靶向干扰CGI-100基因对白血病K562细胞增殖与分化的影响研究
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摘要
目的:本课题通过靶向干扰CGI-100基因表达后,观察人白血病K562细胞增殖、分化的改变,以及对苦参碱和氯化高铁血红素作用的反应性的改变,研究CGI-100基因在K562细胞中的功能和作用,以及K562细胞诱导分化的分子机制,阐明CGI-100基因的功能,探索肿瘤细胞诱导分化的机制,为寻找肿瘤治疗新靶点提供实验依据。
方法:设计及化学合成3对靶向CGI-100基因的短发夹结构寡核苷酸,以及一对组成相同、序列无同源性的阴性对照,分别退火成双链DNA片段,与pGenesil-1质粒连接,构建表达shRNA的重组质粒,用脂质体2 000分别转染到K562细胞中进行表达,经RT-PCR法和斑点杂交法检测转染前后K562细胞中CGI-100基因表达受抑的程度,选择抑制率最高的细胞作为后续实验,命名为K562/shRNA-CGI-100。
利用MTT﹑联苯胺染色﹑流式细胞术、电镜观察等方法研究K562/shRNA-CGI-100细胞增殖、红系分化、细胞周期与细胞超微结构的变化。用0.2mg/ml苦参碱作用K562/shRNA-CGI-100细胞与对照K562细胞(包括K562/shRNA-pGenesil细胞和未转染K562细胞,下同)后,用MTT法检测细胞增殖状态变化、联苯胺染色检测各组细胞的红系分化情况、RT-PCR技术检测红系分化指标GPA与Gfi-1B mRNA表达情况、流式细胞术检测细胞表面分化抗原GPA(又称CD235a)、CD14和CD15表达水平。用30μmol/L氯化高铁血红素作用K562/shRNA-CGI-100细胞与对照K562细胞后,流式细胞术检测细胞表面分化抗原GPA表达水平。
结果:经DNA测序证实3对靶向CGI-100基因的RNA干扰表达载体pGenesil-1-CGI-100与阴性对照载体pGenesil-1-Negative构建成功。分别将上述3对干扰载体、阴性对照载体pGenesil-1-Negative与空载质粒pGenesil-1转染白血病K562细胞,经G418抗性筛选后,荧光倒置显微镜下可以观察到细胞内绿色荧光蛋白(EGFP)表达;RT-PCR与斑点杂交结果表明3对干扰载体均抑制了CGI-100 mRNA的表达,其中第1对pGenesil-1-CGI-100的抑制效率最高,达54%,将此重组细胞命名为K562/shRNA-CGI-100 ,作为后续实验对象。而pGenesil-1-Negative质粒与pGenesil-1质粒对CGI-100 mRNA的表达无影响。
与对照K562细胞相比,K562/shRNA-CGI-100细胞中CGI-100基因mRNA表达降低了54%;细胞常染色质减少,异染色质增加;MTT显示细胞吸光度值下降23%(P<0.05);细胞周期相分布明显改变,G0/G1期细胞由21%增加到35%、S期细胞由56%减少到38%、增殖指数(PI)从78%降低到63%、联苯胺染色阳性率从3%升高到7%(P<0.05)。0.2mg/ml苦参碱作用48h后,MTT显示K562/shRNA-CGI-100细胞增殖抑制率从41%增高到62%(P<0.05),联苯胺染色阳性率由16%升高到23%(P<0.05),红系分化指标GPA mRNA、Gfi-1B mRNA表达明显增高(P<0.05)、流式细胞仪检测红系分化抗原GPA的MFI值增高了31%(P<0.05);单核/巨噬细胞系分化指标CD14在各组细胞均未表达、粒系分化指标CD15虽有表达,但三种细胞之间无统计学意义(P>0.05)。30μmol/L氯化高铁血红素分别处理K562/shRNA-CGI-100与对照K562细胞后,前者细胞表面GPA的MFI升高了20%(P<0.05),MFI值为1485,是0.2mg/ml苦参碱处理组的1.7倍,差异具有统计学意义(P<0.05)。
结论:成功构建了针对CGI-100基因的RNA干扰表达载体,并筛选出1对抑制效率较高的重组干扰载体。CGI-100基因的低表达能抑制K562细胞增殖、使细胞幼稚程度减弱、使K562细胞向红系分化的能力增强,并且增加了K562细胞对苦参碱和氯化高铁血红素的敏感性。氯化高铁血红素的红系诱导分化作用强于苦参碱,但CGI-100基因受抑后,K562细胞对苦参碱的敏感性大于氯化高铁血红素。
Objective: This study aims at explaining the effects on proliferation and differentiation of K562 cells, and the molecular mechanism of induced differertiation of K562 cells after silencing CGI-100 gene expression by RNA interference. Furthermore,we hope to provide experimental basis for seeking for a new target of cancer gene therapy.
