RBM5蛋白的RNA识别区在肿瘤细胞周期调控中作用的研究
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摘要
为分析RNA识别区(RNA recognition motif,RRM)在RBM5蛋白调控肿瘤细胞周期中的作用,本研究利用低表达RBM5的A549细胞系构建了A549/Vector、A549/RBM5-WT、A549/RBM5-△RRM三种稳定细胞株,通过流式细胞术检测,发现仅有A549/RBM5-WT能显著阻滞细胞于G1期,说明RRM区是RBM5抑制细胞周期进程必不可少的区域.利用Western Blot检测细胞中cyclin A与Phospho-Rb(ser 795)的蛋白水平差异,证明了RBM5蛋白的RRM能够参与其介导的pRb去磷酸化以及抑制cyclin A表达,从而抑制细胞周期进程.
In order to analyze the role of RNA recognition region(RRM) in the regulation of tumor cell cycle by RBM5 protein, three stable cell lines A549/Vector, A549/RBM5-WT, A549/RBM5-△RRM were constructed by using A549 cell line with low expression of RBM5. The phase of cell cycle analysis by Flow cytometry showed that only A549/RBM5-WT was arrested in G1 phase, indicating that the RRM region was essential for RBM5 to inhibit cell cycle progression. Using Western Blot to detect the difference between cyclin A and Phospho-Rb(ser 795), it was proved that RRM of RBM5 protein could participate in pRb dephosphorylation mediated by RBM5 and inhibit cyclin A expression, thus inhibiting cell cycle progression.
In order to analyze the role of RNA recognition region(RRM) in the regulation of tumor cell cycle by RBM5 protein, three stable cell lines A549/Vector, A549/RBM5-WT, A549/RBM5-△RRM were constructed by using A549 cell line with low expression of RBM5. The phase of cell cycle analysis by Flow cytometry showed that only A549/RBM5-WT was arrested in G1 phase, indicating that the RRM region was essential for RBM5 to inhibit cell cycle progression. Using Western Blot to detect the difference between cyclin A and Phospho-Rb(ser 795), it was proved that RRM of RBM5 protein could participate in pRb dephosphorylation mediated by RBM5 and inhibit cyclin A expression, thus inhibiting cell cycle progression.
引文
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[22] 陈志红,钱海鑫,马圭,等.p53在结肠癌中的表达及临床意义 [J].江苏大学学报:医学版,2004,14:44.
[23] 许接天,胡兰琳,彭确昆,等.基于表达谱分析筛选肾母细胞瘤关键基因 [J].四川大学学报:自然科学版,2018,55:415.
[24] Song Z,Wu P,Ji P,et al.Solution structure of the second RRM domain of RBM5 and its unusual binding characters for different RNA targets [J].Biochemistry,2012,51:6667.引用本文格式:中文:王迪,吴传芳.RBM5蛋白的RNA识别区在肿瘤细胞周期调控中作用的研究 [J].四川大学学报:自然科学版,2019,56:531.英文:Wang D,Wu C F.Research on the role of RNA recognition motif domains of RBM5 protein in theregulation of tumor cell cycle [J].J Sichuan Univ:Nat Sci Ed,2019,56:531.
[2] 万杨,吕涛,肖智雄.p53突变蛋白对人肺腺癌细胞中E-cadherin的表达调控及细胞迁移的影响 [J].四川大学学报:自然科学版,2016,53:657.
[3] Wang C Y,Xu Z B,Wang J P,et al.Rb deficiency accelerates progression of carcinoma of the urinary bladder in vivo and in vitro through inhibiting autophagy and apoptosis [J].Int J Oncol,2017,50:1221.
[4] Abbas T,Dutta A.p21 in cancer:intricate networks and multiple activities [J].Nat Rev Cancer,2009,9:400.
[5] Feng H,Zhang Z,Qing X,et al.Promoter methylation of APC and RAR-β genes as prognostic markers in non-small cell lung cancer (NSCLC) [J].Exp Mol Pathol,2015,100:109.
