BACKGROUND Metabolic reprogramming has been identified as a core hallmark of cancer.Solute carrier family 2 is a major glucose carrier family.It consists of 14 members,and we mainly study solute carrier family 2 membe...BACKGROUND Metabolic reprogramming has been identified as a core hallmark of cancer.Solute carrier family 2 is a major glucose carrier family.It consists of 14 members,and we mainly study solute carrier family 2 member 1(SLC2A1)and solute carrier family 2 member 2(SLC2A2)here.SLC2A1,mainly existing in human erythrocytes,brain endothelial cells,and normal placenta,was found to be increased in hepatocellular carcinoma(HCC),while SLC2A2,the major transporter of the normal liver,was decreased in HCC.AIM To identify if SLC2A1 and SLC2A2 were associated with immune infiltration in addition to participating in the metabolic reprogramming in HCC.METHODS The expression levels of SLC2A1 and SLC2A2 were tested in HepG2 cells,HepG215 cells,and multiple databases.The clinical characteristics and survival data of SLC2A1 and SLC2A2 were examined by multiple databases.The correlation between SLC2A1 and SLC2A2 was analyzed by multiple databases.The functions and pathways in which SLC2A1,SLC2A2,and frequently altered neighbor genes were involved were discussed in String.Immune infiltration levels and immune marker genes associated with SLC2A1 and SLC2A2 were discussed from multiple databases.RESULTS The expression level of SLC2A1 was up-regulated,but the expression level of SLC2A2 was down-regulated in HepG2 cells,HepG215 cells,and liver cancer patients.The expression levels of SLC2A1 and SLC2A2 were related to tumor volume,grade,and stage in HCC.Interestingly,the expression levels of SLC2A1 and SLC2A2 were negatively correlated.Further,high SLC2A1 expression and low SLC2A2 expression were linked to poor overall survival and relapse-free survival.SLC2A1,SLC2A2,and frequently altered neighbor genes played a major role in the occurrence and development of tumors.Notably,SLC2A1 was positively correlated with tumor immune infiltration,while SLC2A2 was negatively correlated with tumor immune infiltration.Particularly,SLC2A2 methylation was positively correlated with lymphocytes.CONCLUSION SLC2A1 and SLC2A2 are independent therapeutic targets for HCC,and they are quintessential marker molecules for predicting and regulating the number and status of immune cells in HCC.展开更多
背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供...背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。展开更多
Objective·To investigate the role of mitochondrial solute carrier family 25 member 13(SLC25A13)on breast cancer development.Methods·SLC25A13 mRNA and protein expressions in invasive breast cancer tissues and...Objective·To investigate the role of mitochondrial solute carrier family 25 member 13(SLC25A13)on breast cancer development.Methods·SLC25A13 mRNA and protein expressions in invasive breast cancer tissues and normal breast tissues were from The Cancer Genome Atlas(TCGA)breast cancer dataset.Survival analysis was conducted online by Kaplan-Meier software.MCF-7 cell line was used for in vitro cell assay.Knockdown of SLC25A13 and sirtuin 2(SIRT2)were conducted by siRNA transfection.Cell viability was measured with trypan blue exclusion.Cell cycle arrest was determined by flow cytometry.The mRNA expression of SLC25A13 and P27 were detected by quantitative PCR.The protein level of SLC25A13,P27 and SIRT2 were detected by Western blotting.Protein half-life of P27 was assessed by Western blotting after cycloheximide treatment.Results·SLC25A13 was up-regulated in invasive breast cancer tissues.High expression of SLC25A13 correlated with poor overall survival and breast cancer recurrence.SLC25A13 knockdown inhibited MCF-7 cell cycle progression.P27 and SIRT2 both accumulated after SLC25A13 knockdown.P27 accumulation resulted from prolonged protein half-life.Knockdown of SIRT2 restored cell cycle arrest as well as P27 accumulation caused by SLC25A13 silencing.Conclusion·High expression of SLC25A13 may promote cell cycle progression via SIRT2 in breast cancer development.展开更多
基金Supported by National Natural Science Foundation of China,No.81873112Natural Science Foundation of Hebei Province,No.H2020423009+2 种基金Hundred Outstanding Innovative Talents Support Program of Universities in Hebei Province,No.SLRC2019043Basic Scientific Research Project of Hebei Provincial Colleges and Universities,No.JTZ2020005Scientific and Technological Capability Improvement Project of the Hebei University of Chinese Medicine,No.KTZ2019002.