Methods:Three oligonucleotides targeting to CGI-100 gene and a pair of negative control which has the same composition but different sequence, were devised and chemically synthesized. They were annealed to form double-strand DNA and inserted into plasmid pGenesil-1 which digested by BamHI and HindIII. The recombinant vectors were then transfected respectively into K562 cells with lipofectamineTM 2 000 ,The CGI-100 mRNA level in the transfected K562 cells was analyzed by RT-PCR and dot blot hybridization. The recombinated K562 cells with the highest inhibition of CGI-100 expression were chosen for following experiments, named K562/shRNA-CGI-100 cells.
The changes of cell proliferation、cell cycle、erythroid differentiation and ultrastructures of K562/shRNA-CGI-100 cells were detected respectively with MTT、flow cytometry、benzidine staining and electron microscope. K562/shRNA-CGI-100 cells and control K562 cells( including K562/shRNA-pGenesil cells and untransfected K562 cells) were treated respectively with 0.2mg/ml matrine and 30μmol/L hemin.The proliferation were observed by MTT. The expressions of GPA(CD235a)、Gfi-1B mRNA and GPA、CD14、CD15 proteins were detected respectively by RT-PCR and flow cytometry.
Results: The three recombinant plasmids of pGenesil-1-CGI-100 and negative control plasmid—pGenesil-1-Negative were successfully constructed. The three recombinant plasmids of pGenesil-1-CGI-100、pGenesil-1-Negative and blank plasmids of pGenesil-1 were transfected respectively into K562 cells. The modified K562 cells named K562/shRNA-CGI-100、K562/shRNA-Negative、K562/shRNA-pGenesil were selected by G418 pressure ,the fluorescent light might been observed under the inverted fluorescence microscope; The expression of CGI-100mRNA was detected by RT-PCR and dot blot hybridization. One of the modified K562 cells ,which was named K562/shRNA-CGI-100 cells in the following experiments, exhibited the highest inhibition efficiency in CGI-100 mRNA level, up to 54%; however, the pGenesil-1- Negative vector and pGenesil-1 plasmid did not decrease CGI-100 gene expression.
Compared with control K562 cells, K562/shRNA-CGI-100 cells showed CGI-100 gene mRNA expression decrease by 54%,enchromation decreased while heterochromation increased, the absorbance value decreased by 23% with MTT(P<0.05), percentage of G0/G1 phase cells increased from 21% to 35%, While that of S phase decreased from 56% to 38%(P<0.05); PI (proliferation index of cells) decreased from 78% to 63%(P<0.05). The percentage of benzidine-positive cells increased from 3% to 7% (P<0.05). The inhibitory rate of proliferation of K562/shRNA-CGI-100 cells increased from 41% to 62%( P<0.05) after exporing to 0.2mg/ml matrine, while the percentage of benzidine-positive cells increased from 16% to 23%( P<0.05), the mRNA expression of GPA and Gfi-1B were obviously increased( P<0.05),the mean fluorescence intensity(MFI) of GPA was increased by 31%( P<0.05).Expressions of CD14 which was the marker of monocytes andmacrophages were not found. Although the granulocytic marker-CD15expressed, it has no statistical significance( P>0.05).Compared with control cells, the MFI of GPA increased by 20% in K562/shRNA-CGI-100 cells treated with 30μmol/L hemin ( P<0.05). The MFI of GPA in K562/shRNA-CGI-100 cells treated with 30μmol/L hemin was 1.7 times as high as that with 0.2 mg/ml marine(P<0.05).
Conclusion:The RNA interference vectors targeting to CGI-100 gene were successfully constructed and one recombinated interfering vector with highest inhibition efficiency was screened.Low expression of CGI-100 can inhibit the proliferation of K562 cells and reduce the immaturation level. Inhibition of the expression of CGI-100 can induce erythroid differentiation of K562 cells as well as elevate the sensitivity to matrine and hemin. Hemin which was generally accepted as erythroid differentiation inducer have more powerful erythroid differentiation ability than Matrine, while,With CGI-100 knockdown, K562 cells showed higher sensitivity to Matrine than to Hemin.
