Welcome to visit Acta Anatomica Sinica! Today is
Chinese
Down-regulation of tankyrase 1 mediated by RNAi leads to the apoptosis of SH-SY5Y cells
TIAN Xiao-hong BAI Shu-ling* FAN Jun HOU Wei-jian TONG Hao XU He
Acta Anatomica Sinica ›› 2014 ›› Issue (2) : 196-203.
Down-regulation of tankyrase 1 mediated by RNAi leads to the apoptosis of SH-SY5Y cells
Objective To investigate the effect and mechanism of tankyrase 1（TNKS1）down-regulation mediated by RNA interference（RNAi）on the proliferation and apoptosis of human neuroblastoma (NB) SH-SY5Y cells, and to provide experimental basis for the development of a new therapeutical target. Methods According to TNKS1 gene sequences, three short hairpin (shRNA) interference segments with gene-specific were designed and synthesized, and lentiviral vectors were used for constructing RNAi sequence. After SH-SY5Y cells were transfected with lentiviral vector, Real-time PCR assay was used for detecting the expression of TNKS1 gene, and the best interference sequence was screened out for subsequent experiments. The colony forming assay was used to detect the change of cell proliferation after RNAi-TNKS1. The expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein Caspase-3, the key protein β-catenin of Wnt/β-catenin pathway and its downstream target proteins Cyclin D1 and c-Myc were detected by the Western blotting method for exploring the mechanism. Apoptotic morphology was observed by transmission electron microscopy. Results The lentiviral vector was constructed successfully, and the virus concentration was 5×10 11TU/L. The results of Real-time PCR test showed that #3 shRNA was the most effective target sequence. The colony forming assay results showed that colony forming rate decreased significantly after RNAi-TNKS1 compared with that of control group. The results of Western blotting showed that the protein expression of Bcl-2 reduced significantly as well as β-catenin, Cyclin D1 and c-Myc after RNAi-TNKS1, while the protein expression of caspase-3 increased. The transmission electron microscopy results showed significant apoptotic structure and morphology after RNAi-TNKS1. Conclusion The down-regulation of TNKS1 mediated by RNAi leads to the proliferation decrease and the apoptosis of SH-SY5Y cells, and the inhibition of Wnt/β-catenin pathway may play a role in it.
［1］Buechner J, Henriksen JR, Haug BH, et al. Inhibition of mir-21，which is up-regulated during MYCN knockdown-mediated differentiation, does not prevent differentiation of neuroblastoma cells ［J］. Differentiation, 2011, 81(1)：25-34.
［2］London WB, Castel V, Monclair T, et al. Clinical and biologic features predictive of survival after relapse of neuroblastoma: a report from the International Neuroblastoma Risk Group project ［J］. J Clin Oncol, 2011, 29(24): 3286-3292.
［3］Pearson AD, Pinkerton CR, Lewis IJ, et al. High-dose rapid and standard induction chemotherapy for patients aged over 1 year with stage 4 neuroblastoma: a randomised trial［J］. Lancet Oncol, 2008, 9(3): 247-256.
［4］Zage PE, Kletzel M, Murray K, et al. Outcomes of the POG 9340/9341/9342 trials for children with high-risk neuroblastoma: a report from the Children’s Oncology Group ［J］. Pediatr Blood Cancer, 2008, 51(6):747-753.
［5］Coelho T, Adams D, Silva A, et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis ［J］. N Engl J Med, 2013, 369(9): 819-829.
［6］Hao J, Shi H, Zhao S, et al. Effects of SREBP-1 targeted RNAi on lipid droplet formation in HKC cells under stimulation of high glucose ［J］. Chinese Pharmacological Bulletin, 2009, 25(3)：371-377. (in Chinese)
郝军，石宏，赵松，等. 靶向抑制人SREBP-1基因对高糖刺激下人肾小管上皮细胞脂滴形成的影响［J］. 中国药理学通报，2009，25(3)：371-377.
［7］Chen S, Ge X, Chen Y, et al. Advances with RNA interference in Alzheimer’s disease research ［J］. Drug Des Devel Ther, 2013, 7(2):117-125.
［8］Voronkov A, Krauss S. Wnt/beta-catenin signaling and small molecule inhibitors［J］. Curr Pharm Des, 2012, 19(4): 634-664.
