中山大学中山医学院干细胞与组织工程研究中心,广东 广州 510080
巫孟师,在读硕士研究生,研究方向:干细胞与再生医学,E-mail:wwArmy@outlook.com
纸质出版日期:2021-01-20,
收稿日期:2020-10-13,
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巫孟师,熊敏敏,彭丹等.构建MS2-RIP方法用于鉴定lncRNA DANCR在肿瘤细胞中的相互作用分子[J].中山大学学报(医学科学版),2021,42(01):24-32.
WU Meng-shi,XIONG Min-min,PENG Dan,et al.MS2-RIP Assay for Identifying the Interaction Partners of lncRNA DANCR in Tumor Cells[J].Journal of Sun Yat-sen University(Medical Sciences),2021,42(01):24-32.
目的
2
为了探讨lncRNA DANCR在肿瘤细胞中的作用机制,本研究拟构建一种新的MS2-RIP方法用于鉴定DANCR的相互作用分子。
方法
2
利用MS2噬菌体衣壳蛋白可与噬菌体复制酶编码基因5’端一段由19个碱基组成的RNA茎环结构序列,即MS2结合位点(MS2 Binding Site,MS2 BS)高效结合的特点,构建串联表达MS2 BS和DANCR的过量表达质粒,通过MS2蛋白与MS2 BS的亲和作用,使DANCR得到富集,并且同步捕获与DANCR相互作用的分子,用于进一步分析鉴定。
结果
2
成功构建串联表达MS2 BS和DANCR的过量表达质粒用于MS2-RIP实验,该实验可显著富集DANCR以及作为阳性对照的DANCR结合蛋白EZH2。
结论
2
基于MS2蛋白与MS2结合位点的高效结合特性,我们针对lncRNA DANCR建立了MS2-RIP方法。该方法可有效富集DANCR以及与DANCR结合的生物活性分子,为研究DANCR的作用机制提供一种新的技术手段。
Objective
2
To explore the functional mechanism of lncRNA DANCR in tumor cells, a MS2-RIP method was designed and conducted to identify the molecules that interact with DANCR.
Methods
2
The specific binding of MS2 bacteriophage capsid protein and MS2 binding site (MS2 BS), a 19-base RNA stem-loop structure located at the 5′ terminus of the MS2 bacteriophage replicase gene, was applied to construct a plasmid for tandemly overexpressing DANCR and MS2 BS. DANCR was enriched and its associated molecules were further identified and analyzed.
Results
2
The plasmid for tandemly overexpressing DANCR and MS2 BS was successfully constructed for the MS2-RIP experiment, which could significantly enrich DANCR and DANCR-binding protein EZH2 as a positive control.
Conclusion
2
Based on the high affinity binding between MS2 protein and MS2 BS, the MS2-RIP method was established for lncRNA DANCR, which could effectively capture DANCR as well as its associated molecules, providing a new technology for studying the functional mechanism of DANCR.
MS2RNA结合蛋白免疫沉淀长链非编码RNADANCR
MS2RNA-binding protein immunoprecipitation(RIP)long non-coding RNA(lncRNA)DANCR
Kretz M,Webster DE,Flockhart RJ,et al. Suppression of progenitor differentiation requires the long noncoding RNA ANCR[J]. Genes Dev, 2012, 26(4):338-343.
Chen ZY,Chen X,Xie RH,et al. DANCR promotes metastasis and proliferation in bladder cancer cells by enhancing IL-11-STAT3 signaling and CCD1 expression[J]. Mol Ther, 2019, 27(2):326-341.
Li ZW,Hou PF,Fan DM,et al. The degradation of EZH2 mediated by lnCRNA ANCR attenuated the invasion and metastasis of breast cancer[J]. Cell Death Differ, 2017, 24(1):59-71.
Lu YQ,Hu ZY,Mangala LS,et al. MYC targeted long noncoding RNA DANCR promotes cancer in part by reducing p21 levels[J]. Cancer Res, 2018, 78(1):64-74.
Wen X,Liu X,Mao YP,et al. Long non-coding RNA DANCR stabilizes HIF-1α and promotes metastasis by interacting with NF90/NF45 complex in nasopharyngeal carcinoma[J]. Theranostics, 2018, 8(20):5676-5689.
Yuan SX,Wang J,Yang F,et al. Long noncoding RNA DANCR increases stemness features of hepatocellular carcinoma by derepression of CTNNB1[J]. Hepatology, 2016, 63(2):499-511.
Zhan YH,Chen ZC,Li YF,et al. Long non-coding rna dancr promotes malignant phenotypes of bladder cancer cells by modulating the miR-149/MSI2 axis as a ceRNA[J]. J Exp Clin Cancer Res, 2018, 37(1):273-284.
Cesana M,Cacchiarelli D,Legnini I,et al. A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA[J]. Cell, 2011, 147(2):358-369.
Poliseno L,Salmena L,Zhang J,et al. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology[J]. Nature, 2010, 465(7301):1033-1038.
Tsai MC,Manor O,Wan Y,et al. Long noncoding RNA as modular scaffold of histone modification complexes[J]. Science, 2010, 329(5992):689-693.
Zhu L,Xu PC. Downregulated lncRNA-ANCR promotes osteoblast differentiation by targeting EZH2 and regulating RUNX2 expression[J]. Biochem Biophys Res Commun, 2013, 432(4):612-617.
Witherell GW,Gott JM,Uhlenbeck OC. Specific interaction between RNA phage coat proteins and RNA[J]. Prog Nucleic Acid Res Mol Biol, 1991, 40:185-220. Doi: 10.1016/s0079-6603(08)60842-9http://dx.doi.org/10.1016/s0079-6603(08)60842-9
Li J,Zhou L. Overexpression of lncRNA DANCR positively affects progression of glioma via activating WNT/β-catenin signaling[J]. Biomed Pharmacother, 2018, 102:602-607. Doi: 10.1016/j.biopha.2018.03.116http://dx.doi.org/10.1016/j.biopha.2018.03.116
Lin XJ,Yang F,Qi XR,et al. LncRNA DANCR promotes tumor growth and angiogenesis in ovarian cancer through direct targeting of miR-145[J]. Mol Carcinog, 2019, 58(12):2286-2296.
Yao ZC,Chen QY,Ni ZL,et al. Long non-coding RNA differentiation antagonizing nonprotein coding RNA (DANCR) promotes proliferation and invasion of pancreatic cancer by sponging miR-214-5p to regulate E2F2 expression[J]. Med Sci Monit, 2019, 25:4544-4552. Doi: 10.12659/MSM.916960http://dx.doi.org/10.12659/MSM.916960
胡建兰,安玉玲.长链非编码RNA与肝癌[J].器官移植,2017,8(3):242-245.
Hu JL, An YL. Long non coding RNA and hepatocellular carcinoma[J]. Organ Transplan,2017,8(3):242-245.
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