MASPIN真核表达载体的构建及对胃癌细胞SGC7901凋亡与增殖的影响
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摘要
目的:
1.构建MASPIN基因真核表达载体,转染至胃癌细胞株SGC7901,检测其对SGC7901细胞MASPIN表达的影响。
2.检测转染MASPIN对胃癌细胞SGC7901细胞增殖、细胞周期和凋亡的影响。
3.检测凋亡相关基因Bax/Bcl-2基因表达变化,探讨过表达MASPIN诱导胃癌细胞凋亡的机制。
方法:
1.RT-PCR扩增MASPIN基因全长,载体PCR2.1用HindⅢ/XbaI双酶切,二者用T4连接酶构建MASPIN/PCR2.1表达载体,转染胃癌细胞株SGC7901。
2.琼脂糖凝胶电泳检测凋亡特征性DNA梯形带.
3.采用AnnexinV-FIFC/PI方法检测转染前后﹑使用凋亡诱导剂TRATL(50ng/ml)前后凋亡率变化。
4.RT-PCR和Western bolt检测MASPIN基因、Bax/Bcl-2基因表达变化。
5.采用四甲基偶氮唑盐(MTT)法和流式细胞术(FCM),检测过表达MASPIN对SGC7901细胞增殖及细胞周期的影响。
结果:
1.酶切证实成功构建真核表达质粒MASPIN/PCR2.1;转染重组质粒组MASPIN mRNA(33.62±1.2)和蛋白水平(23.36±1.6)的表达明显高于未处理组(13.72±2.0、12.01±1.3)和空质粒组(15.01±1.5、12.32±1.5, P<0.05)。
2.凝胶电泳观察到特征性凋亡梯带。
3.各组细胞均有凋亡率的增加且呈时间依赖性:转染重组质粒/TRAIL作用组最高,12h、24h、48h分别达到8.0%、16.3%、25.8%,转染重组质粒组为3.0%、8.2%、14.4%,TRAIL作用组为4.1%、9.8%、15.9%(P<0.05)。
4.48h转染重组质粒?TRAIL组Bax mRNA和蛋白表达(55.32±1.4、75.38±1.3)高于转染重组质粒组(34.32±1.2、40.67±1.8)和TRAIL作用组(43.25±1.8、36.18±1.3,P<0.05);三组Bcl-2 mRNA表达为28.33±2.5、34.31±1.2、32.84±2.1(P<0.05),蛋白表达为17.43±1.5、45.11±2.1、42.84±1.5(P<0.05)。
5.流式细胞仪检测显示和与转染空载体组相比,过表达MASPIN的SGC7901细胞G0/G1期百分比增高(70.3%vs55.6%),S期细胞减少(19.8% vs 34.9%,P<0.05)。
6.MTT实验显示,转染MASPIN/PCR2.1组细胞增殖随时间延长而减慢,第12h、24h、48h、72h的细胞增殖率分别为0.98%、6.93%、18.03%和33.5%,明显低于转染空载体组0.04%、3.01%、4.21%、4.41%(P<0.05)。
结论:
1.成功构建MASPIN/PCR2.1表达载体。转染胃癌细胞株SGC7901后上调了MASIPIN基因在mRNA和蛋白水平表达。
4.过表达MASPIN能诱导SGC7901细胞发生凋亡并显著增加胃癌细胞对凋亡刺激信号的敏感性,时间越长效应越显著。其机制可能通过上调Bax,下调Bcl-2基因的表达诱导胃癌细胞凋亡。
5.过表达MASPIN能诱导SGC7901细胞发生增殖抑制和G0/G1期阻滞。
Objective:
1. To construct the recombinant eukaryotic vector expressing MASPIN cDNA and detect its effect on MASPIN expression of SGC7901.
2. To detect the influence of cell proliferation, cell cycle and apoptosis of SGC7901 before and after transfection.
3. To detect the expression changes of Bax/Bcl-2 gene and investgate the molecular mechanism of apoptosis of SGC7901induced by MASPIN overexpresing.
