宫颈癌中DNMT1的功能与作用机制研究
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摘要
目的：
研究宫颈癌中DNMT1基因与肿瘤恶性表型以及肿瘤相关基因异常甲基化、基因表达之间的关系,进一步探讨DNMT1在宫颈癌发生发展中的功能,以及肿瘤相关基因表达调控的机制。
方法：
脂质体法将DNMT1-siRNA转染进入Hela、Siha细胞中,应用RT-PCR、荧光定量PCR, Western blot检测DNMT1的mRNA和蛋白质的表达变化,评价基因干扰效果。MTT、Annexin V-FITC/PI双染法流式细胞术检测细胞的增殖、周期与凋亡变化。MeDIP-qPCR, qPCR分别检测沉默DNMT1的Hela细胞中7个肿瘤相关基因CCNA、CHFR、PAX1、SFRP4、TSLC1、PTEN、FHIT的去甲基化情况与mRNA重表达水平。
结果：
转染DNMT1-siRNA72h后,Hela、Siha细胞DNMT1基因的mRNA表达抑制率分别为52.54%、41.58%；Hela、Siha细胞中转染组DNMT1蛋白表达的抑制率分别为为50.31%、99.76%。MTT实验表明,转染组转染24、48、72、96h时Siha细胞存活率分别为90.45%、84.16%、71.09%、60.47%,Hela细胞存活率分别为91.47%、86.74%、78.92%、48.98%。转染48h后,Siha, Hela细胞的凋亡率达(19.4±2.90)%、(25.7±3.92)%；Siha细胞S期细胞比例由空白组(21.81±1.39)%降低到转染组的(17.54±1.84)%,Hela细胞相应的从(29.34±1.14)%降低到(17.67±1.29)%,均具有显著性差异(P<0.01)。在Hela细胞中(未经任何处理),各基因具有不同程度的甲基化,PAX1、SFRP4、TSLC1甲基化程度较高,FHIT、PTEN甲基化程度较低；各基因相对表达量亦有差异,PTEN、FHIT表达量较高,CCNA1、CHFR表达量较低。沉默DNMT1后,5个甲基化基因CCNA1、CHFR、PAX1、SFRP4、TSLC1启动子区域甲基化程度明显降低,mRNA表达量显著增加,而对PTEN、FHIT却没有影响。甲基化酶抑制剂5-aza-dC处理Hela:细胞后结果与沉默DNMT1相一致。
结论：
1.沉默DNMT1基因可逆转宫颈癌细胞Siha、Hela的恶性表型。
2. Hela细胞中,沉默DNMT1基因能促使7个基因中的5个(CCNA1、CHFR、PAX1、SFRP4、TSLC1)启动子区域甲基化程度明显降低,基因表达量显著增加,而对PTE、FHIT却没有影响。
3. CCNA1、CHFR, PAX1、SFRP4、TSLC1是通过启动子区域高甲基化实现低表达的,DNMT1是其甲基化的关键因素；而FHIT、PTEN基因通过低甲基化实现高表达的,与DNMT1没有显著关联。
4. DNMT1是治疗宫颈癌的一个有效靶点。
Objective:
To investigate the relationship between DNMT1 and abnormal methylation of tumor-related genes, malignant phenotype in cervical cancer, and further explore the function of DNMT1 in development of cervical carcinoma, as well as the specific regulatory mechanisms of tumor suppressor genes expression.
Methods:
DNMT1-siRNA was transfected into Hela, Siha cells with Lipofection method, RT-PCR, quantitative PCR(qPCR), Western blot were applied to detect DNMT1 mRNA and protein expression, evaluating gene interference effect. Cell proliferation and cell cycle, apoptosis were analyzed by MTT, Annexin V-FITC/PI double staining flow cytometry. MeDIP-qPCR, qPCR were used to measure de-methylation and mRNA re-expression of 7 tumor-related genes (CCNA, CHFR, PAX1, SFRP4, TSLC1, PTEN, FHIT) in Hela cells silenced DNMT1.
