RNAi沉默Notch1、Notch2对人胶质瘤U251细胞增殖的影响及其机制
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摘要
神经胶质瘤是最常见的颅内肿瘤,高级别胶质瘤的预后通常很差,因此,探讨胶质瘤的分子机制,研究新的靶向治疗手段就显得十分重要。Notch信号是胶质瘤发病机制的新研究靶点。为深入了解Notch信号通路在神经胶质瘤发生中的作用,探讨沉默Notch治疗神经胶质瘤这一可能的基因治疗方法,本研究利用Notch1-shRNA慢病毒感染人胶质瘤U251细胞,筛选获得Notch1基因稳定沉默的胶质瘤细胞单克隆,对其体内外增殖能力进行研究,并采用基因表达谱芯片技术,分析沉默Notch1对胶质瘤U251细胞的基因表达谱的影响。此外,本研究合成了Notch2的特异siRNA序列,并采用慢病毒介导的RNAi技术研究Notch2基因沉默对胶质瘤U251细胞的体外增殖能力影响,以比较Notch1与Notch2在胶质瘤中功能的异同点。
本研究的第一部分采用实时定量PCR筛选有效的Notch2 siRNA序列,构建Notch2-shRNA慢病毒载体,进行病毒包装,结果成功包装出表达Notch2-shRNA和阴性对照shRNA的慢病毒,滴度分别为3.7×10~5TU/ml和2.5×10~5TU/ml。
本研究的第二部分采用本实验室原有的Notch1-shRNA慢病毒和第一部分生产的Notch2-shRNA慢病毒感染人胶质瘤U251细胞,筛选稳定低表达Notch1和Notch2的细胞单克隆,实时定量PCR检测Notch1和Notch2 mRNA的表达,Western-blot法检测细胞Notch1和Notch2蛋白的表达;结果成功筛选得到Notch1和Notch2稳定沉默的U251细胞单克隆。
本研究的第三部分采用CCK-8法测定细胞生长;平板克隆形成实验和软琼脂克隆形成实验检测细胞的集落形成能力;裸鼠种植瘤模型观察Notch1沉默对种植瘤生长的影响。结果显示Notch1稳定沉默组的增殖与对照组相比明显减慢;Notch1沉默组的平板克隆形成率为(18.6±2.5)%,低于对照组(37.0±3.3)%;Notch1沉默组软琼脂集落形成率(51±5)%,低于对照组(80±4)%;Notch1沉默组裸鼠腋下种植瘤生长速度减慢,30天后瘤重(0.31±0.09)g,明显低于对照组(2.35±0.71)g;统计学分析均有显著意义。而Notch2沉默对细胞增殖无影响。
本研究的第四部分采用基因芯片技术,分析Notch1稳定沉默对U251基因表达谱的影响。结果显示Notch1稳定沉默细胞与对照细胞间的差异表达基因共有775个,涉及细胞增殖与分化、细胞周期、细胞通讯与粘附、糖脂代谢、MAPK信号转导、TGF-β信号转导等多个方面,而Notch1沉默抑制肿瘤的机制可能和细胞周期调控以及TGF-β信号有关。
Gliomas are the most common intracranial tumors. The prognosis for patients with high-grade gliomas is generally poor. It is very important to analyze the molecular mechanisms of gliomas and to develop new treatments. Recently, the relationship of Notch signaling and gliomas was concerned and the Notch signaling was seemed to be a new target of glioma therapies. Therefore, we had interest of the role of Notch signaling in gliomas. We knocked down the Notch1 by lentivirus-mediated RNA interference technology and screened the stable transduced cell clones. After that we measured the cell proliferation of Notch1 knockdown clones in vitro and in vivo. We analyzed the different gene expression between the Notch1 knockdown clone and the control clone by cDNA microarray technology. We also knocked down the Notch2 expression by lentivirus-mediated RNA interference and measured the cell proliferation of Notch2 knockdown clones in vitro, in order to compare the Notch1 and Notch2 function in gliomas.
