肿瘤干细胞中受H3K9me2调控的干性基因的筛选

生物技术通报 ›› 2016, Vol. 32 ›› Issue (12): 195-202.

肿瘤干细胞中受H3K9me2调控的干性基因的筛选

陶虹¹,张绍平²，张琳娜¹,张漫¹,扈启宽¹，²

1. 宁夏医科大学基础医学院生理学系，银川 750004；
2. 宁夏颅脑疾病重点实验室国家重点实验室培育基地，银川 750004

收稿日期:2016-06-06 出版日期:2016-12-25 发布日期:2016-12-07
作者简介:陶虹，女，硕士，副教授，研究方向：神经生理；E-mail：kellywar2003@163.com
基金资助:
国家自然科学基金（31260246，31460300），宁夏医科大学校级项目（XY201406）

Screening of Stem Genes Regulated by H3K9me2 in Tumor Stem Cells

TAO Hong¹,ZHANG Shao-ping²,ZHANG Lin-na¹,ZHANG Man¹,HU Qi-kuan ¹，²

1.The Department of Physiology，Ningxia Medical University，Yinchuan 750004；
2. Ningxia Key Lab of Cerebrocranial Diseases，the National Key Laboratory Incubation Base，Yinchuan 750004

Received:2016-06-06 Published:2016-12-25 Online:2016-12-07

摘要/Abstract

摘要： 为了分析比较甲基转移酶G9a和组蛋白H3K9me2修饰在胶质瘤干细胞与非干细胞中存在的差异，筛选出维持胶质瘤干细胞干性的相关基因。通过G9a抑制剂促进U87细胞成球和过表达G9a促进U87细胞分化的方法，培养了成球的胶质瘤干细胞和贴壁的非干细胞，这两种细胞的CD133表达差异明显。再利用H3K9me2抗体通过ChIP-seq技术比较H3K9me2修饰在干细胞组与非干细胞组中的差异，在存在差异的基因中，对TSS±2 000 bp范围内的基因进行了GO分析，并随机选出10个转录因子进行QPCR验证，结果与ChIP-seq实验基本一致。

关键词: ChIP-seq, G9a, H3K9me2, Bix01294, U87细胞株

Abstract: In order to analyze and compare the modification differences of the methyltransferase G9a and histone（H3K9me2）between in glioma stem cells and non-stem cells，the related genes maintaining the stem of glioma stem cells were screened. By using G9a inhibitor to facilitate U87 cells into spheres，and G9a overexpression to promote the cell differentiation，the sphered glioma stem cells and monolayer non-stem cells were cultured，and there were significant differences in CD133 expression between the two cell models. Then we used H3K9me2 antibody to compare the differences of H3K9me2 modification between glioma stem cells and non-stem cells via ChIP-seq assay. Genes in the range of TSS ± 2 000 bp among the genes with differences were selected for GO analysis. Moreover，10 transcription factors were randomly selected to confirm the expression level by QPCR，and the result of which was almost consistent with that by ChIP-seq assay.

Key words: ChIP-seq, G9a, H3K9me2, Bix01294, U87 cell line

中图分类号:

10.13560/j.cnki.biotech.bull.1985.2016.12.030

陶虹,张绍平，张琳娜,张漫,扈启宽，. 肿瘤干细胞中受H3K9me2调控的干性基因的筛选[J]. 生物技术通报, 2016, 32(12): 195-202.

TAO Hong,ZHANG Shao-ping,ZHANG Lin-na,ZHANG Man,HU Qi-kuan,. Screening of Stem Genes Regulated by H3K9me2 in Tumor Stem Cells[J]. Biotechnology Bulletin, 2016, 32(12): 195-202.

