摘要
目的探讨siRNA技术沉默DNA甲基转移酶1(DNMT1)基因表达对前列腺癌细胞(LNCap)增殖、凋亡的影响,初步分析其对谷胱甘肽S转移酶P1(GSTP1)、矮小同源盒基因2(SHOX2)、凋亡相关蛋白激酶(DAPK)甲基化状态的影响。方法体外培养人前列腺癌细胞LNCap、正常前列腺上皮细胞RWPE-1,采用实时荧光定量PCR(qRTPCR)及蛋白印迹(WB)检测细胞中DNMT1 mRNA及蛋白表达水平。实验分为3组:空白对照组(未经任何处理LNCap细胞); si-NC组(转染DNMT1阴性对照质粒的LNCap细胞); si-SH2B1组(转染si-DNMT1的LNCap细胞)。采用CCK-8法检测三组细胞增殖抑制率;流式细胞仪检测细胞凋亡水平。采用甲基化特异性PCR (MSP)检测GSTP1、SHOX2、DAPK基因甲基化状态; qRT-PCR与WB法分别检测各组LNCap细胞DNMT1、GSTP1、SHOX2、DAPK mRNA及蛋白表达情况。结果与正常组细胞相比,前列腺癌组细胞中DNMT1 mRNA及蛋白表达水平均显著升高(P <0. 05);si-DNMT1组DNMT1 mRNA及蛋白表达水平均显著低于空白对照组、si-NC组(P <0. 05);与空白对照组、si-NC组相比,si-DNMT1组LNCap细胞增殖抑制率、细胞凋亡率、GSTP1、SHOX2、DAPK mRNA及蛋白表达水平均显著升高(P<0. 05);与空白对照组、si-NC组相比,si-DNMT1组GSTP1、DAPK、SHOX2基因甲基化水平均显著降低(P <0. 05)。结论沉默DNMT1基因后可明显抑制LNCap细胞增殖并促使其凋亡,其可能通过逆转GSTP1、DAPK、SHOX2甲基化状态并上调其表达而发挥作用。
Objective To investigate the effects of siRNA silencing of DNA methyltransferase 1(DNMT1)gene expression on proliferation and apoptosis of prostate cancer cells(LNCap),and to preliminarily analyze its effects on methylation status of glutathione S-transferase P1(GSTP1),short stature homobox2(SHOX2)and death-associated protein kinase(DAPK).Methods Human prostate cancer cell line LNCap and normal prostatic epithelial cell line RWPE-1 were cultured in vitro.The expression of DNMT1 was detected by real-time fluorescence quantitative PCR(qRT-PCR)and Western Blot(WB).The experiment was divided into three groups:blank control group(untreated LNCap cells);si-NC group(LNCap cells transfected with DNMT1 negative control plasmid);si-SH2B1 group(LNCap cells transfected with si-DNMT1).CCK-8 method was used to detect the inhibition rate of cell proliferation,and flow cytometry was used to detect the level of cell apoptosis.Methylation-specific PCR(MSP)was used to detect the methylation status of GSTP1,SHOX2 and DAPK genes.The expression of DNMT1,GSTP1,SHOX2 and DAPK in LNCap cells was detected by qRT-PCR and WB methods,respectively.Results Compared with normal cells,the expression of DNMT1 in prostate cancer cells increased significantly(P<0.05).The levels of DNMT1 gene and protein in si-DNMT1 group were significantly lower than those in blank control group and si-NC group(P<0.05).Compared with the blank control group and the si-NC group,the proliferation inhibition rate,apoptotic rate,GSTP1,SHOX2,DAPK gene and protein expression levels of LNCap cells in the si-DNMT1 group were significantly increased(P<0.05).Compared with the blank control group and the si-NC group,the methylation levels of GSTP1,DAPK and SHOX2 genes in the si-DNMT1 group were significantly lower(P<0.05).Conclusion DNMT1 gene silencing can significantly inhibit the proliferation of LNCap cells and promote their apoptosis,which may play a role by reversing the methylation status of GSTP1,DAPK and SHOX2 and up-regulating their expression.
作者
邢红宇
朱明月
郑俊
吴翔
郑才玲
苏民
XING Hong-yu;ZHU Ming-yue;ZHENG Jun(Department of Clinical Laboratory,Hainan Provincial Hospital of Traditional Chinese Medicine,Haikou Hainan 570203,China;Hainan Medical College (Key Laboratory of Cancerogenesis and Intervention,Hainan Province,Haikou Hainan 570203,China;Department of Clinical Laboratory,Haikou Fourth People's Hospital,Haikou Hainan 570203,China)
出处
《临床和实验医学杂志》
2019年第10期1029-1034,共6页
Journal of Clinical and Experimental Medicine
基金
海南省自然科学基金支持项目(编号:20158301)