In the first part of our study we screened effective Notch2 siRNA sequence by real-time PCR technology and we constructed Notch2-shRNA lentiviral vector for virus packaging. We succeeded in packing the Notch2-knockdown and negative control lentivirus. The titer of the viruses was 3.7×10~5 TU/ml and 2.5×10~5 TU/ml respectively.
In the second part of our study we screened the cell clones of Notch1-knockdown and Notch2-knockdown. The human glioma U251 cells were infected with lentivirus expressing Notch1-shRNA or Notch2-shRNA. The knockdown effects of Notch1 or Notch2 of transduced U251 cell clones was detected by real-time PCR and Western blot.
In the third part of our study we investigated the effects of Notch1 knockdown by RNA interference on proliferation of human glioma U251 cells. The cell growth viability was evaluated by CCK-8 assay. Colony formation assay and soft-agar colony formation were used to measure the colony formation ability of stably transduced cells. The influence of Notch1-knockdown on the implanted tumor growth was observed. The ability of growth in Notch1-knockdown cells was decreased significantly. The plate colony formation rate of Notch1-knockdown cells was (18.6±2.5)%, whereas the rate of control cells was (37.0±3.3)%. The soft-agar colony formation rate of Notch1-knockdown cells was (51±5)%, whereas the rate of control cells was (80±4)%. Implanted glioma mouse model was successively established. The tumor weight in Notch1-knockdown group was markedly lower than that in control group(0.31±0.09g vs 2.35±0.71g, P<0.01). But the Notch2 knockdown had no effect on cell proliferation.
In the fourth part of our study we analyzed the different gene expression between the Notch1 knockdown clone and the control clone by cDNA microarray technology. The results showed that there are total of 775 differentially expressed genes related to cell proliferation and differentiation, cell cycle, cell communication and adhesion, lipid metabolism, MAPK signal transduction, TGF-βsignal transduction, etc. The inhibition of cell proliferation of Notch1 knockdown cell clone might be mediated by cell cycle regulation and TGF-βsignaling.
本研究的第一部分采用实时定量PCR筛选有效的Notch2 siRNA序列,构建Notch2-shRNA慢病毒载体,进行病毒包装,结果成功包装出表达Notch2-shRNA和阴性对照shRNA的慢病毒,滴度分别为3.7×10~5TU/ml和2.5×10~5TU/ml。
本研究的第二部分采用本实验室原有的Notch1-shRNA慢病毒和第一部分生产的Notch2-shRNA慢病毒感染人胶质瘤U251细胞,筛选稳定低表达Notch1和Notch2的细胞单克隆,实时定量PCR检测Notch1和Notch2 mRNA的表达,Western-blot法检测细胞Notch1和Notch2蛋白的表达;结果成功筛选得到Notch1和Notch2稳定沉默的U251细胞单克隆。
本研究的第三部分采用CCK-8法测定细胞生长;平板克隆形成实验和软琼脂克隆形成实验检测细胞的集落形成能力;裸鼠种植瘤模型观察Notch1沉默对种植瘤生长的影响。结果显示Notch1稳定沉默组的增殖与对照组相比明显减慢;Notch1沉默组的平板克隆形成率为(18.6±2.5)%,低于对照组(37.0±3.3)%;Notch1沉默组软琼脂集落形成率(51±5)%,低于对照组(80±4)%;Notch1沉默组裸鼠腋下种植瘤生长速度减慢,30天后瘤重(0.31±0.09)g,明显低于对照组(2.35±0.71)g;统计学分析均有显著意义。而Notch2沉默对细胞增殖无影响。
本研究的第四部分采用基因芯片技术,分析Notch1稳定沉默对U251基因表达谱的影响。结果显示Notch1稳定沉默细胞与对照细胞间的差异表达基因共有775个,涉及细胞增殖与分化、细胞周期、细胞通讯与粘附、糖脂代谢、MAPK信号转导、TGF-β信号转导等多个方面,而Notch1沉默抑制肿瘤的机制可能和细胞周期调控以及TGF-β信号有关。
Gliomas are the most common intracranial tumors. The prognosis for patients with high-grade gliomas is generally poor. It is very important to analyze the molecular mechanisms of gliomas and to develop new treatments. Recently, the relationship of Notch signaling and gliomas was concerned and the Notch signaling was seemed to be a new target of glioma therapies. Therefore, we had interest of the role of Notch signaling in gliomas. We knocked down the Notch1 by lentivirus-mediated RNA interference technology and screened the stable transduced cell clones. After that we measured the cell proliferation of Notch1 knockdown clones in vitro and in vivo. We analyzed the different gene expression between the Notch1 knockdown clone and the control clone by cDNA microarray technology. We also knocked down the Notch2 expression by lentivirus-mediated RNA interference and measured the cell proliferation of Notch2 knockdown clones in vitro, in order to compare the Notch1 and Notch2 function in gliomas.