参考文献

[1] Li YF, Xiao B, Tu SF, et al. Cultivation and identification of colon cancer stem cell-derived spheres from the Colo205 cell line[J] . Braz J Med Biol Res, 2012, 45（3）：197-204.
[2] Nakanishi Y, Seno H, Fukuoka A, et al. Dclk1 distinguishes between tumor and normal stem cells in the intestine[J] . Nat Genet, 2013, 45（1）：98-103.
[3] Chen J, Li YJ, Yu TS, et al. A restricted cell population propagates glioblastoma growth after chemotherapy[J] . Nature, 2012, 488（7412）：522-526.
[4] Machado HL, Kittrell FS, Edwards D, et al. Separation by cell size enriches for mammary stem cell repopulation activity[J] . Stem Cells Transl Med, 2013, 2（3）：199-203.
[5] Driessens G, Beck B, Caauwe A, et al. Defining the mode of tumour growth by clonal analysis[J] . Nature, 2012, 488（7412）：527-530.
[6] Hinohara K, Kobayashi S, Kanauchi H, et al. ErbB receptor tyrosine kinase/NF-κB signaling controls mammosphere formation in human breast cancer[J] . Proc Natl Acad Sci USA, 2012, 109（17）：6584-6589.
[7] Liao WT, Ye YP, Deng YJ, et al. Metastatic cancer stem cells：from the concept to therapeutics[J] . Am J Stem Cells, 2014, 3（2）：46-62.
[8] Sharma S, Kelly TK, Jones PA. Epigenetics in cancer[J] . Carcinogenesis, 2010, 31（1）：27-36.
[9] Wen B, Wu H, Shinkai Y, et al. Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells[J] . Nat Genet, 2009, 41（2）：246-250.
[10] Zhang J, Gao Q, Li P, et al. S phase-dependent interaction with DNMT1 dictates the role of UHRF1 but not UHRF2 in DNA methylation maintenance[J] . Cell Res, 2011, 21（12）：1723-1739.
[11] Black JC, Van Rechem C, Whetstine JR. Histone lysine methylation dynamics：establishment, regulation and biological impact[J] . Mol cell, 2012, 48（4）：491-507.
[12] Mosammaparast N, Shi Y. Reversal of histone methylation-bioche-mical and molecular mechanisms of histone demethylases[J] . Annu Rev Biochem, 2010, 79（7）：155-179.
[13] Tao H, Li H, Su Y, et al. Histone methyltransferase G9a and H3K9 dimethylation inhibit the self-renewal of glioma cancer stem cells[J] . Mol Cell Biochem, 2014, 394（1-2）：23-30.
[14] Sánchez Alvarado A, Yamanaka S. Rethinking differentiation：stem cells, regeneration and plasticity[J] . Cell, 2014, 157（1）：110-119.
[15] Kreso A, Dick JE. Evolution of the cancer stem cell model[J] . Cell Stem Cell, 2014, 14（3）：275-291.
[16] Park TS, Donnenberg VS, Donnenberg AD, et al. Dynamic interactions between cancer stem cells and their stromal partners[J] . Current Pathobiology Reports, 2014, 1（2）：41-52.
[17] Islam F, Qiao B, Smith RA, et al. Cancer stem cell：fundamental experimental pathological concepts and updates[J] . Exp Mol Pathol, 2015, 98（2）：184-191.
[18] Hanahan D, Weinberg RA. Hallmarks of cancer：the next generation[J] . Cell, 2011, 144（5）：646-674.
[19] Dawson MA, Kouzarides T. Cancer epigenetics：from mechanism to therapy[J] . Cell, 2012, 150（1）：12-27.
[20] Simon C, Chagraoui J, Krosl J, et al. A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia[J] . Genes Dev, 2012, 26（7）：651-656.
[21] Hu S, Yu LL, Li ZM, et al. Overexpression of EZH2 contributes to acquired cisplatin resistance in ovarian cancer cells in vitro and in vivo[J] . Cancer Biology & Therapy, 2010, 10（8）：788-795.
[22] Mallen-St Clair J, Soydaner-Azeloglu R, Lee KE, et al. EZH2 couples pancreatic regeneration to neoplastic progression[J] . Genes Dev, 2012, 26（5）：439-444.
[23] Au SLK, Wong CCL, Lee JMF, et al. Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis[J] . Hepatology, 2012, 56（2）：622-631.
[24] Cai MY, Hou JH, Rao HL, et al. High expression of H3K27me3 in human hepatocellular carcinomas correlates closely with vascular invasion and predicts worse prognosis in patients[J] . Mol Med, 2011, 17（1-2）：12-20.
[25] Fussbroich B, Wagener N, Macher-Goeppinger S, et al. EZH2 depletion blocks the proliferation of colon cancer cells[J] . PLoS One, 2011, 6（7）：e21651.
[26] Qi W, Chan H, Teng L, et al. Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation[J] . Proc Natl Acad Sci USA, 2012, 109（52）：21360-21365.
[27] Chase A, Cross NCP. Aberrations of EZH2 in cancer[J] . Clin Cancer Res, 2011, 17（9）：2613-2618.