方法:设计及化学合成3对靶向CGI-100基因的短发夹结构寡核苷酸,以及一对组成相同、序列无同源性的阴性对照,分别退火成双链DNA片段,与pGenesil-1质粒连接,构建表达shRNA的重组质粒,用脂质体2 000分别转染到K562细胞中进行表达,经RT-PCR法和斑点杂交法检测转染前后K562细胞中CGI-100基因表达受抑的程度,选择抑制率最高的细胞作为后续实验,命名为K562/shRNA-CGI-100。
利用MTT﹑联苯胺染色﹑流式细胞术、电镜观察等方法研究K562/shRNA-CGI-100细胞增殖、红系分化、细胞周期与细胞超微结构的变化。用0.2mg/ml苦参碱作用K562/shRNA-CGI-100细胞与对照K562细胞(包括K562/shRNA-pGenesil细胞和未转染K562细胞,下同)后,用MTT法检测细胞增殖状态变化、联苯胺染色检测各组细胞的红系分化情况、RT-PCR技术检测红系分化指标GPA与Gfi-1B mRNA表达情况、流式细胞术检测细胞表面分化抗原GPA(又称CD235a)、CD14和CD15表达水平。用30μmol/L氯化高铁血红素作用K562/shRNA-CGI-100细胞与对照K562细胞后,流式细胞术检测细胞表面分化抗原GPA表达水平。
结果:经DNA测序证实3对靶向CGI-100基因的RNA干扰表达载体pGenesil-1-CGI-100与阴性对照载体pGenesil-1-Negative构建成功。分别将上述3对干扰载体、阴性对照载体pGenesil-1-Negative与空载质粒pGenesil-1转染白血病K562细胞,经G418抗性筛选后,荧光倒置显微镜下可以观察到细胞内绿色荧光蛋白(EGFP)表达;RT-PCR与斑点杂交结果表明3对干扰载体均抑制了CGI-100 mRNA的表达,其中第1对pGenesil-1-CGI-100的抑制效率最高,达54%,将此重组细胞命名为K562/shRNA-CGI-100 ,作为后续实验对象。而pGenesil-1-Negative质粒与pGenesil-1质粒对CGI-100 mRNA的表达无影响。
与对照K562细胞相比,K562/shRNA-CGI-100细胞中CGI-100基因mRNA表达降低了54%;细胞常染色质减少,异染色质增加;MTT显示细胞吸光度值下降23%(P<0.05);细胞周期相分布明显改变,G0/G1期细胞由21%增加到35%、S期细胞由56%减少到38%、增殖指数(PI)从78%降低到63%、联苯胺染色阳性率从3%升高到7%(P<0.05)。0.2mg/ml苦参碱作用48h后,MTT显示K562/shRNA-CGI-100细胞增殖抑制率从41%增高到62%(P<0.05),联苯胺染色阳性率由16%升高到23%(P<0.05),红系分化指标GPA mRNA、Gfi-1B mRNA表达明显增高(P<0.05)、流式细胞仪检测红系分化抗原GPA的MFI值增高了31%(P<0.05);单核/巨噬细胞系分化指标CD14在各组细胞均未表达、粒系分化指标CD15虽有表达,但三种细胞之间无统计学意义(P>0.05)。30μmol/L氯化高铁血红素分别处理K562/shRNA-CGI-100与对照K562细胞后,前者细胞表面GPA的MFI升高了20%(P<0.05),MFI值为1485,是0.2mg/ml苦参碱处理组的1.7倍,差异具有统计学意义(P<0.05)。
结论:成功构建了针对CGI-100基因的RNA干扰表达载体,并筛选出1对抑制效率较高的重组干扰载体。CGI-100基因的低表达能抑制K562细胞增殖、使细胞幼稚程度减弱、使K562细胞向红系分化的能力增强,并且增加了K562细胞对苦参碱和氯化高铁血红素的敏感性。氯化高铁血红素的红系诱导分化作用强于苦参碱,但CGI-100基因受抑后,K562细胞对苦参碱的敏感性大于氯化高铁血红素。
Objective: This study aims at explaining the effects on proliferation and differentiation of K562 cells, and the molecular mechanism of induced differertiation of K562 cells after silencing CGI-100 gene expression by RNA interference. Furthermore,we hope to provide experimental basis for seeking for a new target of cancer gene therapy.
