HIC-1基因在宫颈癌Hela细胞中的表达及其甲基化调控
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摘要
目的:检测甲基化抑制剂处理前后宫颈癌Hela细胞增殖、凋亡改变,及HIC-1、HPV表达的变化,初步探讨去甲基化对宫颈癌中HIC-1基因及HPV表达的影响。
方法:分别用不同浓度5-氮杂脱氧胞苷(5-Aza-CdR)干预细胞5d。提取细胞的RNA,采用逆转录聚合酶链反应(RT-PCR)方法检测HIC-1mRNA、P53mRNA、HPV18E6mRNA在干预后表达变化情况;用直接记数法和四甲基偶氮唑盐(MTT)显色法检测细胞生长曲线与细胞增殖实验。
结果:
(1)凝胶图像分析仪分析RT-PCR产物:实验组5-Aza-CdR(终浓度分别为5μmol/L、10μmol/L、20μmol/L)作用Hela细胞5d前后,①各实验组和对照组HIC-1 mRNA/β-actin分别为0.486±0.025、0.548±0.014、0.864±0.032、0.927±0.031,随着5-Aza-CdR浓度的升高,其表达随之增加,经方差分析对照组和实验组之间差异具有显著性(F=701.430,P＜0.05)。②实验组和对照组P53 mRNA/β-actin分别为0.063±0.026、0.207±0.028、0.598±0.030、0.807±0.029。经方差分析多重比较结果显示试验组与对照组有显著性差异(F=446.673,P＜0.05)。③实验组和对照组HPV18E6mRNA/β-actin分别为0.405±0.022、0.373±0.021、0.377±0.015、0.408±0.012,各实验组和对照组数据之间差异无显著性(F=2.900,P＞0.05),即5-Aza-CdR干预前后,HPV18E6mRNA表达无明显改变。
(2)经终浓度分别为5μmol/L、10μmol/L、20μmol/L的5-Aza-CdR处理HeLa细胞5d后,MTT检测各实验组细胞活力分别为(A490值)65.08%±4.06%、49.68%±1.49%、38.80%±3.00%,与对照组(A490值)91.22%±2.7%,比较有明显差异(F=208.39 P＜0.05)。在所检测的浓度范围内随着5-Aza-CdR浓度的增加,对HeLa细胞作用随之增加,呈量一效关系(相关度比较R=-0.969 P＜0.05)。
(3)5-Aza-CdR处理前后Hela细胞形态学出现以下改变:倒置显微镜下观察,对照组细胞生长良好,细胞伸展、透亮,呈不规则对角型,随时间推移,细胞逐渐增多,细胞接触紧密。实验组20μmol/L的5-Aza-CdR作用5d后Hela细胞细胞密度降低,汇合度约为对照组的70%,细胞形态未见明显改变。(见图1、2)
结论:
1.HIC-1基因甲基化是HIC-1基因表达下调的重要机制之一;
2.HIC-1基因甲基化可能在宫颈癌Hela细胞增殖中发挥作用;
3.5-Aza-CdR可抑制Hela细胞增殖,并在一定浓度范围内呈量效关系。
Objective To detect the expression of HPV and HIC-1 gene and their relations and study proliferative changes in HeLa cells with the effects of 5-Aza-2'-deoxycytidine (5-Aza-CdR).Then to investigate the promoter methylation status of HIC-1 and the potential mechanism of its antitumorigenesis in cervical cancer.
Methods Human cervical cancer cell line Hela was treated with 5-Aza-CdR (5, 10 and 20μmol/L ), a specific demethylation agent for 5 days. The expression of HIC-1 mRNA and HPV18E6 mRNA was observed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The anti- proliferative effect was measured by methabenzthiazuron (MTT) assay.
The data was analyzed with SPSS11.5 statistical software.
Results After 5-Aza-CdR was added into HeLa cells with different dose for 5 days, our results were spread out below:
1、After 5,10and20μmol/L 5-Aza-CdR treatment, the level of HIC-1 mRNA and P53 mRNA expression were increased, and they were significantly correlated with the concentration of 5-Aza-CdR. The expression of HPV18E6 mRNA was remaining unchanged in all of the cases.
2、Hela cells treated with 5-Aza-CdR displayed a slow growth rate in comparison with that of the control cells, The cell vitalities of treated group were 91.22%±2.7%、65.08%±4.06%、49.68%±1.49%、38.80 %±3.00% individually compared with the control group and the inhibiting effect of 5-Aza-CdR was dose-dependent.
3、5-Aza-CdR could reduce the density of Hela cells ,and the convergence of team was about 70 percent of the control group, the morphologic changes in cells was no significant.
Conclusion
1、Aberrant methylation of the HIC-1gene seems to be an important mechanism underlying its down-regulation of expression.
