iASPP对不同p53背景的胃癌细胞顺铂化疗敏感性的影响
摘要
目的研究不同p53背景的胃癌细胞p53凋亡刺激蛋白（ASPP）的表达与顺铂化疗敏感性的关系以及下调iASPP的表达，对不同p53背景的胃癌细胞顺铂化疗敏感性的影响。
方法（1）采用MTT法检测不同浓度的顺铂对胃癌细胞AGS（p53野生型）及SGC-7901（p53突变型）在24h、48h、72h时的生长抑制作用。RT-PCR技术检测胃癌细胞AGS和SGC-7901ASPP家族的表达水平，并分析该家族与顺铂化疗敏感性的关系。
（2）构建针对iASPP基因的短发卡RNA干扰（iASPP-shRNA）质粒，并转染胃癌细胞AGS和SGC-7901；利用RT-PCR技术检测iASPPmRNA表达水平的变化。
（3）下调胃癌细胞AGS及SGC-7901的iASPP的表达后，MTT法检测顺铂对其生长抑制作用及流式细胞技术检测凋亡率。
结果（1）胃癌细胞AGS、SGC-7901经不同浓度的顺铂化疗不同时间后，细胞的增殖与空白对照组相比均受到抑制，差异均有统计学意义（P<0.05）。相同浓度的顺铂化疗相同时间，除24h6.00μg/ml及72h3.00μg/ml、6.00μg/ml、12.00μg/ml组之外，顺铂对AGS抑制率低于对SGC-7901的抑制率，差异有统计学意义（P<0.05）；顺铂化疗24h、48h、72h后，AGS半效抑制浓度(IC50)均高于SGC-7901，差异均有统计学意义（P<0.05）；AGS与SGC-7901ASPP家族的表达水平的比较，ASPP2的表达AGS低于SGC-7901，差异有统计学意义（P<0.05）；iASPP的表达AGS高于SGC-7901，差异有统计学意义（P<0.05）；ASPP1的表达差异无统计学意义（P>0.05）。
（2）针对iASPP基因的短发卡RNA干扰（pGenesil-2.1-iASPP-shRNA）质粒构建成功，并设有pGenesil-2.1-HK阴性对照和pGenesil-2.1-KB空白对照；RT-PCR法检测iASPP表达变化，转染iASPP-shRNA的胃癌细胞AGS及SGC-7901，与对应的转染空载体pGenesil-2.1的细胞相比，iASPP表达均下调（P<0.05）。
（3）以3.00μg/ml顺铂作用24h后，AGS-iASPP-shRNA的生长抑制率（88.2±3.7）高于AGS-KB（54.3±1.8），差异有统计学意义（P<0.05）；SGC-7901-iASPP-shRNA的生长抑制率（68.3±2.3）高于SGC-7901-KB（63.2±1.5），差异有统计学意义（P<0.05）。以3.00μg/ml顺铂作用24h后， AGS-iASPP-shRNA的凋亡率（68.7±3.7）高于AGS-KB（21.5±1.9），差异有统计学意义（P<0.05）；SGC-7901-iASPP-shRNA的凋亡率（38.5±2.9）高于SGC-7901-KB（32.4±2.2），差异有统计学意义（P<0.05）。AGS-iASPP-shRNA与AGS-KB凋亡率差值高于SGC-7901-iASPP-shRNA与SGC-7901-KB凋亡率差值，差异有统计学意义（P<0.05）。
结论p53野生型胃癌细胞AGS对顺铂化疗的敏感性低于p53突变型胃癌细胞SGC-7901；相比SGC-7901的ASPP家族的表达水平，AGS细胞ASPP2低表达、iASPP高表达，而ASPP1无差异；iASPP表达水平的变化可以影响胃癌细胞的化疗敏感性，其表达越高，胃癌细胞的顺铂化疗敏感性越低，对AGS的影响更为显著。
Objective To approach the association between the mRNA expression ofASPP family genes and cisplatin chemotherapy sensitivity in gastric cancercells of different p53genetic background. To approach the influence ofcisplatin chemotherapy sensitivity to gastric cancer cells of different p53genetic background after the mRNA expression of iASPP weredownregulated.
Methods （1）Gastric cancer cell lines AGS (wild-type p53) andSGC-7901(mutant-type p53) with different p53genetic background weretreated with different concentrations of cisplatin, and measured the cell linesinhibition ratio after24hours,48hours and72hours by MTT assay. ThemRNA expression of ASPP family genes in gastric cancer cell lines AGS andSGC-7901were detected by RT-PCR,and analysed the association betweenthe mRNA expression of ASPP family genes and cisplatin chemotherapysensitivity.
（2）Short hairpin RNAs (shRNAs) that target iASPP were constructed,and transfected into gastric cancer cell lines AGS and SGC-7901. Theexpression alteration of iASPP were detected by RT-PCR.
