胰腺癌干细胞Oct4、Nanog基因生物学特性的研究
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摘要
目的
研究胚胎干性相关基因Oct4和Nanog在人胰腺癌肿瘤干细胞(PCSCs)和普通肿瘤细胞中表达情况，然后再通过特异shRNA沉默PCSCs细胞中Oct4和Nanog的表达，研究PCSCs干性特征的变化及其机制。
方法
本实验分为两大部分。第一部分：用流式细胞仪在人胰腺癌Panc-1细胞系中以CD44+CD24+ESA+为表面标记筛选PCSCs，RT-PCR法和细胞免疫荧光法检测PCSCs细胞和Panc-1细胞中Oct4和Nanog基因的表达情况。用CCK8法检测其体外增殖能力，描制生长曲线，用吉西他滨分别干预普通肿瘤细胞和PCSCs细胞，研究PCSCs的干性特征。第二部分：用慢病毒载体介导的shRNA沉默PCSCs中Oct4和Nanog的表达，并用FQRT-PCR和Western blot检测干扰效率。单克隆形成实验、Transwell小室模型和CCK8法检测Oct4和Nanog基因对PCSCs增殖、迁移、侵袭和药物敏感性的影响；FQRT-PCR和Western blot探讨其作用机制。通过NOD/SCID小鼠致瘤实验观察Oct4和Nanog对PCSCs致瘤性的影响。
结果
分选出的CD44+CD24+ESA+三阳性PCSCs细胞约占细胞总量的0.1%-0.8%，Oct4及Nanog基因在PCSCs细胞中表达高于Panc-1细胞，Oct4在PCSCs细胞中的表达高于Panc-1细胞8.141±1.378倍；Nanog在PCSCs细胞中的表达高于Panc-1细胞7.945±0.652倍。Oct4和Nanog基因在两种干细胞中均为核表达。Panc-1细胞PCSCs细胞体外培养，Panc-1细胞第2天进入对数生长期，肿瘤干细胞体外无血清培养后第5天仍未进入对数生长期，呈干细胞球样缓慢生长。吉西他滨刺激后细胞生长被抑制，第48H，测Panc-1细胞IC50值为982μg/mL，PCSCs细胞IC50值为1981μg/mL。第72H测Panc-1细胞IC50值为670μg/mL，PCSCs细胞IC50值为1484μg/mL，差异有统计学意义(P<0.05)；慢病毒载体介导shRNA可明显降低PCSCs中Oct4、Nanog的表达(P<0.05)。沉默Oct4和Nanog后，PCSCs的单克隆形成、增殖、迁移、侵袭力较对照组显著下降，药物敏感性增强并诱导凋亡(P<0.05)。致瘤实验表明PCSCs致瘤性在沉默Oct4和Nanog基因后，较对照组明显下降(P<0.05)。
结论
1．通过流式细胞仪在胰腺癌Panc-1细胞株中分选出CD44+CD24+ESA+三阳性细胞，在体外培养中表现缓慢生长及为耐受化疗药物的干细胞特性。
2．Oct4和Nanog基因在CD44+CD24+ESA+阳性PCSCs细胞中的表达高于普通胰腺癌细胞，说明这两种基因与胰腺癌干细胞的干性特征相关。
3．慢病毒介导的shRNA可以沉默PCSCs中Oct4和Nanog基因的表达，可显著抑制其干细胞性特征，增强药物敏感性并诱导凋亡。
Objective
To study the gene expression of Oct4and Nanog that related toembryonic stem cell in human pancreatic cancer stem cells（PCSCs）and study the proliferation and drug-resistence ability of pancreaticcancer stem cell in vitro. Silence Oct4and Nanog expression in PCSCsby specific shRNA, study the biological characteristics of PCSCs andthe related mechanisms.
Methods
Pancreatic cancer stem cells of CD44+CD24+ESA+phenotypesisolated by a FACS Aria II in human cell line Panc-1. Detect theexpression of Oct4and Nanog gene expression in Panc-1cells andPCSCs cells by immunofluorescence and RT-PCR. To study theproliferation ability of cancer stem cell, we draw a growth curve bythe ways of CCK8staining. To detect the ability of drug-resistence ofPCSCs, we stimulated both cells by gemcitabine and study thedifference of their IC50. Nanog and Oct4in PCSCs silenced byLentiviral vector-GFP(LV) mediated shRNA. Observe themigration,invasion, proliferation and the drug sensitivity of PCSCsinhibited by Oct4and Nanog by monoclonal formation as well as theCCK8assay, transwell chamber model. Investigate related mechanismsby FQRT-PCR and Western blot and NOD/SCID mice tumorigenicityexperiments was done to study the tumorigenic potential of PCSCsinfluenced by Oct4and Nanog.
