melan-α and M14.The results showed that BCAT1 was relatively highly expressed in B16 cells.3.Lentivirus transfection into B16 cells induced a decrease in BCAT1 expression.Cell function experiments showed that knocking down BCAT1 inhibited the proliferation,migration and G2/M cell cycle of B16 cells.4.The results of glycolysis stress test showed that the ECAR value(extracellular acidification rate)of B16-BCAT1-sh2 cells increased after exogenous glucose supplementation and the glycolysis process was enhanced compared with the control.After adding oligomycin,the function of ATP synthase was inhibited,and the ECAR value did not change,indicating that knocking down BCAT1 had no effect on the maximum value of glycolysis.Adding 2-DG inhibits hexokinase activity,and the ECAR value decreases,indicating that the BCAT1 reserve value for inhibiting glycolysis is knocked down.5.The mitochondrial stress test showed that knockdown of BCAT1 inhibited the mitochondrial oxygen consumption of the B16 cell and resulted in a significant decrease in the basal value,ATP content,maximum value and reserve value of the mitochondrial stress test.6.Targeted-energy metabolic substance detection found that knocking down BCAT1 resulted in a significant reduction in acetyl-Co A metabolism and adenosine metabolism in the mitochondrial TCA cycle,as well as a significant reduction in FMN and NAD content during oxidative phosphorylation.7.Western blot results showed that mitochondrial function-related enzymes such as CS(citrate synthase),PDH(pyruvate dehydrogenase),SDH(succinate dehydrogenase)and other key enzymes decreased.Conclusions1.BCAT1 expression in malignant melanoma tissues is higher than normal tissues,knocking down BCAT1 inhibits B16 cell proliferation,migration,and G2 / M cell cycle.2.Knockdown of BCAT1 increases the glycolytic function of B16 cells but inhibits mitochondrial metabolism,and inhibits the metabolism levels of Acetyl-Co A,adenosine,FMN and NAD in B16 cells.3.Knockdown of BCAT1 increases E-cadherin expression in B16 cells and inhibits Vimentin expression,thereby inhibiting the EMT pathway.4.Knockdown of BCAT1 inhibits the expression of mitochondrial function-related proteins in B16 cells and thus inhibits mitochondrial metabolic activity.