（3）The mRNA expression of iASPP in gastric cancer cell lines AGSand SGC-7901were downregulated，and the cell lines were treated withcisplatin,then the inhibition rate were measured by MTT assay,the apoptosisrate by flow cytometry.
Results（1）Compared with the control group,the cell proliferation wasslowed down in gastric cancer cell lines AGS and SGC-7901when treatedwith different levels of cisplatin(P<0.05). The cell lines treated with cisplatinafter24hours,48hours and72hours,the inhibition ratios of AGS were lowerthan SGC-7901（P<0.05）,the IC50of AGS were higher than SGC-7901（P<0.05）. Compared with the mRNA expression of ASPP family genes inAGS and SGC-7901, the mRNA expression of ASPP2in AGS was lower thanin SGC-7901（P<0.05）, the mRNA expression of iASPP in AGS was higherthan in SGC-7901（P<0.05）, the mRNA expression of ASPP1wasundifferentiated（P>0.05）.
（2）Recombinant plasmid vector expressing iASPP was successfullyestablished，compared to the group of HK and KB，the mRNA expressioniASPP identified by RT-PCR were significantly inhibited by iASPP-shRNA inAGS and SGC-7901cells（P<0.05）.
（3）The cell lines treated with cisplatin（3.00μg/ml）after24hours, theinhibition rate of AGS-iASPP-shRNA was higher than that of AGS-KB,whilethe inhibition rate of SGS-7901-iASPP-shRNA was higher than that of SGC-7901-KB（P<0.05）.The cell lines treated with cisplatin（3.00μg/ml）after24hours, the apoptosis rate of AGS-iASPP-shRNA was higher thanthat of AGS-KB, while the apoptosis rate of SGC-7901-iASPP-shRNA washigher than that of SGS-7901-KB（P<0.05）. The apoptosis rate D-valuebetween AGS-iASPP-shRNA and AGS-KB was significantly higher than thatbetween SGC-7901-iASPP-shRNA and SGS-7901-KB（P<0.05）.
Conclusion The cisplatin chemotherapy sensitivity of AGS was lowerthan that of SGC-7901.Compared to the mRNA expression of ASPP familygenes in SGC-7901, ASPP2in AGS was lower,iASPP in AGS was higher,ASPP1was undifferentiated. The iASPP expression level can influence thecisplatin resistance of gastric cancer cells.The iASPP expression level ishigher,the cisplatin sensitivity is the lower,more significantly in AGS.
方法（1）采用MTT法检测不同浓度的顺铂对胃癌细胞AGS（p53野生型）及SGC-7901（p53突变型）在24h、48h、72h时的生长抑制作用。RT-PCR技术检测胃癌细胞AGS和SGC-7901ASPP家族的表达水平，并分析该家族与顺铂化疗敏感性的关系。
（2）构建针对iASPP基因的短发卡RNA干扰（iASPP-shRNA）质粒，并转染胃癌细胞AGS和SGC-7901；利用RT-PCR技术检测iASPPmRNA表达水平的变化。
（3）下调胃癌细胞AGS及SGC-7901的iASPP的表达后，MTT法检测顺铂对其生长抑制作用及流式细胞技术检测凋亡率。
结果（1）胃癌细胞AGS、SGC-7901经不同浓度的顺铂化疗不同时间后，细胞的增殖与空白对照组相比均受到抑制，差异均有统计学意义（P<0.05）。相同浓度的顺铂化疗相同时间，除24h6.00μg/ml及72h3.00μg/ml、6.00μg/ml、12.00μg/ml组之外，顺铂对AGS抑制率低于对SGC-7901的抑制率，差异有统计学意义（P<0.05）；顺铂化疗24h、48h、72h后，AGS半效抑制浓度(IC50)均高于SGC-7901，差异均有统计学意义（P<0.05）；AGS与SGC-7901ASPP家族的表达水平的比较，ASPP2的表达AGS低于SGC-7901，差异有统计学意义（P<0.05）；iASPP的表达AGS高于SGC-7901，差异有统计学意义（P<0.05）；ASPP1的表达差异无统计学意义（P>0.05）。
（2）针对iASPP基因的短发卡RNA干扰（pGenesil-2.1-iASPP-shRNA）质粒构建成功，并设有pGenesil-2.1-HK阴性对照和pGenesil-2.1-KB空白对照；RT-PCR法检测iASPP表达变化，转染iASPP-shRNA的胃癌细胞AGS及SGC-7901，与对应的转染空载体pGenesil-2.1的细胞相比，iASPP表达均下调（P<0.05）。
（3）以3.00μg/ml顺铂作用24h后，AGS-iASPP-shRNA的生长抑制率（88.2±3.7）高于AGS-KB（54.3±1.8），差异有统计学意义（P<0.05）；SGC-7901-iASPP-shRNA的生长抑制率（68.3±2.3）高于SGC-7901-KB（63.2±1.5），差异有统计学意义（P<0.05）。以3.00μg/ml顺铂作用24h后， AGS-iASPP-shRNA的凋亡率（68.7±3.7）高于AGS-KB（21.5±1.9），差异有统计学意义（P<0.05）；SGC-7901-iASPP-shRNA的凋亡率（38.5±2.9）高于SGC-7901-KB（32.4±2.2），差异有统计学意义（P<0.05）。AGS-iASPP-shRNA与AGS-KB凋亡率差值高于SGC-7901-iASPP-shRNA与SGC-7901-KB凋亡率差值，差异有统计学意义（P<0.05）。