Methods:Three oligonucleotides targeting to CGI-100 gene and a pair of negative control which has the same composition but different sequence, were devised and chemically synthesized. They were annealed to form double-strand DNA and inserted into plasmid pGenesil-1 which digested by BamHI and HindIII. The recombinant vectors were then transfected respectively into K562 cells with lipofectamineTM 2 000 ,The CGI-100 mRNA level in the transfected K562 cells was analyzed by RT-PCR and dot blot hybridization. The recombinated K562 cells with the highest inhibition of CGI-100 expression were chosen for following experiments, named K562/shRNA-CGI-100 cells.
The changes of cell proliferation、cell cycle、erythroid differentiation and ultrastructures of K562/shRNA-CGI-100 cells were detected respectively with MTT、flow cytometry、benzidine staining and electron microscope. K562/shRNA-CGI-100 cells and control K562 cells( including K562/shRNA-pGenesil cells and untransfected K562 cells) were treated respectively with 0.2mg/ml matrine and 30μmol/L hemin.The proliferation were observed by MTT. The expressions of GPA(CD235a)、Gfi-1B mRNA and GPA、CD14、CD15 proteins were detected respectively by RT-PCR and flow cytometry.
Results: The three recombinant plasmids of pGenesil-1-CGI-100 and negative control plasmid—pGenesil-1-Negative were successfully constructed. The three recombinant plasmids of pGenesil-1-CGI-100、pGenesil-1-Negative and blank plasmids of pGenesil-1 were transfected respectively into K562 cells. The modified K562 cells named K562/shRNA-CGI-100、K562/shRNA-Negative、K562/shRNA-pGenesil were selected by G418 pressure ,the fluorescent light might been observed under the inverted fluorescence microscope; The expression of CGI-100mRNA was detected by RT-PCR and dot blot hybridization. One of the modified K562 cells ,which was named K562/shRNA-CGI-100 cells in the following experiments, exhibited the highest inhibition efficiency in CGI-100 mRNA level, up to 54%; however, the pGenesil-1- Negative vector and pGenesil-1 plasmid did not decrease CGI-100 gene expression.
Compared with control K562 cells, K562/shRNA-CGI-100 cells showed CGI-100 gene mRNA expression decrease by 54%,enchromation decreased while heterochromation increased, the absorbance value decreased by 23% with MTT(P<0.05), percentage of G0/G1 phase cells increased from 21% to 35%, While that of S phase decreased from 56% to 38%(P<0.05); PI (proliferation index of cells) decreased from 78% to 63%(P<0.05). The percentage of benzidine-positive cells increased from 3% to 7% (P<0.05). The inhibitory rate of proliferation of K562/shRNA-CGI-100 cells increased from 41% to 62%( P<0.05) after exporing to 0.2mg/ml matrine, while the percentage of benzidine-positive cells increased from 16% to 23%( P<0.05), the mRNA expression of GPA and Gfi-1B were obviously increased( P<0.05),the mean fluorescence intensity(MFI) of GPA was increased by 31%( P<0.05).Expressions of CD14 which was the marker of monocytes andmacrophages were not found. Although the granulocytic marker-CD15expressed, it has no statistical significance( P>0.05).Compared with control cells, the MFI of GPA increased by 20% in K562/shRNA-CGI-100 cells treated with 30μmol/L hemin ( P<0.05). The MFI of GPA in K562/shRNA-CGI-100 cells treated with 30μmol/L hemin was 1.7 times as high as that with 0.2 mg/ml marine(P<0.05).
Conclusion:The RNA interference vectors targeting to CGI-100 gene were successfully constructed and one recombinated interfering vector with highest inhibition efficiency was screened.Low expression of CGI-100 can inhibit the proliferation of K562 cells and reduce the immaturation level. Inhibition of the expression of CGI-100 can induce erythroid differentiation of K562 cells as well as elevate the sensitivity to matrine and hemin. Hemin which was generally accepted as erythroid differentiation inducer have more powerful erythroid differentiation ability than Matrine, while,With CGI-100 knockdown, K562 cells showed higher sensitivity to Matrine than to Hemin.
引文
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