LncRNA-ANCR介导的组蛋白甲基化酶EZH2对结直肠癌侵袭与转移的调控
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摘要
目的探讨lnc RNA-ANCR与组蛋白甲基转移酶EZH2的表达及其与结直肠癌细胞侵袭与转移的关系。方法培养结肠癌SW620细胞系,转染ANCR-siRNA至细胞,采用实时荧光定量PCR(RT-PCR)检测ANCR和EZH2表达,Western blot检测EZH2蛋白表达水平,采用RNA pull-down和免疫共沉淀的方法检测ANCR与EZH2的相互作用关系,Transwell实验和划痕实验检测细胞侵袭和转移能力。结果结肠癌SW620细胞中ANCR和EZH2的表达均显著高于正常结肠上皮细胞(P<0.05)。转染ANCR-siRNA的SW620细胞中ANCR的表达较转染阴性对照质粒的SW620细胞显著降低,且EZH2的mRNA和蛋白表达水平均显著降低(P<0.05)。RNA pull-down与免疫共沉淀的结果显示ANCR可与EZH2特异性结合。而Transwell实验和划痕实验显示转染ANCR-siRNA的SW620细胞的侵袭和迁移能力均显著低于转染阴性对照质粒的SW620细胞(P<0.05)。结论结直肠癌细胞中ANCR与EZH2的表达均上调,ANCR可能通过特异性结合EZH2调节结直肠癌细胞的侵袭和转移。
Objective To investigate the expression of anti-differentiation noncoding RNA(lnc RNA-ANCR) and histone methyltransferase enhancer of zeste homolog 2(EZH2) and their relationship with the invasion and metastasis of colorectal cancer cells. MethodsColorectal cancer cell line SW620 was cultured and transfected with ANCR-siRNA. The ANCR and EZH2 expression were detected by RT-PCR(real-time PCR) and the expression level of EZH2 protein was detected by Western blot. RNA pull-down and co-immunoprecipitation were used to detect the interaction between ANCR and EZH2. The Transwell assay and scratch test were applied to detect the cell ability of invasion and metastasis. Results The expression of ANCR and EZH2 were higher in SW620 cell than in normal colonic epithelial cell(P< 0.05). Compared with SW620 cells transfected with negative control plasmid, the expression level of ANCR decreased in SW620 cells transfected with ANCR-siRNA. Moreover, the mRNA and protein expression of EZH2 were decreased(all P< 0.05). RNA pull-down and co-immunoprecipitation results indicated that ANCR could specifically bind to EZH2. Transwell assay and scratch test showed that the invasion and migration ability of SW620 cells transfected with ANCR-siRNA were significantly attenuated(P< 0.05). Conclusion The expression levels of ANCR and EZH2 in colorectal cancer were significantly upregulated, and ANCR can specifically bind to EZH2 to regulate the invasion and metastasis process of colorectal cancer cells.
Objective To investigate the expression of anti-differentiation noncoding RNA(lnc RNA-ANCR) and histone methyltransferase enhancer of zeste homolog 2(EZH2) and their relationship with the invasion and metastasis of colorectal cancer cells. MethodsColorectal cancer cell line SW620 was cultured and transfected with ANCR-siRNA. The ANCR and EZH2 expression were detected by RT-PCR(real-time PCR) and the expression level of EZH2 protein was detected by Western blot. RNA pull-down and co-immunoprecipitation were used to detect the interaction between ANCR and EZH2. The Transwell assay and scratch test were applied to detect the cell ability of invasion and metastasis. Results The expression of ANCR and EZH2 were higher in SW620 cell than in normal colonic epithelial cell(P< 0.05). Compared with SW620 cells transfected with negative control plasmid, the expression level of ANCR decreased in SW620 cells transfected with ANCR-siRNA. Moreover, the mRNA and protein expression of EZH2 were decreased(all P< 0.05). RNA pull-down and co-immunoprecipitation results indicated that ANCR could specifically bind to EZH2. Transwell assay and scratch test showed that the invasion and migration ability of SW620 cells transfected with ANCR-siRNA were significantly attenuated(P< 0.05). Conclusion The expression levels of ANCR and EZH2 in colorectal cancer were significantly upregulated, and ANCR can specifically bind to EZH2 to regulate the invasion and metastasis process of colorectal cancer cells.
引文
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[2]Austin H,Henley SJ,King J,et al.Changes in colorectal cancer incidence rates in young and older adults in the United States:what does it tell us about screening[J].Cancer Causes Control,2014,25(2):191-201.doi:10.1007/s10552-013-0321-y.