Results
The isolated PCSCs is0.1%-0.8%of all cells. Oct4and Nanoggenes have a higher expression in sorted cells than unsorted ones.The△CT of Nanog in PCSCs and Panc-1cell is7.945±0.652. The△CT of Oct4in PCSCs and Panc-1cell is8.141±1.378; Both twogenes were expressed in nucleus and were restrained by gemcitabine.The48th hour’s IC50of Panc-1cell is982μg/mL, IC50of PCSCsis1981μg/mL. The72th hour’s IC50of Panc-1cell is670μg/mL,IC50of PCSCs cell is1484μg/mL.LV mediated shRNA have stronginhibitory effects for Nanog and Oct4expression(P<0.05).Compared tothe control group,The proliferation, monoclonal formation, invasionability and migration of PCSCs decreased significantly if silencing Oct4and Nanog expression, while enhance drug sensitivity and induceapoptosis(P<0.05). Tumorigenicity experiments showed PCSCstumorigenic capacity decreased significantly than that of control groupif Oct4and Nanog gene silenced.(P<0.05).
Conclusions
1. Cancer stem cell perform a stem cell-like characteristic of highlydrug-resistence and declined proliferation.
2. Oct4and Nanog gene have a higher expression in PCSCs thanin Panc-1cells.
3. LV mediated shRNA effectively reduce the expression of Oct4and Nanog gene in PCSCs.
4. Silence Nanog and Oct4can inhibit the biological characteristicsof PCSCs，while enhance drug sensitivity and induce apoptosis.
研究胚胎干性相关基因Oct4和Nanog在人胰腺癌肿瘤干细胞(PCSCs)和普通肿瘤细胞中表达情况，然后再通过特异shRNA沉默PCSCs细胞中Oct4和Nanog的表达，研究PCSCs干性特征的变化及其机制。
方法
本实验分为两大部分。第一部分：用流式细胞仪在人胰腺癌Panc-1细胞系中以CD44+CD24+ESA+为表面标记筛选PCSCs，RT-PCR法和细胞免疫荧光法检测PCSCs细胞和Panc-1细胞中Oct4和Nanog基因的表达情况。用CCK8法检测其体外增殖能力，描制生长曲线，用吉西他滨分别干预普通肿瘤细胞和PCSCs细胞，研究PCSCs的干性特征。第二部分：用慢病毒载体介导的shRNA沉默PCSCs中Oct4和Nanog的表达，并用FQRT-PCR和Western blot检测干扰效率。单克隆形成实验、Transwell小室模型和CCK8法检测Oct4和Nanog基因对PCSCs增殖、迁移、侵袭和药物敏感性的影响；FQRT-PCR和Western blot探讨其作用机制。通过NOD/SCID小鼠致瘤实验观察Oct4和Nanog对PCSCs致瘤性的影响。
结果
分选出的CD44+CD24+ESA+三阳性PCSCs细胞约占细胞总量的0.1%-0.8%，Oct4及Nanog基因在PCSCs细胞中表达高于Panc-1细胞，Oct4在PCSCs细胞中的表达高于Panc-1细胞8.141±1.378倍；Nanog在PCSCs细胞中的表达高于Panc-1细胞7.945±0.652倍。Oct4和Nanog基因在两种干细胞中均为核表达。Panc-1细胞PCSCs细胞体外培养，Panc-1细胞第2天进入对数生长期，肿瘤干细胞体外无血清培养后第5天仍未进入对数生长期，呈干细胞球样缓慢生长。吉西他滨刺激后细胞生长被抑制，第48H，测Panc-1细胞IC50值为982μg/mL，PCSCs细胞IC50值为1981μg/mL。第72H测Panc-1细胞IC50值为670μg/mL，PCSCs细胞IC50值为1484μg/mL，差异有统计学意义(P<0.05)；慢病毒载体介导shRNA可明显降低PCSCs中Oct4、Nanog的表达(P<0.05)。沉默Oct4和Nanog后，PCSCs的单克隆形成、增殖、迁移、侵袭力较对照组显著下降，药物敏感性增强并诱导凋亡(P<0.05)。致瘤实验表明PCSCs致瘤性在沉默Oct4和Nanog基因后，较对照组明显下降(P<0.05)。
结论
1．通过流式细胞仪在胰腺癌Panc-1细胞株中分选出CD44+CD24+ESA+三阳性细胞，在体外培养中表现缓慢生长及为耐受化疗药物的干细胞特性。
2．Oct4和Nanog基因在CD44+CD24+ESA+阳性PCSCs细胞中的表达高于普通胰腺癌细胞，说明这两种基因与胰腺癌干细胞的干性特征相关。
3．慢病毒介导的shRNA可以沉默PCSCs中Oct4和Nanog基因的表达，可显著抑制其干细胞性特征，增强药物敏感性并诱导凋亡。
Objective
To study the gene expression of Oct4and Nanog that related toembryonic stem cell in human pancreatic cancer stem cells（PCSCs）and study the proliferation and drug-resistence ability of pancreaticcancer stem cell in vitro. Silence Oct4and Nanog expression in PCSCsby specific shRNA, study the biological characteristics of PCSCs andthe related mechanisms.