结论p53野生型胃癌细胞AGS对顺铂化疗的敏感性低于p53突变型胃癌细胞SGC-7901；相比SGC-7901的ASPP家族的表达水平，AGS细胞ASPP2低表达、iASPP高表达，而ASPP1无差异；iASPP表达水平的变化可以影响胃癌细胞的化疗敏感性，其表达越高，胃癌细胞的顺铂化疗敏感性越低，对AGS的影响更为显著。
Objective To approach the association between the mRNA expression ofASPP family genes and cisplatin chemotherapy sensitivity in gastric cancercells of different p53genetic background. To approach the influence ofcisplatin chemotherapy sensitivity to gastric cancer cells of different p53genetic background after the mRNA expression of iASPP weredownregulated.
Methods （1）Gastric cancer cell lines AGS (wild-type p53) andSGC-7901(mutant-type p53) with different p53genetic background weretreated with different concentrations of cisplatin, and measured the cell linesinhibition ratio after24hours,48hours and72hours by MTT assay. ThemRNA expression of ASPP family genes in gastric cancer cell lines AGS andSGC-7901were detected by RT-PCR,and analysed the association betweenthe mRNA expression of ASPP family genes and cisplatin chemotherapysensitivity.
（2）Short hairpin RNAs (shRNAs) that target iASPP were constructed,and transfected into gastric cancer cell lines AGS and SGC-7901. Theexpression alteration of iASPP were detected by RT-PCR.
（3）The mRNA expression of iASPP in gastric cancer cell lines AGSand SGC-7901were downregulated，and the cell lines were treated withcisplatin,then the inhibition rate were measured by MTT assay,the apoptosisrate by flow cytometry.
Results（1）Compared with the control group,the cell proliferation wasslowed down in gastric cancer cell lines AGS and SGC-7901when treatedwith different levels of cisplatin(P<0.05). The cell lines treated with cisplatinafter24hours,48hours and72hours,the inhibition ratios of AGS were lowerthan SGC-7901（P<0.05）,the IC50of AGS were higher than SGC-7901（P<0.05）. Compared with the mRNA expression of ASPP family genes inAGS and SGC-7901, the mRNA expression of ASPP2in AGS was lower thanin SGC-7901（P<0.05）, the mRNA expression of iASPP in AGS was higherthan in SGC-7901（P<0.05）, the mRNA expression of ASPP1wasundifferentiated（P>0.05）.
（2）Recombinant plasmid vector expressing iASPP was successfullyestablished，compared to the group of HK and KB，the mRNA expressioniASPP identified by RT-PCR were significantly inhibited by iASPP-shRNA inAGS and SGC-7901cells（P<0.05）.
（3）The cell lines treated with cisplatin（3.00μg/ml）after24hours, theinhibition rate of AGS-iASPP-shRNA was higher than that of AGS-KB,whilethe inhibition rate of SGS-7901-iASPP-shRNA was higher than that of SGC-7901-KB（P<0.05）.The cell lines treated with cisplatin（3.00μg/ml）after24hours, the apoptosis rate of AGS-iASPP-shRNA was higher thanthat of AGS-KB, while the apoptosis rate of SGC-7901-iASPP-shRNA washigher than that of SGS-7901-KB（P<0.05）. The apoptosis rate D-valuebetween AGS-iASPP-shRNA and AGS-KB was significantly higher than thatbetween SGC-7901-iASPP-shRNA and SGS-7901-KB（P<0.05）.
Conclusion The cisplatin chemotherapy sensitivity of AGS was lowerthan that of SGC-7901.Compared to the mRNA expression of ASPP familygenes in SGC-7901, ASPP2in AGS was lower,iASPP in AGS was higher,ASPP1was undifferentiated. The iASPP expression level can influence thecisplatin resistance of gastric cancer cells.The iASPP expression level ishigher,the cisplatin sensitivity is the lower,more significantly in AGS.
引文
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