［9］Davar D, Beumer JH, Hamieh L, et al. Role of PARP inhibitors in cancer biology and therapy ［J］. Curr Med Chem, 2012, 19(23): 3907-3921.
［10］Klapper W, Krams M, Qian W, et al. Telomerase activity in B-cell non-Hodgkin lymphomas is regulated by hTERT transcription and correlated with telomere-binding protein expression but uncoupled from proliferation［J］. Br J Cancer, 2003, 89(4): 713-719.
［11］Gelmini S, Poggesi M, Distante V, et al. Tankyrase, a positive regulator of telomere elongation, is over expressed in human breast cancer ［J］. Cancer Lett, 2004, 216(1):81-87.
［12］Gelmini S, Poggesi M, Pinzani P, et al. Distribution of tankyrase-1 mRNA expression in colon cancer and its prospective correlation with progression stage ［J］. Oncol Rep, 2006,16 (6):1261-1266.
［13］Gelmini S, Quattrone S, Malentacchi F, et al. Tankyrase-1 mRNA expression in bladder cancer and paired urine sediment: preliminary experience ［J］. Clin Chem Lab Med, 2007, 45(7):862-866.
［14］Shervington A, Patel R, Lu C, et al. Telomerase subunits expression variation between biopsy samples and cell lines derived from malignant glioma ［J］. Brain Res, 2007, 1134(1):45-52.
［15］Tang ChZh, Zhang WX, Gao DW, et al. Changes of the ultrastructure of colon mucosa after partial hepatectomy in rats ［J］. Acta Anatomica Sinica, 2012,43（4）：545-550. (in Chinese)
唐超智，张文学，高道文，等. 部分肝切除后大鼠结肠黏膜层超微结构的变化［J］. 解剖学报，2012,43（4）：545-550.
［16］Tan ChY. The progresses of diagnosis and treatment for neuroblastoma［J］. The Journal of Chinese Birth Health and Heredity, 2011，19（1）：132-134. (in Chinese)
谭春英. 神经母细胞瘤的诊治进展［J］. 中国优生与遗传杂志，2011，19（1）：132-134.
［17］Smith MA, Seibeil NL, Altekruse SF, et al. Outcomes for children and adolescents with cancer: challenges for the twenty-first century ［J］. J Clin Oncol, 2010, 28 (15): 2625-2634.
［18］Muramatsu Y, Ohishi T, Sakamoto M, et al. Cross-species difference in telomeric function of tankyrase 1［J］. Cancer Sci, 2007, 98(6): 850-857.
［19］Huang SM, Mishina YM, Liu S, et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt sianaling［J］. Nature, 2009, 461(7264): 614-620.
［20］Busch AM, Johnson KC, Stan RV, et al. Evidence for tankyrases as antineoplastic targets in lung cancer［J］. BMC Cancer, 2013, 13(4):211.
［21］Bao R, Christova T, Song S, et al. Inhibition of tankyrases induces Axin stabilization and blocks Wnt signalling in breast cancer cells ［J］. PLoS One, 2012, 7(11): e48670.
［22］Waaler J, Machon O, Tumova L, et al. A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice ［J］. Cancer Res, 2012, 72(11): 2822-2832.
［23］Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Coenorhabditis elegans ［J］. Nature, 1998, 391(6669): 806-811.
［24］Fish RJ, Kruithof EK. Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors ［J］. BMC Mol Biol, 2004, 5(8): 9.
［25］Yang W, Kang J, Wang X, et al. Comparison between lentivirus and plasmid as shRNA vector targeting RhoA gene of ovary cancer cell line HO8910［J］. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2013, 29(5): 473-476, 480.
［26］Zhao X, Zhu DM, Gan WJ, et al. Lentivirus-mediated shRNA interference targeting vascular endothelial growth factor inhibits angiogenesis and progression of human pancreatic carcinoma ［J］. Oncol Rep, 2013, 29(3):1019-1026.
［27］Pang WJ, Xiong Y, Zhang Z, et al. Lentivirus-mediated Sirt1 shRNA and resveratrol independently induce porcine preadipocyte apoptosis by canonical apoptotic pathway ［J］. Mol Biol Rep, 2013, 40(1):129-139.
［28］Clevers H. Wnt/beta-catenin signaling in development and disease ［J］. Cell, 2005, 127(3): 469-680.
/
| 〈 |
|
〉 |