2、Aberrant methylation of the HIC-1 seems to be one of the role in the proliferation of Hela cells .
3、5-Aza-CdR can inhibit the proliferation of Hela cells with dose-dependent relationgship of its effect.
方法:分别用不同浓度5-氮杂脱氧胞苷(5-Aza-CdR)干预细胞5d。提取细胞的RNA,采用逆转录聚合酶链反应(RT-PCR)方法检测HIC-1mRNA、P53mRNA、HPV18E6mRNA在干预后表达变化情况;用直接记数法和四甲基偶氮唑盐(MTT)显色法检测细胞生长曲线与细胞增殖实验。
结果:
(1)凝胶图像分析仪分析RT-PCR产物:实验组5-Aza-CdR(终浓度分别为5μmol/L、10μmol/L、20μmol/L)作用Hela细胞5d前后,①各实验组和对照组HIC-1 mRNA/β-actin分别为0.486±0.025、0.548±0.014、0.864±0.032、0.927±0.031,随着5-Aza-CdR浓度的升高,其表达随之增加,经方差分析对照组和实验组之间差异具有显著性(F=701.430,P＜0.05)。②实验组和对照组P53 mRNA/β-actin分别为0.063±0.026、0.207±0.028、0.598±0.030、0.807±0.029。经方差分析多重比较结果显示试验组与对照组有显著性差异(F=446.673,P＜0.05)。③实验组和对照组HPV18E6mRNA/β-actin分别为0.405±0.022、0.373±0.021、0.377±0.015、0.408±0.012,各实验组和对照组数据之间差异无显著性(F=2.900,P＞0.05),即5-Aza-CdR干预前后,HPV18E6mRNA表达无明显改变。
(2)经终浓度分别为5μmol/L、10μmol/L、20μmol/L的5-Aza-CdR处理HeLa细胞5d后,MTT检测各实验组细胞活力分别为(A490值)65.08%±4.06%、49.68%±1.49%、38.80%±3.00%,与对照组(A490值)91.22%±2.7%,比较有明显差异(F=208.39 P＜0.05)。在所检测的浓度范围内随着5-Aza-CdR浓度的增加,对HeLa细胞作用随之增加,呈量一效关系(相关度比较R=-0.969 P＜0.05)。
(3)5-Aza-CdR处理前后Hela细胞形态学出现以下改变:倒置显微镜下观察,对照组细胞生长良好,细胞伸展、透亮,呈不规则对角型,随时间推移,细胞逐渐增多,细胞接触紧密。实验组20μmol/L的5-Aza-CdR作用5d后Hela细胞细胞密度降低,汇合度约为对照组的70%,细胞形态未见明显改变。(见图1、2)
结论:
1.HIC-1基因甲基化是HIC-1基因表达下调的重要机制之一;
2.HIC-1基因甲基化可能在宫颈癌Hela细胞增殖中发挥作用;
3.5-Aza-CdR可抑制Hela细胞增殖,并在一定浓度范围内呈量效关系。
Objective To detect the expression of HPV and HIC-1 gene and their relations and study proliferative changes in HeLa cells with the effects of 5-Aza-2'-deoxycytidine (5-Aza-CdR).Then to investigate the promoter methylation status of HIC-1 and the potential mechanism of its antitumorigenesis in cervical cancer.
Methods Human cervical cancer cell line Hela was treated with 5-Aza-CdR (5, 10 and 20μmol/L ), a specific demethylation agent for 5 days. The expression of HIC-1 mRNA and HPV18E6 mRNA was observed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The anti- proliferative effect was measured by methabenzthiazuron (MTT) assay.
The data was analyzed with SPSS11.5 statistical software.
Results After 5-Aza-CdR was added into HeLa cells with different dose for 5 days, our results were spread out below:
1、After 5,10and20μmol/L 5-Aza-CdR treatment, the level of HIC-1 mRNA and P53 mRNA expression were increased, and they were significantly correlated with the concentration of 5-Aza-CdR. The expression of HPV18E6 mRNA was remaining unchanged in all of the cases.
2、Hela cells treated with 5-Aza-CdR displayed a slow growth rate in comparison with that of the control cells, The cell vitalities of treated group were 91.22%±2.7%、65.08%±4.06%、49.68%±1.49%、38.80 %±3.00% individually compared with the control group and the inhibiting effect of 5-Aza-CdR was dose-dependent.
3、5-Aza-CdR could reduce the density of Hela cells ,and the convergence of team was about 70 percent of the control group, the morphologic changes in cells was no significant.
Conclusion
1、Aberrant methylation of the HIC-1gene seems to be an important mechanism underlying its down-regulation of expression.
2、Aberrant methylation of the HIC-1 seems to be one of the role in the proliferation of Hela cells .
3、5-Aza-CdR can inhibit the proliferation of Hela cells with dose-dependent relationgship of its effect.
引文
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