RSL1D1在肿瘤细胞中的功能及其分子机制研究

发布时间：2018-10-18 12:01

【摘要】：RSL1D1(ribosomal L1-domain-containing 1),即细胞衰老抑制基因(CSIG,cellular senescence-inhibited gene),编码一种核糖体蛋白,属核糖体L1p/L10e家族。RSL1D1蛋白主要定位于核仁,在其N端和C端分别含有核糖体L1结构域和赖氨酸富集区。研究表明,RSL1D1具有抑制细胞衰老、调节细胞凋亡和细胞增殖的功能。最近,本课题组发现RSL1D1可能与p53之间存在相互作用,推测RSL1D1可能在肿瘤的发生发展过程中发挥重要作用。本研究旨在阐明RSL1D1在肿瘤细胞中的功能及其与p53之间的相互作用,为最终解析RSL1D1与肿瘤发生发展之间的关系提供重要实验依据。首先,本项目研究了 RSL1D1表达水平的下调对肿瘤细胞增殖速率的影响。利用常规分子生物学技术构建慢病毒重组质粒pLKO.1-shRNA,包装用于敲低RSL1D1的慢病毒颗粒,感染人直结肠癌细胞HCT116,经抗生素筛选,获得敲低RSL1D1的稳定HCT116细胞株,并通过绘制生长曲线,分析RSL1D1的敲低对HCT116增殖速率的影响。结果发现,与对照组相比较,RSL1D1被敲低后,HCT116的增殖速率明显下降。接着,利用流式细胞术分析了 RSL1D1的表达下调对细胞周期的影响。将上述制备的RSL1D1被敲低的HCT116细胞经乙醇固定,利用PI染色法进行染色,然后利用流式细胞仪分析细胞周期。结果发现,和对照组相比较,RSL1D1的敲低导致Sub-G1的明显增加,表明RSL1D1的敲低促进细胞凋亡,提示RSL1D1具有促进肿瘤细胞生存的功能。然后,研究了 RSL1D1的表达下调与细胞凋亡信号途径关键分子Bax和Puma表达水平之间的关系。收集上述制备的RSL1D1被敲低的HCT116细胞,利用实时荧光定量PCR和免疫印迹法检测Bax和Puma的mRNA和蛋白质水平。结果显示,RSL1D1的表达下调后,作为p53下游靶基因的促凋亡因子Puma的mRNA和蛋白质水平明显升高,而Bax的蛋白质水平则没有明显变化,尽管其mRNA水平略有上升。本研究还发现RSL1D1的敲低导致p53负调控因子HDM2的蛋白质水平明显上升。提示RSL1D1可能通过抑制HDM2,从而调节p53的胞内水平和活性,并进一步通过抑制Puma促进肿瘤细胞的生存。最后,研究了 RSL1D1与p53之间的相互作用。通过基因重组技术将编码全长RSL1D1、RSL1D1(1-281)、RSL1D1(282-485)、RSL1D1(387-490)的核苷酸序列克隆至原核表达载体pGEX-5X-1或pGEX-6P-1,同时将全长p53基因克隆至另一原核表达载体pET-32a。将测序正确的重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达,并利用谷胱甘肽琼脂糖凝胶或镍琼脂糖凝胶纯化融合蛋白,用于研究RSL1D1与p53之间的相互作用。结果发现,RSL1D1能在体外与p53直接结合,并确定RSL1D1(387-490)参与结合p53。综上所述,本项目的一系列研究结果揭示,RSL1D1可能通过直接与p53结合,调节p53的活性及Puma的胞内水平,从而调节肿瘤细胞的增殖与生存。这对于深入理解RSL1D1和p53在肿瘤发生发展中的作用,为肿瘤治疗提供新思路与新靶点具有重要意义。
[Abstract]:RSL1D1 (ribosomal L1-domain-containing 1), a cell senescence suppressor gene (CSIG,cellular senescence-inhibited gene), encodes a ribosomal protein, which belongs to ribosomal L1p/L10e family. RSL1D1 protein is mainly located in nucleoli and contains ribosomal L1 domain and lysine rich region at its N-terminal and C-terminal, respectively. RSL1D1 can inhibit cell senescence, regulate cell apoptosis and cell proliferation. Recently, our team found that there may be interaction between RSL1D1 and p53, suggesting that RSL1D1 may play an important role in tumorigenesis and development. The purpose of this study was to elucidate the function of RSL1D1 in tumor cells and its interaction with p53 in order to provide an important experimental basis for the final analysis of the relationship between RSL1D1 and tumorigenesis and development. First, we studied the effect of down-regulation of RSL1D1 expression on the proliferation rate of tumor cells. The lentivirus recombinant plasmid pLKO.1-shRNA, packaging was constructed by conventional molecular biology technique to knock down the lentivirus particles of RSL1D1. The stable HCT116 cell line with low RSL1D1 was obtained by antibiotic screening after infection with HCT116, and the growth curve was plotted. The effect of RSL1D1 knockout on the proliferation rate of HCT116 was analyzed. The results showed that compared with the control group, the proliferation rate of HCT116 decreased significantly after RSL1D1 was knocked down. Then, flow cytometry was used to analyze the effect of down-regulation of RSL1D1 expression on cell cycle. The RSL1D1 prepared above was immobilized by ethanol on the knockout HCT116 cells, stained by PI staining, and then the cell cycle was analyzed by flow cytometry. The results showed that compared with the control group, the knockout of RSL1D1 resulted in the increase of Sub-G1, which indicated that the knockout of RSL1D1 promoted cell apoptosis, suggesting that RSL1D1 could promote the survival of tumor cells. Then, the relationship between the down-regulation of RSL1D1 and the expression levels of Bax and Puma, the key molecules of apoptotic signaling pathway, was studied. The RSL1D1 knockout HCT116 cells were collected and the mRNA and protein levels of Bax and Puma were detected by real-time fluorescence quantitative PCR and Western blotting. The results showed that after down-regulation of RSL1D1 expression, the mRNA and protein levels of apoptosis-promoting factor Puma, the downstream target gene of p53, increased significantly, but the protein level of Bax did not change, although the mRNA level increased slightly. This study also found that RSL1D1 knockout significantly increased the protein level of p53 negative regulatory factor HDM2. It is suggested that RSL1D1 may regulate the intracellular level and activity of p53 by inhibiting HDM2, and further promote the survival of tumor cells by inhibiting Puma. Finally, the interaction between RSL1D1 and p53 was studied. The nucleotide sequences encoding full-length RSL1D1,RSL1D1 (1-281), RSL1D1 (282-485) and RSL1D1 (387-490) were cloned into the prokaryotic expression vector pGEX-5X-1 or pGEX-6P-1, by gene recombination and the full-length p53 gene was cloned into another prokaryotic expression vector pET-32a.. The recombinant plasmid was transformed into E. coli BL21 (DE3) and expressed by IPTG. The fusion protein was purified by glutathione agarose gel or nickel agarose gel to study the interaction between RSL1D1 and p53. The results showed that RSL1D1 could bind p53 directly in vitro, and confirmed that RSL1D1 (387-490) was involved in p53 binding. In conclusion, a series of results of this study suggest that RSL1D1 may regulate the proliferation and survival of tumor cells by directly binding to p53, regulating the activity of p53 and the intracellular level of Puma. It is of great significance to understand the role of RSL1D1 and p53 in tumorigenesis and development and to provide new ideas and targets for tumor therapy.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.2

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