转运RNA来源的分子片段770靶向细胞骨架相关蛋白2对乳腺癌细胞增殖的影响  

Effect of transfer RNA-derived molecule fragment 770 on proliferation of breast cancer cells by targeting cytoskeletal-associated protein 2

作　　者：汤珣 王勇[2] 严枫[1] Tang Xun;Wang Yong;Yan Feng(Department of Clinical Laboratory,the Affiliated Cancer Hospital of Nanjing Medical University,Jiangsu Cancer Hospital,Jiangsu Institute of Cancer Research,Jiangsu Key Laboratory of Cancer Molecular Biology and Translational Medicine,Nanjing 210009,China;State Key Laboratory of Analytical Chemistry for Life Science,Medical School,Nanjing University,Jiangsu Key Laboratory of Molecular Medicine,Nanjing 210093,China)

机构地区：[1]南京医科大学附属肿瘤医院,江苏省肿瘤医院,江苏省肿瘤防治研究所检验科,江苏省恶性肿瘤分子生物学及转化医学重点实验室,南京210009 [2]南京大学医学院生命分析化学国家重点实验室,江苏省医学分子技术重点实验室,南京210093

出　　处：《肿瘤研究与临床》2022年第11期801-806,共6页Cancer Research and Clinic

基　　金：国家自然科学基金(82003077);江苏省卫生健康委员会科研项目(H2019070)。

摘　　要：目的探讨转运RNA来源的小分子片段770(tRF-770)调控细胞骨架相关蛋白2(CKAP2)对乳腺癌细胞增殖的影响。方法使用MINTbase数据库v2.0序列与基因组中成熟的tRNA进行比对,确认tRF-770的染色体定位;TA克隆实验鉴定tRF-770;利用TargetScan和miRBase数据库分析预测tRF-770的靶基因;利用癌症基因组图谱(TCGA)数据库数据分析CKAP2在乳腺癌中的表达情况。选择乳腺癌细胞株MDB-MA-231和MCF7,分别分为空白对照组(不予任何处理)、tRF-770过表达组(转染tRF-770过表达序列)及阴性对照组(转染阴性对照序列);另外设立tRF-770过表达+CKAP2-HA组(共转染tRF-770过表达序列与CKAP2过表达序列)。采用实时荧光定量聚合酶链反应(qRT-PCR)检测乳腺癌细胞中tRF-770的相对表达量;CCK-8法检测乳腺癌细胞增殖能力并进行挽救实验;双荧光素酶报告基因实验验证tRF-770的靶基因;蛋白印迹法检测CKAP2-ERK2信号通路相关蛋白的表达情况。结果tRF-770与9类tRNA剪切修饰的5’’端完全匹配,TA克隆测序验证结果表明产物大小以及碱基与预期tRF-770序列一致。基于TCGA数据库数据分析发现CKAP2在乳腺癌组织中高表达(t=7.21,P<0.05)。qRT-PCR检测结果显示,在MDA-MB-231细胞中,空白对照组、阴性对照组、tRF-770过表达组tRF-770相对表达量分别为1.00±0.00、2.42±0.11、3.75±0.01,差异有统计学意义(F=1395.00,P<0.001);在MCF7细胞中,3组tRF-770相对表达量分别为1.00±0.00、2.45±0.21、3.26±0.16,差异有统计学意义(F=169.30,P<0.001);两种细胞中,与空白对照组及阴性对照组比较,tRF-770过表达组中tRF-770相对表达量均升高(均P<0.05)。双荧光素酶报告基因实验结果表明,tRF-770与CKAP2 mRNA 3’’UTR结合。CCK-8法检测结果显示,在MDA-MB-231、MCF7细胞中,第3天和第4天tRF-770过表达组细胞增殖能力均低于空白对照组和阴性对照组(均P<0.05);第3天和第4天阴性对照组与tRF-770过表达组细胞增殖能力�Objective To explore the effect of transfer RNA-derived small molecule fragment 770(tRF-770)on proliferation of breast cancer cells through regulating cytoskeletal-associated protein 2(CKAP2).Methods Chromosome localization of tRF-770 was identified using the MINTbase database v2.0 sequence alignment with mature tRNA in the genome.TA cloning assay was used to identify tRF-770;TargetScan and miRBase database were used to analyze and predict the target genes of tRF-770.The expression of CKAP2 in breast cancer was analyzed by using the data from The Cancer Genome Atlas(TCGA)database.Breast cancer cell lines MDB-MA-231 and MCF7 were selected and divided into three groups:blank control group(without any treatment),tRF-770 overexpression group(transfected with tRF-770 overexpression sequence)and negative control group(transfected with negative control sequence).In addition,tRF-770 overexpression+CKAP2-HA group was established(co-transfected with tRF-770 overexpression sequence and CKAP2 overexpression sequence).Real-time quantitative fluorescence polymerase chain reaction(qRT-PCR)was used to detect the relative expression of tRF-770 in breast cancer cells.CCK-8 assay was used to detect the proliferation of breast cancer cells and perform rescue experiment.Dual luciferase reporter gene assay was used to verify the target genes of tRF-770.The protein expression of CKAP2-ERK2 signaling pathway was detected by Western blotting.Results tRF-770 completely matched 5'UTR spliced and modified by 9 kinds of tRNA.TA clone sequencing verification results showed that the product size and bases were consistent with the expected tRF-770 sequences.CKAP2 was highly expressed in breast cancer tissues based on analysis of the data from TCGA database(t=7.21,P<0.05).qRT-PCR showed that the relative expressions of tRF-770 in MDA-MB-231 cells of blank control group,negative control group and tRF-770 overexpression group were 1.00±0.00,2.42±0.11 and 3.75±0.01,respectively,and the difference was statistically significant(F=1395.00,P<0.001).

关 键 词：乳腺肿瘤 转运RNA 细胞增殖 细胞骨架相关蛋白2 

分 类 号：R737.9[医药卫生—肿瘤]