Results:
Relative to control, the inhibitory rates of DNMT1 mRNA levels in Hela, Siha cells were 52.54%,41.58% respectively after DNMT1-siRNA transfection 72h. The inhibitory rates of DNMT1 protein levels Hela, Siha cells were 50.31%,99.76% respectively.MTT results show that, after DNMT1-siRNA effect 24、48、72、96h, Siha cells survival rates decreased to 90.45%、84.16%、71.09%、60.47%respectively, and those of Hela cells 91.47%、86.74%、78.92%、48.98% respectively. After transfection 48h, Siha, Hela cells apoptosis rates were (19.4±2.90)%, (25.7±3.92)% respectively.The proportion of cells in S phase decreased from the control group (21.81±1.39)% to transfected group (17.54±1.84)% in Siha cells, and that of in Hela cells from the control group (29.34±1.14)% to transfected group (17.67± 1.29)%, there were significant differences (P<0.01)betweeii two groups. In Hela cells (without any treatment), all genes methylated with different degrees, enrichment of methylation of PAX1, SFRP4, TSLClwere higher than that of PTEN, FHIT. Concerning gene expression, PTEN mRNA level was higher than that of any others. After depletion DNMT1, percentage of promoter methylation decreased in five methylated genes (CCNA1, CHFR, PAX1, SFRP4, TSLC1), mRNA expression increased significantly, whereas there was no significant effect of DNMT1-siRNA on PTEN, FHIT gene. Transfection group and 5-aza-dC treatment showed the similar effect on gene demethylation and re-expression.
Conclusions:
1. Siha, Hela cells could reverse their malignant phenotype for DNMT1 gene depletion.
2. In Hela cells, DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CCNA1, CHFR, PAX1, SFRP4, TSLC1) out of seven tumor-related genes examined.
3. In Hela cells, CCNA1, CHFR, PAX1, SFRP4, TSLC1 genes presented low-expression by promoter region hypermethylation, and their methylation mainly depend on DNMT1. FHIT, PTEN genes might be present high-expression by promoter hypomethylation. There was probably no relationship between DNA methylation and DNMT1 in FHIT, PTEN gene.
4. DNMT1 is an effective target for treatment of cervical cancer.
研究宫颈癌中DNMT1基因与肿瘤恶性表型以及肿瘤相关基因异常甲基化、基因表达之间的关系,进一步探讨DNMT1在宫颈癌发生发展中的功能,以及肿瘤相关基因表达调控的机制。
方法：
脂质体法将DNMT1-siRNA转染进入Hela、Siha细胞中,应用RT-PCR、荧光定量PCR, Western blot检测DNMT1的mRNA和蛋白质的表达变化,评价基因干扰效果。MTT、Annexin V-FITC/PI双染法流式细胞术检测细胞的增殖、周期与凋亡变化。MeDIP-qPCR, qPCR分别检测沉默DNMT1的Hela细胞中7个肿瘤相关基因CCNA、CHFR、PAX1、SFRP4、TSLC1、PTEN、FHIT的去甲基化情况与mRNA重表达水平。
结果：
转染DNMT1-siRNA72h后,Hela、Siha细胞DNMT1基因的mRNA表达抑制率分别为52.54%、41.58%；Hela、Siha细胞中转染组DNMT1蛋白表达的抑制率分别为为50.31%、99.76%。MTT实验表明,转染组转染24、48、72、96h时Siha细胞存活率分别为90.45%、84.16%、71.09%、60.47%,Hela细胞存活率分别为91.47%、86.74%、78.92%、48.98%。转染48h后,Siha, Hela细胞的凋亡率达(19.4±2.90)%、(25.7±3.92)%；Siha细胞S期细胞比例由空白组(21.81±1.39)%降低到转染组的(17.54±1.84)%,Hela细胞相应的从(29.34±1.14)%降低到(17.67±1.29)%,均具有显著性差异(P<0.01)。在Hela细胞中(未经任何处理),各基因具有不同程度的甲基化,PAX1、SFRP4、TSLC1甲基化程度较高,FHIT、PTEN甲基化程度较低；各基因相对表达量亦有差异,PTEN、FHIT表达量较高,CCNA1、CHFR表达量较低。沉默DNMT1后,5个甲基化基因CCNA1、CHFR、PAX1、SFRP4、TSLC1启动子区域甲基化程度明显降低,mRNA表达量显著增加,而对PTEN、FHIT却没有影响。甲基化酶抑制剂5-aza-dC处理Hela:细胞后结果与沉默DNMT1相一致。
结论：
1.沉默DNMT1基因可逆转宫颈癌细胞Siha、Hela的恶性表型。
2. Hela细胞中,沉默DNMT1基因能促使7个基因中的5个(CCNA1、CHFR、PAX1、SFRP4、TSLC1)启动子区域甲基化程度明显降低,基因表达量显著增加,而对PTE、FHIT却没有影响。
