印记基因IGF2在胰腺癌干细胞中的印记表达研究
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摘要
目的研究印记基因IGF2在胰腺癌干细胞中的印记表达情况。方法利用流式细胞分选技术通过CD133抗体从胰腺癌细胞株ASPC分离出胰腺癌干细胞;流式细胞术检测分离前后CD133~+细胞的比例;采用无血清悬浮培养法富集胰腺癌干细胞;通过成球实验、免疫荧光染色、流式细胞术测定肿瘤干细胞(cancer stem cells,CSCs)标志蛋白表达,以及通过软琼脂克隆形成实验等方法鉴定ASPC胰腺癌干细胞;以测序法分析IGF2在胰腺癌干细胞中的印记表达情况;应用qRT-PCR法研究胰腺癌干细胞中IGF2在基因水平上的表达;应用Western blot方法研究胰腺癌干细胞中IGF2在蛋白水平上的表达。结果利用流式细胞分选技术通过CD133抗体标记,从胰腺癌细胞ASPC中分离出胰腺癌干细胞,其可在干细胞培养液中成球培养,连续消化传代后仍保持与原代相同的形态特征;免疫荧光检测发现CSCs标志物CD133和乙醛脱氢酶(aldehyde dehydrogenase,ALDH)在胰腺癌干细胞表面持续阳性表达;软琼脂克隆形成实验显示CSCs的克隆形成能力增强;对分选前后肿瘤细胞CD133表达检测,显示超过90%的成球细胞为CD133阳性;通过测序法分析IGF2在胰腺癌干细胞中的印记表达情况,结果显示胰腺癌干细胞存在IGF2印记丢失(loss of imprinting,LOI),而与其对应的非干性肿瘤细胞(non-stem cancer cells,nSCCs)的印记状态为印记保持(maintance of imprinting,MOI);应用qRT-PCR法研究胰腺癌干细胞中IGF2在基因水平上的表达,结果显示与nSCCs相比,IGF2在CSCs中的表达明显升高;应用Western blot方法研究CSCs中IGF2在蛋白水平上的表达,结果显示与nSCCs相比,IGF2在CSCs中的表达明显升高。结论胰腺癌干细胞存在IGF2印记丢失,从而导致IGF2的高表达,IGF2印记丢失可能是胰腺癌干细胞的标志性特征之一。
Objective To investigate the expression of imprinted gene IGF2 in pancreatic cancer stem cells(CSCs).MethodsPancreatic CSCs were isolated from pancreatic cancer cell line ASPC by flow cytometry sorting technology using CD133 antibody.The proportion of CD133~+ cells before and after separation was measured by flow cytometry.Pancreatic CSCs were enriched by serum-free suspension culture.CSCs sphere culturing,immunofluorescence staining and flow cytometry were used to measure the expression of CSCs markers,and pancreatic CSCs were identified by soft agar colony formation assay.The IGF2 imprinting in pancreatic CSCs was analyzed by sequencing.qRT-PCR and Western blotting were used to detect the expression of IGF2 in pancreatic CSCs at mRNA and protein levels.Results The pancreatic CSCs were successfully isolated from pancreatic cancer cells ASPC by flow cytometric sorting using CD133 antibody.They could be cultured in stem cell media and grew to spheroidal cells,and maintained the same morphological characteristics as the primary generation after continuous digestion and passage.Immunofluorescence staining showed that CSC markers CD133 and aldehyde dehydrogenase(ALDH)continued to be positive on the surface of pancreatic CSCs.Soft agar clone formation experiments showed that the colony forming ability of CSCs was enhanced.Detection of CD133 expression in tumor cells before and after sorting showed that more than 90% of the spheroidal cells were CD133-positive.Sequencing showed the IGF2 imprinting loss(LOI)in pancreatic CSCs,and the maintenance of imprinting(MOI)in non-stem cancer cells(nSCCs).qRT-PCR and Western blotting showed that the expression of IGF2 was significantly increased at mRNA and protein levels in pancreatic CSCs as compared with nSCCs.Conclusion Loss of IGF2 imprinting is present in pancreatic CSCs,which results in a significantly upregulated expression of IGF2.IGF2 LOI may be a common feature of pancreatic cancer stem cells.