Methods
Pancreatic cancer stem cells of CD44+CD24+ESA+phenotypesisolated by a FACS Aria II in human cell line Panc-1. Detect theexpression of Oct4and Nanog gene expression in Panc-1cells andPCSCs cells by immunofluorescence and RT-PCR. To study theproliferation ability of cancer stem cell, we draw a growth curve bythe ways of CCK8staining. To detect the ability of drug-resistence ofPCSCs, we stimulated both cells by gemcitabine and study thedifference of their IC50. Nanog and Oct4in PCSCs silenced byLentiviral vector-GFP(LV) mediated shRNA. Observe themigration,invasion, proliferation and the drug sensitivity of PCSCsinhibited by Oct4and Nanog by monoclonal formation as well as theCCK8assay, transwell chamber model. Investigate related mechanismsby FQRT-PCR and Western blot and NOD/SCID mice tumorigenicityexperiments was done to study the tumorigenic potential of PCSCsinfluenced by Oct4and Nanog.
Results
The isolated PCSCs is0.1%-0.8%of all cells. Oct4and Nanoggenes have a higher expression in sorted cells than unsorted ones.The△CT of Nanog in PCSCs and Panc-1cell is7.945±0.652. The△CT of Oct4in PCSCs and Panc-1cell is8.141±1.378; Both twogenes were expressed in nucleus and were restrained by gemcitabine.The48th hour’s IC50of Panc-1cell is982μg/mL, IC50of PCSCsis1981μg/mL. The72th hour’s IC50of Panc-1cell is670μg/mL,IC50of PCSCs cell is1484μg/mL.LV mediated shRNA have stronginhibitory effects for Nanog and Oct4expression(P<0.05).Compared tothe control group,The proliferation, monoclonal formation, invasionability and migration of PCSCs decreased significantly if silencing Oct4and Nanog expression, while enhance drug sensitivity and induceapoptosis(P<0.05). Tumorigenicity experiments showed PCSCstumorigenic capacity decreased significantly than that of control groupif Oct4and Nanog gene silenced.(P<0.05).
Conclusions
1. Cancer stem cell perform a stem cell-like characteristic of highlydrug-resistence and declined proliferation.
2. Oct4and Nanog gene have a higher expression in PCSCs thanin Panc-1cells.
3. LV mediated shRNA effectively reduce the expression of Oct4and Nanog gene in PCSCs.
4. Silence Nanog and Oct4can inhibit the biological characteristicsof PCSCs，while enhance drug sensitivity and induce apoptosis.
引文
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[1] Rebecca Siegel, Elizabeth Ward, et al. Cancer Statistics,2011. CA Cancer J Clin.2011,61(4):212~234.
[2] George P, Stathopoulos, Nikos Androulakis, John Souglakos, et al. PresentTreatment and Future Expectations in Advanced Pancreatic Cancer. AnticancerResearch.2008,28(2B):1303~1308.
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[4] Zhou J, Wang CY, Liu T, Wu B, Zhou F, Xiong JX, et al. Persistence of sidepopulation cells with high drug efflux capacity in pancreatic cancer. World JGastroenterol,2008,14(6):925~930.
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[13]Chenwei Li, David G Heidt, Piero Dalerba, Charles F. Burant, Lanjing Zhang, etal. Simeone. Identification of Pancreatic Cancer Stem Cells. Cancer Res.2007,67:(3):1030~1037.
[14]Olempska M, Eisenach PA, Ammerpohl O, et al. Detection of tumor stem cellmarkers in pancreatic carcinoma cell lines. Hepatobiliary Pancreat Dis Int.2007,6(1):92~97.
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[17]Hongxi Zhao, Yang Li, Haifeng Jin. Rapid and efficient reprogramming ofhuman amnion-derived cells into pluripotency by three factorsOCT4/SOX2/NANOG. Differentiation.2010,80(2-3):123~129.
[18]J Silva, J Nichols, TW Theunissen, G Guo, AL van Oosten, et al. Nanog is thegateway to the pluripotent ground state. Cell.2009,138(4):722~737.
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[26]Y Atlasi, SJ Mowla, SA Ziaee, et al. OCT-4, an embryonic stem cell marker, ishighly expressed in bladder cancer. Int J Cancer.2007,120(7):1598~1602.
[27]Guangyu Gu, Jialing Yuan, Marcia Wills, et al. Prostate cancer cells with stemcell characteristics reconstitute the original human tumor in vivo. Cancer Res.2007,67(10):4807~4815.
[28]Ittai Ben-Porath, Matthew W Thomson, Vincent J Carey, et al. An embryonicstem cell-like gene expression signature in poorly differentiated aggressivehuman tumors. Nat Genet.2008,40(5):499~507.
[29]杜志勇,魏翠凤,胡均,王敏,秦仁义。胚胎干细胞标志物Oct-4在胰腺癌的表达.中华胰腺病杂志.2007,7(6):357~359
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[32]Zeng G, Germinaro M, Micsenyi A, Monga NK, Bell A, Sood A, et al. AberrantWnt/beta-catenin signaling in pancreatic adenocarcinoma. Neoplasia.2006,8(4):279~289
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