特异性沉默hTERT基因表达对卵巢癌细胞增殖、凋亡及p53、p21的表达的影响
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摘要
目的探索特异性沉默hTERT基因表达对卵巢癌细胞增殖凋亡及p53和p21表达的影响,为临床诊治提供参考依据。方法采用分子克隆技术构建表达hTERTsh RNA的载体,特异性沉默hTERT基因表达; Westen Blot法、MTT比色法、流式细胞仪检测抑制hTERT基因表达对卵巢癌细胞p53表达及其增殖和凋亡的影响。结果 p SIREN-hTERTsh RNA转染有效阻抑A2780细胞中hTERT蛋白表达,抑制率为77%;随着A2780细胞中hTERT蛋白表达降低,p53和p21表达均明显增加;与空白对照及转染p SIREN-Con对照质粒相比,p SIREN-hTERT转染的A2780细胞生长速度明显降低; p SIREN-hTERT转染48 h A2780细胞凋亡率为(26. 76±7. 42)%,明显高于对照质粒p SIREN-Con转染组(3. 73±0. 78)%及空白对照组(1. 33±0. 15)%。结论 hTERTsh RNA重组质粒可特异性抑制卵巢癌细胞hTERT的表达,诱导细胞增殖抑制和凋亡,且可能与上调p53和p21的表达有关。
Objective To explore the effect of special inhibition of hTERT gene expression on proliferation and apoptosis of ovarian cancer cells and expressions of p53 and p21,provide a reference basis for clinical diagnosis and treatment of ovarian cancer. Methods The vector expressing hTERT sh RNA was constructed by molecular cloning technique,hTERT gene expression was inhibited specifically. Westen Blot,MTT assay,and flow cytometry were used to detect the effect of special inhibition of hTERT gene expression on proliferation and apoptosis of ovarian cancer cells and expressions of p53 and p21. Results p SIREN-hTERT sh RNA transfection effectively inhibited hTERT protein expression in A2780 cells,the inhibition rate was 77%. With the decrease of hTERT protein expression in A2780 cells,the expression levels of p53 and p21 increased significantly. Compared with blank control group and p SIREN-Con transfection group,the growth rate of A2780 cells transfected by p SIREN-hTERT decreased significantly. At 48 hours after p SIREN-hTERT transfection,the apoptotic rate of A2780 cells was( 26. 76± 7. 42) %,which was significantly higher than those in p SIREN-Con transfection group and blank control group[( 3. 73±0. 78) % and( 1. 33±0. 15) %]. Conclusion hTERT sh RNA recombinant plasmid can specially inhibit hTERT expression in ovarian cancer cells,induce apoptosis,which may be correlated with upregulation of p53 and p21 expressions.
Objective To explore the effect of special inhibition of hTERT gene expression on proliferation and apoptosis of ovarian cancer cells and expressions of p53 and p21,provide a reference basis for clinical diagnosis and treatment of ovarian cancer. Methods The vector expressing hTERT sh RNA was constructed by molecular cloning technique,hTERT gene expression was inhibited specifically. Westen Blot,MTT assay,and flow cytometry were used to detect the effect of special inhibition of hTERT gene expression on proliferation and apoptosis of ovarian cancer cells and expressions of p53 and p21. Results p SIREN-hTERT sh RNA transfection effectively inhibited hTERT protein expression in A2780 cells,the inhibition rate was 77%. With the decrease of hTERT protein expression in A2780 cells,the expression levels of p53 and p21 increased significantly. Compared with blank control group and p SIREN-Con transfection group,the growth rate of A2780 cells transfected by p SIREN-hTERT decreased significantly. At 48 hours after p SIREN-hTERT transfection,the apoptotic rate of A2780 cells was( 26. 76± 7. 42) %,which was significantly higher than those in p SIREN-Con transfection group and blank control group[( 3. 73±0. 78) % and( 1. 33±0. 15) %]. Conclusion hTERT sh RNA recombinant plasmid can specially inhibit hTERT expression in ovarian cancer cells,induce apoptosis,which may be correlated with upregulation of p53 and p21 expressions.
引文
[1]张汉英,黎丹戎,李力,等.端粒酶与卵巢上皮性癌化疗的相关性研究[J].中国癌症防治杂志,2013,27(3):221-227
[2]林燕真,程通,张雅丽,等.肿瘤细胞特异启动子hTERT介导的靶向mdr1基因的RNAi用于逆转卵巢癌细胞MDR的研究[J].中国医学创新,2015,8(34):8-12
[3]常青,胡耀杰,陈春悠,等.人端粒酶逆转录酶与p53蛋白在老年人甲状腺癌组织中的表达及临床意义[J].中华老年医学杂志,2016,35(2):171-173.
[4]张桂香,李春辉,齐洁敏,等. PTEN p53及h TERT在Ⅰ型子宫内膜癌中的表达[J].河北医学,2013,19(12):1787-1790.