In the first part of our study we screened effective Notch2 siRNA sequence by real-time PCR technology and we constructed Notch2-shRNA lentiviral vector for virus packaging. We succeeded in packing the Notch2-knockdown and negative control lentivirus. The titer of the viruses was 3.7×10~5 TU/ml and 2.5×10~5 TU/ml respectively.
In the second part of our study we screened the cell clones of Notch1-knockdown and Notch2-knockdown. The human glioma U251 cells were infected with lentivirus expressing Notch1-shRNA or Notch2-shRNA. The knockdown effects of Notch1 or Notch2 of transduced U251 cell clones was detected by real-time PCR and Western blot.
In the third part of our study we investigated the effects of Notch1 knockdown by RNA interference on proliferation of human glioma U251 cells. The cell growth viability was evaluated by CCK-8 assay. Colony formation assay and soft-agar colony formation were used to measure the colony formation ability of stably transduced cells. The influence of Notch1-knockdown on the implanted tumor growth was observed. The ability of growth in Notch1-knockdown cells was decreased significantly. The plate colony formation rate of Notch1-knockdown cells was (18.6±2.5)%, whereas the rate of control cells was (37.0±3.3)%. The soft-agar colony formation rate of Notch1-knockdown cells was (51±5)%, whereas the rate of control cells was (80±4)%. Implanted glioma mouse model was successively established. The tumor weight in Notch1-knockdown group was markedly lower than that in control group(0.31±0.09g vs 2.35±0.71g, P<0.01). But the Notch2 knockdown had no effect on cell proliferation.
In the fourth part of our study we analyzed the different gene expression between the Notch1 knockdown clone and the control clone by cDNA microarray technology. The results showed that there are total of 775 differentially expressed genes related to cell proliferation and differentiation, cell cycle, cell communication and adhesion, lipid metabolism, MAPK signal transduction, TGF-βsignal transduction, etc. The inhibition of cell proliferation of Notch1 knockdown cell clone might be mediated by cell cycle regulation and TGF-βsignaling.
引文
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7. Bolo′s V, Grego-Bessa J, Luis de la Pompa J. Notch Signaling in Development and Cancer[J]. Endocr Rev. 2007; 28(3): 339–363
8. Iso T, Kedes L, Hamamori Y. HES and HERP families: multiple effectors of the Notch signaling pathway[J]. J Cell Physiol. 2003; 3: 237–255.
9. Kageyama R, Ohtsuka T, Hatakeyama J, et al. Roles of bHLH genes in neural stem cell differentiation[J]. Exp Cell Res. 2005; 2: 343–348.
10. Rangarajan A, Talora C, Okuyama R, et al. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation[J]. EMBO J. 2001; 13: 3427–3436.
11. Ge W, Martinowich K, Wu X, et al. Notch signaling promotes astrogliogenesis via direct CSL-mediated glial gene activation[J]. J Neurosci Res. 2002; 6: 848–860.
12. Ronchini C, Capobianco AJ. Induction of cyclin D1 transcription and CDK2 activity by Notch(ic): implication for cell cycle disruption in transformation byNotch(ic)[J]. Mol Cell Biol. 2001; 17: 5925–5934.
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