[3]Wan DS.Epidemiologic trend of and strategies for colorectal cancer[J].Chin JCancer,2009,28(9):897-902.[万德森.结直肠癌流行趋势及其对策[J].癌症,2009,28(9):897-902.]doi:10.3321/j.issn:1000-467X.2009.09.001.
[4]Wang N,Sun TT,Zheng RS,et al.An Analysis of Inci-dence and Mortality of Colorectal Cancer in China,2009[J].China Cancer,2013,22(7):515-520.[王宁,孙婷婷,郑荣寿,等.中国2009年结直肠癌发病和死亡资料分析[J].中国肿瘤,2013,22(7):515-520.]doi:10.11735/j.issn1004-0242.2013.07.A001.
[5]Chen Q,Liu ZC,Cheng LP,et al.An Analysis of Incidence and Mortality of Colorectal Cancer in China,2003~2007[J].China Cancer,2012,21(3):179-182.[陈琼,刘志才,程兰平,等.2003~2007年中国结直肠癌发病与死亡分析[J].中国肿瘤,2012,21(3):179-182.]
[6]Li Z,Hou P,Fan D,et al.The degradation of EZH2 mediated by lnc RNA ANCR attenuated the invasion and metastasis of breast cancer[J].Cell Death Differ,2017,24(1):59-71.doi:10.1038/cdd.2016.95.
[7]Li Z,Hou P,Fan D,et al.The degradation of EZH2 mediated by lnc RNA ANCR attenuated the invasion and metastasis of breast cancer[J].Cell Death&Differentiation,2016.
[8]Tsang DP,Cheng AS.Epigenetic regulation of signaling pathways in cancer:role of the histone methyltransferase EZH2[J].J Gastroenterol Hepatol,2011,26(1):19-27.doi:10.1111/j.1440-1746.2010.06447.x.
[9]Chen GB,Subedi K,Mi J,et al.Histone methyltransferase Ezh2 is critical for activation-induced CD8 T cell proliferation and survival(LYM7P.615)(LYM7P.615)[J].J Immunol,2015,194(1 Suppl):200.7.
[10]Zhou XL,Wang JJ,Zhang HX,et al.Research progressand function of longnon-coding RNA in nervous system diseases[J].Chin JNeuroimmunol Neurol,2016,23(5):356-359.[周雪灵,王健健,张荟雪,等.长链非编码RNA在神经系统疾病中的功能及研究进展[J].中国神经免疫学和神经病学杂志,2016,23(5):356-359.]doi:10.3969/j.issn.1006-2963.2016.05.014.
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[12]Tao KX,Cai M.Application of laparoscopic technique in colorectal cancer surgery[J].Journal of Abdom Surg,2010,23(1):8-9.[陶凯雄,蔡明.腹腔镜技术在结直肠癌外科治疗中的应用[J].腹部外科,2010,23(1):8-9.]doi:10.3969/j.issn.1003-5591.2010.01.003.
[13]Zhao G,Xiao G,Huang MX,et al.Effect of laparoscopic radical operation on systemic immunity in patients with colorectal cancer[J].Chin JGastrointest Surg,2005,8(5):407-409.
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[21]Angrand P O,Vennin C,Bourhis X L,et al.The role of long non-coding RNAs in genome formatting and expression[J].Frontiers in Genetics,2015,6:165.
[22]Li Z,Hou P,Fan D,et al.The degradation of EZH2 mediated by lnc RNA ANCR attenuated the invasion and metastasis of breast cancer[J].Cell Death Differ,2017,24(1):59-71.doi:10.1038/cdd.2016.95.
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[24]Hirata H,Hinoda Y,Shahryari V,et al.Long Noncoding RNA MALAT1 Promotes Aggressive Renal Cell Carcinoma through Ezh2 and Interacts with mi R-205[J].Cancer Res,2015,75(7):1322-1331.doi:10.1158/0008-5472.CAN-14-2931
[25]Cerase A,Smeets D,Tang YA,et al.Spatial separation of Xist RNA and polycomb proteins revealed by superresolution microscopy[J].Proc Natl Acad Sci U S A,2014,111(6):2235-2240.doi:10.1073/pnas.1312951111.
[26]Jing L,Ren JL,Ji Q,et al.Research progresson the mechanism of lnc RNAregulating the invasion and metastasis of colorectal cancer[J].Int JDig Dis,2015,35(5):334-337.[靖琳,任建琳,季青,等.长链非编码RNA调控结直肠癌侵袭与转移机制的研究进展[J].国际消化病杂志,2015,35(5):334-337.]doi:10.3969/j.issn.1673-534X.2015.05.012.