Objective To investigate the expression of imprinted gene IGF2 in pancreatic cancer stem cells(CSCs).MethodsPancreatic CSCs were isolated from pancreatic cancer cell line ASPC by flow cytometry sorting technology using CD133 antibody.The proportion of CD133~+ cells before and after separation was measured by flow cytometry.Pancreatic CSCs were enriched by serum-free suspension culture.CSCs sphere culturing,immunofluorescence staining and flow cytometry were used to measure the expression of CSCs markers,and pancreatic CSCs were identified by soft agar colony formation assay.The IGF2 imprinting in pancreatic CSCs was analyzed by sequencing.qRT-PCR and Western blotting were used to detect the expression of IGF2 in pancreatic CSCs at mRNA and protein levels.Results The pancreatic CSCs were successfully isolated from pancreatic cancer cells ASPC by flow cytometric sorting using CD133 antibody.They could be cultured in stem cell media and grew to spheroidal cells,and maintained the same morphological characteristics as the primary generation after continuous digestion and passage.Immunofluorescence staining showed that CSC markers CD133 and aldehyde dehydrogenase(ALDH)continued to be positive on the surface of pancreatic CSCs.Soft agar clone formation experiments showed that the colony forming ability of CSCs was enhanced.Detection of CD133 expression in tumor cells before and after sorting showed that more than 90% of the spheroidal cells were CD133-positive.Sequencing showed the IGF2 imprinting loss(LOI)in pancreatic CSCs,and the maintenance of imprinting(MOI)in non-stem cancer cells(nSCCs).qRT-PCR and Western blotting showed that the expression of IGF2 was significantly increased at mRNA and protein levels in pancreatic CSCs as compared with nSCCs.Conclusion Loss of IGF2 imprinting is present in pancreatic CSCs,which results in a significantly upregulated expression of IGF2.IGF2 LOI may be a common feature of pancreatic cancer stem cells.
引文
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[13] Rainier S,Johnson L A,Dobry C J,et al.Relaxation of imprinted genes in human cancer.Nature,1993,362(6422):747-749.
[14] Chen H L,Li T,Qiu X W,et al.Correction of aberrant imprinting of IGF2 in human tumors by nuclear transfer-induced epigenetic reprogramming[J].EMBO J,2006,25(22):5329-5338.
[15] Li T,Chen H,Li W,et al.Promoter histone H3 K27 methylation in the control of IGF2 imprinting in human tumor cell lines[J].Hum Mol Genet,2014,23(1):117-128.
[16] Vu T H,Nguyen A H,Hoffman A R.Loss of IGF2 imprinting is associated with abrogation of long-range intrachromosomal interactions in human cancer cells[J].Hum Mol Genet,2010,19(5):901-919.
[2] 刘永忠,杨本海,祝怀平,等.急性髓细胞白血病中胰岛素样生长因子-2基因印迹的研究[J].中华医学遗传学杂志,2000,17(1):39-41.
[3] O'Dell S D,Day I N.Insulin-like growffth factor Ⅱ(IGF-Ⅱ)[J].Int J Biochem Cell Biol,1998,30(7):767-771.
[4] Schneider M,Huber J,Hadaschik B,et al.Characterization of colon cancer cells:a functional approach characterizing CD133 as a potential stem cell marker[J].BMC Cancer,2012,12:96.
[5] Fan X,Ouyang N,Teng H,et al.Isolation and characterization of spheroid cells from the HT29 colon cancer cell line[J].Int J Colorectal Dis,2011,26(10):1279-1285.
[6] Li D,Xie K,Wolff R,et al.Pancreatic cancer[J].Lancet,2004,363(9414):1049-1057.
[7] Li C,Heidt D G,Dalerba P,et al.Identification of pancreatic cancer stem cells[J].Cancer Res,2007,67(3):1030-1037.
[8] Hermann P C,Huber S L,Herrler T,et al.Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer[J].Cell Stem Cell,2007,1(3):313-323.
[9] 宋文庆,俞岚,周蕾,等.肿瘤干细胞标记物CD133在食管鳞状细胞癌中的表达及其与血管生成拟态的关系[J].华中科技大学学报:医学版,2018,47(3):335-338.
[10] Church D N,Phillips B R,Stuckey D J,et al.Igf2 ligand dependency of Pten(+/-)developmental and tumour phenotypes in the mouse.Oncogene,2012,31(31):3635-3646.
[11] Ogawa O,Eccles M R,Szeto J,et al.Relaxation of insulin-like growth factor Ⅱ gene imprinting implicated in Wilms' tumour[J].Nature,1993,362(6422):749-751.
[12] Ohlsson R,Nystrom A,Pfeifer O S,et al.IGF2 is parentally imprinted during human embryogenesis and in the Beckwith-Wiedemann syndrome[J].Nat Genet,1993,4(1):94-97.
[13] Rainier S,Johnson L A,Dobry C J,et al.Relaxation of imprinted genes in human cancer.Nature,1993,362(6422):747-749.
[14] Chen H L,Li T,Qiu X W,et al.Correction of aberrant imprinting of IGF2 in human tumors by nuclear transfer-induced epigenetic reprogramming[J].EMBO J,2006,25(22):5329-5338.
[15] Li T,Chen H,Li W,et al.Promoter histone H3 K27 methylation in the control of IGF2 imprinting in human tumor cell lines[J].Hum Mol Genet,2014,23(1):117-128.
[16] Vu T H,Nguyen A H,Hoffman A R.Loss of IGF2 imprinting is associated with abrogation of long-range intrachromosomal interactions in human cancer cells[J].Hum Mol Genet,2010,19(5):901-919.