[6] Timmer T,Terpstra P,Van d B A,et al.A comparison of genomic structures and expression patterns of two closely related flanking genes in a critical lung cancer region at 3p21.3 [J].Eur J Hum Genet,1999,7:478.
[7] Lerman M I,Minna J D.The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3:identification and evaluation of the resident candidate tumor suppressor genes [J].Cancer Res,2000,60:6116.
[8] Mourtadamaarabouni M,Keen J,Clark J,et al.Candidate tumor suppressor LUCA-15/RBM5/H37 modulates expression of apoptosis and cell cycle genes [J].Exp Cell Res,2006,312:1745.
[9] Oh J J,Razfar A,Delgado I,et al.3p21.3 tumor suppressor gene H37/Luca15/RBM5 inhibits growth of human lung cancer cells through cell cycle arrest and apoptosis [J].Cancer Res,2006,66:3419.
[10] Bonnal S,Martínez C,F?rch P,et al.RBM5/Luca-15/H37 regulates fas alternative splice site pairing after exon definition [J].Mol Cell,2008,32:81.
[11] Fushimi K,Ray P,Kar A,et al.Up-Regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor,RBM5 [J].Proc Natl Acad Sci U S A,2008,105:15708.
[12] Kobayashi T,Ishida J,Musashi M,et al.p53 transactivation is involved in the antiproliferative activity of the putative tumor suppressor RBM5 [J].Int J Cancer,2011,128:304.
[13] Ji L,Minna J D,Roth J A.3p21.3 tumor suppressor cluster:prospects for translational applications [J].Future Oncol,2005,1:79.
[14] Zhao L,Li R,Shao C,et al.3p21.3 tumor suppressor gene RBM5 inhibits growth of human prostate cancer PC-3 cells through apoptosis [J].World J Surg Oncol,2012,10:247.
[15] Hong L,Jie Z,Chen S,et al.Differential expression of RBM5,EGFR and KRAS mRNA and protein in non-small cell lung cancer tissues [J].J Exp Clin Cancer Res,2012,31:36.
[16] Mour?o A,Bonnal S,Soni K,et al.Structural basis for the recognition of spliceosomal SmN/B/B’ proteins by the RBM5 OCRE domain in splicing regulation [J].Elife,2016,5:e14707.
[17] Soucek T,Pusch O,Hengstschl?gerottnad E,et al.Deregulated expression of E2F-1 induces cyclin A- and E-associated kinase activities independently from cell cycle position [J].Oncogene,1997,14:2251.
[18] Mudryj M,Devoto S H,Hiebert S W,et al.Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A [J].Cell,1991,65:1243.
[19] Dyson N.The regulation of E2F by pRB-family proteins [J].Genes Dev,1998,12:2245.
[20] Van d H S,Dyson N J.Conserved functions of the pRB and E2F families [J].Nat Rev Mol Cell Biol,2008,9:713.
[21] Cao L,Peng B,Yao L,et al.The ancient function of RB-E2F pathway:insights from its evolutionary history [J].Biol Direct,2010,5:55.
[22] 陈志红,钱海鑫,马圭,等.p53在结肠癌中的表达及临床意义 [J].江苏大学学报:医学版,2004,14:44.
[23] 许接天,胡兰琳,彭确昆,等.基于表达谱分析筛选肾母细胞瘤关键基因 [J].四川大学学报:自然科学版,2018,55:415.
[24] Song Z,Wu P,Ji P,et al.Solution structure of the second RRM domain of RBM5 and its unusual binding characters for different RNA targets [J].Biochemistry,2012,51:6667.引用本文格式:中文:王迪,吴传芳.RBM5蛋白的RNA识别区在肿瘤细胞周期调控中作用的研究 [J].四川大学学报:自然科学版,2019,56:531.英文:Wang D,Wu C F.Research on the role of RNA recognition motif domains of RBM5 protein in theregulation of tumor cell cycle [J].J Sichuan Univ:Nat Sci Ed,2019,56:531.