3. CCNA1、CHFR, PAX1、SFRP4、TSLC1是通过启动子区域高甲基化实现低表达的,DNMT1是其甲基化的关键因素；而FHIT、PTEN基因通过低甲基化实现高表达的,与DNMT1没有显著关联。
4. DNMT1是治疗宫颈癌的一个有效靶点。
Objective:
To investigate the relationship between DNMT1 and abnormal methylation of tumor-related genes, malignant phenotype in cervical cancer, and further explore the function of DNMT1 in development of cervical carcinoma, as well as the specific regulatory mechanisms of tumor suppressor genes expression.
Methods:
DNMT1-siRNA was transfected into Hela, Siha cells with Lipofection method, RT-PCR, quantitative PCR(qPCR), Western blot were applied to detect DNMT1 mRNA and protein expression, evaluating gene interference effect. Cell proliferation and cell cycle, apoptosis were analyzed by MTT, Annexin V-FITC/PI double staining flow cytometry. MeDIP-qPCR, qPCR were used to measure de-methylation and mRNA re-expression of 7 tumor-related genes (CCNA, CHFR, PAX1, SFRP4, TSLC1, PTEN, FHIT) in Hela cells silenced DNMT1.
Results:
Relative to control, the inhibitory rates of DNMT1 mRNA levels in Hela, Siha cells were 52.54%,41.58% respectively after DNMT1-siRNA transfection 72h. The inhibitory rates of DNMT1 protein levels Hela, Siha cells were 50.31%,99.76% respectively.MTT results show that, after DNMT1-siRNA effect 24、48、72、96h, Siha cells survival rates decreased to 90.45%、84.16%、71.09%、60.47%respectively, and those of Hela cells 91.47%、86.74%、78.92%、48.98% respectively. After transfection 48h, Siha, Hela cells apoptosis rates were (19.4±2.90)%, (25.7±3.92)% respectively.The proportion of cells in S phase decreased from the control group (21.81±1.39)% to transfected group (17.54±1.84)% in Siha cells, and that of in Hela cells from the control group (29.34±1.14)% to transfected group (17.67± 1.29)%, there were significant differences (P<0.01)betweeii two groups. In Hela cells (without any treatment), all genes methylated with different degrees, enrichment of methylation of PAX1, SFRP4, TSLClwere higher than that of PTEN, FHIT. Concerning gene expression, PTEN mRNA level was higher than that of any others. After depletion DNMT1, percentage of promoter methylation decreased in five methylated genes (CCNA1, CHFR, PAX1, SFRP4, TSLC1), mRNA expression increased significantly, whereas there was no significant effect of DNMT1-siRNA on PTEN, FHIT gene. Transfection group and 5-aza-dC treatment showed the similar effect on gene demethylation and re-expression.
Conclusions:
1. Siha, Hela cells could reverse their malignant phenotype for DNMT1 gene depletion.
2. In Hela cells, DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CCNA1, CHFR, PAX1, SFRP4, TSLC1) out of seven tumor-related genes examined.
3. In Hela cells, CCNA1, CHFR, PAX1, SFRP4, TSLC1 genes presented low-expression by promoter region hypermethylation, and their methylation mainly depend on DNMT1. FHIT, PTEN genes might be present high-expression by promoter hypomethylation. There was probably no relationship between DNA methylation and DNMT1 in FHIT, PTEN gene.
4. DNMT1 is an effective target for treatment of cervical cancer.
引文
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