文摘BACKGROUND Metabolic reprogramming has been identified as a core hallmark of cancer.Solute carrier family 2 is a major glucose carrier family.It consists of 14 members,and we mainly study solute carrier family 2 member 1(SLC2A1)and solute carrier family 2 member 2(SLC2A2)here.SLC2A1,mainly existing in human erythrocytes,brain endothelial cells,and normal placenta,was found to be increased in hepatocellular carcinoma(HCC),while SLC2A2,the major transporter of the normal liver,was decreased in HCC.AIM To identify if SLC2A1 and SLC2A2 were associated with immune infiltration in addition to participating in the metabolic reprogramming in HCC.METHODS The expression levels of SLC2A1 and SLC2A2 were tested in HepG2 cells,HepG215 cells,and multiple databases.The clinical characteristics and survival data of SLC2A1 and SLC2A2 were examined by multiple databases.The correlation between SLC2A1 and SLC2A2 was analyzed by multiple databases.The functions and pathways in which SLC2A1,SLC2A2,and frequently altered neighbor genes were involved were discussed in String.Immune infiltration levels and immune marker genes associated with SLC2A1 and SLC2A2 were discussed from multiple databases.RESULTS The expression level of SLC2A1 was up-regulated,but the expression level of SLC2A2 was down-regulated in HepG2 cells,HepG215 cells,and liver cancer patients.The expression levels of SLC2A1 and SLC2A2 were related to tumor volume,grade,and stage in HCC.Interestingly,the expression levels of SLC2A1 and SLC2A2 were negatively correlated.Further,high SLC2A1 expression and low SLC2A2 expression were linked to poor overall survival and relapse-free survival.SLC2A1,SLC2A2,and frequently altered neighbor genes played a major role in the occurrence and development of tumors.Notably,SLC2A1 was positively correlated with tumor immune infiltration,while SLC2A2 was negatively correlated with tumor immune infiltration.Particularly,SLC2A2 methylation was positively correlated with lymphocytes.CONCLUSION SLC2A1 and SLC2A2 are independent therapeutic targets for HCC,and they are quintessential marker molecules for predicting and regulating the number and status of immune cells in HCC.
基金Morning Star Prograrm of Shanghai Municipal Science and Technology Commission,11QA1403700Shanghai College Experimental Technology Team Building PlanK.C.Wong Medical Fellowship Fund,Shanghai Jiao Tong University~~
文摘Objective·To investigate the role of mitochondrial solute carrier family 25 member 13(SLC25A13)on breast cancer development.Methods·SLC25A13 mRNA and protein expressions in invasive breast cancer tissues and normal breast tissues were from The Cancer Genome Atlas(TCGA)breast cancer dataset.Survival analysis was conducted online by Kaplan-Meier software.MCF-7 cell line was used for in vitro cell assay.Knockdown of SLC25A13 and sirtuin 2(SIRT2)were conducted by siRNA transfection.Cell viability was measured with trypan blue exclusion.Cell cycle arrest was determined by flow cytometry.The mRNA expression of SLC25A13 and P27 were detected by quantitative PCR.The protein level of SLC25A13,P27 and SIRT2 were detected by Western blotting.Protein half-life of P27 was assessed by Western blotting after cycloheximide treatment.Results·SLC25A13 was up-regulated in invasive breast cancer tissues.High expression of SLC25A13 correlated with poor overall survival and breast cancer recurrence.SLC25A13 knockdown inhibited MCF-7 cell cycle progression.P27 and SIRT2 both accumulated after SLC25A13 knockdown.P27 accumulation resulted from prolonged protein half-life.Knockdown of SIRT2 restored cell cycle arrest as well as P27 accumulation caused by SLC25A13 silencing.Conclusion·High expression of SLC25A13 may promote cell cycle progression via SIRT2 in breast cancer development.