[5]张汉英,黎丹戎,李力,等.端粒酶与卵巢上皮性癌化疗的相关性研究[J].中国癌症防治杂志,2013,27(3):221-226,227.
[6] Janowski BA,Kaihatsu K,Huffman KE,et al. Inhibiting transcription of chromosomal DNA with antigene peptide nucleic acids[J]. Nat Chem Biol,2005,1(4):210-215.
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[8]智慧,李通化.应用新的基于知识编码方法及双层SVM识别人类PolⅡ启动子[J].哈尔滨医科大学学报,2012,46(1):23-26.
[9]王善凤,刘尧.腺病毒介导Ad-HSV-TK/GCV自杀基因对人卵巢癌细胞体内杀伤效应研究[J].中国现代医学杂志,2013,23(36):21-24.
[10]李雨聪,王冬. h TERT启动子调控的PUMA腺病毒转染卵巢癌COC1细胞的研究[J].重庆医学, 2014, 43(26):3452-3454.
[11]朱宝菊,乔玉环,李印,等.含hTERT基因核心启动子和canstatin基因重组腺病毒载体的构建及其对卵巢上皮性癌的抑制作用[J].中华妇产科杂志,2009,44(3):214-218
[12]王会敏,何柯新,徐建华,等.利用RNAi阻抑hTERT和Bi-1双基因表达的RNAi作用效果的研究[J].重庆医学,2015,44(8):1012-1016.
[13]董一楠,孔凡铭,张新伟,等.癌基因i ASPP-SV参与乳腺癌的形成[J].中国癌症杂志,2016,26(10):831-839.
[14]张妍,张艳君,张业,等.结肠癌细胞HCT116中p53的R213突变对靶基因p21表达的影响[J].基础医学与临床,2017,37(5):663-667.
[15]仲智勇,时保军,周辉,等.重组人p53腺病毒注射液对肾母细胞瘤细胞增殖、凋亡及自噬的影响[J].中国药房,2017,28(7):889-892.
[2]林燕真,程通,张雅丽,等.肿瘤细胞特异启动子hTERT介导的靶向mdr1基因的RNAi用于逆转卵巢癌细胞MDR的研究[J].中国医学创新,2015,8(34):8-12
[3]常青,胡耀杰,陈春悠,等.人端粒酶逆转录酶与p53蛋白在老年人甲状腺癌组织中的表达及临床意义[J].中华老年医学杂志,2016,35(2):171-173.
[4]张桂香,李春辉,齐洁敏,等. PTEN p53及h TERT在Ⅰ型子宫内膜癌中的表达[J].河北医学,2013,19(12):1787-1790.
[5]张汉英,黎丹戎,李力,等.端粒酶与卵巢上皮性癌化疗的相关性研究[J].中国癌症防治杂志,2013,27(3):221-226,227.
[6] Janowski BA,Kaihatsu K,Huffman KE,et al. Inhibiting transcription of chromosomal DNA with antigene peptide nucleic acids[J]. Nat Chem Biol,2005,1(4):210-215.
[7] Janowski BA,Huffman KE,Schwartz JC,et al. Inhibiting gene expression attranscription start sites in chromosomal DNA with antigene RNAs[J]. Nat Chem Biol,2005,1(4):216-222.
[8]智慧,李通化.应用新的基于知识编码方法及双层SVM识别人类PolⅡ启动子[J].哈尔滨医科大学学报,2012,46(1):23-26.
[9]王善凤,刘尧.腺病毒介导Ad-HSV-TK/GCV自杀基因对人卵巢癌细胞体内杀伤效应研究[J].中国现代医学杂志,2013,23(36):21-24.
[10]李雨聪,王冬. h TERT启动子调控的PUMA腺病毒转染卵巢癌COC1细胞的研究[J].重庆医学, 2014, 43(26):3452-3454.
[11]朱宝菊,乔玉环,李印,等.含hTERT基因核心启动子和canstatin基因重组腺病毒载体的构建及其对卵巢上皮性癌的抑制作用[J].中华妇产科杂志,2009,44(3):214-218
[12]王会敏,何柯新,徐建华,等.利用RNAi阻抑hTERT和Bi-1双基因表达的RNAi作用效果的研究[J].重庆医学,2015,44(8):1012-1016.
[13]董一楠,孔凡铭,张新伟,等.癌基因i ASPP-SV参与乳腺癌的形成[J].中国癌症杂志,2016,26(10):831-839.
[14]张妍,张艳君,张业,等.结肠癌细胞HCT116中p53的R213突变对靶基因p21表达的影响[J].基础医学与临床,2017,37(5):663-667.
[15]仲智勇,时保军,周辉,等.重组人p53腺病毒注射液对肾母细胞瘤细胞增殖、凋亡及自噬的影响[J].中国药房,2017,28(7):889-892.