Methods:
1. The fragment of MASPIN gene was amplified by polymerase chain reaction(RT-PCR).A eukaryotic expression vector MASIPIN/PCR2.1 was constructed with PCR2.1 and transfected into the SGC7901.
2. The characteristic DNA ladders of apoptosis were determined by agarose gel electrophoresis (AGE).
3.TNF-related apoptosis inducing ligand (TRAIL) was used to induce apoptosis during the different time (50ng/ml). Cell apoptosis rate was measured by FCM.
4. RT-PCR and Western blot methods were used to detect the expression changes of MASPIN、Bax/Bcl-2 gene.
5. The effects of MASPIN overexpressing on the cell proliferation, cell cycle of SGC7901 were detected by MTT and FCM.
Results:
1. Recombinant plasmid MASIPIN/PCR2.1 was successfully constructed and transfected into SGC7901 cells. The mRNA and protein level of MASPIN were significantly higher in the MASPIN/PCR2.1 group (33.62±1.2, 23.36±1.6)than in the PCR2.1 group(15.01±1.5、12.32±1.5)and the untreated group(13.72±2.0、12.01±1.3, P<0.05).
2. DNA ladders appeared by AGE. A time-dependent apoptosis inducing effect was demonstrated in each group: The apoptosis rate was the highest in the MASPIN/PCR2.1 group with TRAIL inducing and the rates at 12h、24h、48h were 8%、16.3%、25.8%; the MASPIN/PCR2.1 group’s were 3.0%、8.2%、14.4%; the TRAIL inducing group’s were 4.1%、9.8%、15.9%(P<0.05).
3.The Bax mRNA and protein expressing level at 48h of MASPIN/PCR2.1 vector group with TRAIL inducing(55.32±、75.38±1.3) were significantly higher than the PCR2.1 group (34.32±1.2、40.67±1.8)and the TRAIL inducing group(43.25±1.8、36.18±1.3,P<0.05). The Bcl-2 mRNA expression were 28.33±2.5、34.31±1.2、32.84±2.1(P<0.05)and the protein expression were 17.43±1.5、45.11±2.1、42.84±1.5(P<0.05).
4. The proliferation of SGC7901 cells was significantly lower(93.07%、81.97%、66.5%) in the MASPIN/PCR2.1 group than in the PCR2.1 group(95.98%、95.79%、95.59%,P < 0.05) at 24h、48h、72h. the G0/G1 phase proportion of the MASPIN/PCR2.1 group at 72h was significantly higher than in the PCR2.1 group(70.3%vs55.6%,P < 0.05).the S phase proportion was 19.8% and 34.9%(P<0.05)respectively .
Conclusions:
1. The expression vector MASPIN/PCR2.1 is constructed successfully and can be expressed in eukaryotic cells.
2. The MASPIN overexpression can induce apoptosis and significantly enhance the sensibility to the apoptotic inducer in gastric carcinoma. The onset of apoptosis in gastric carcinoma cell lines is related to the up-regulation of Bax and down-regulation of Bcl-2 in mRNA and protein level that induced by the MASPIN overexpression.
3. MASPIN overexpressing can suppress the proliferation of gastric carcinoma cells SGC7901.
1.构建MASPIN基因真核表达载体,转染至胃癌细胞株SGC7901,检测其对SGC7901细胞MASPIN表达的影响。
2.检测转染MASPIN对胃癌细胞SGC7901细胞增殖、细胞周期和凋亡的影响。
3.检测凋亡相关基因Bax/Bcl-2基因表达变化,探讨过表达MASPIN诱导胃癌细胞凋亡的机制。
方法:
1.RT-PCR扩增MASPIN基因全长,载体PCR2.1用HindⅢ/XbaI双酶切,二者用T4连接酶构建MASPIN/PCR2.1表达载体,转染胃癌细胞株SGC7901。
2.琼脂糖凝胶电泳检测凋亡特征性DNA梯形带.
3.采用AnnexinV-FIFC/PI方法检测转染前后﹑使用凋亡诱导剂TRATL(50ng/ml)前后凋亡率变化。
4.RT-PCR和Western bolt检测MASPIN基因、Bax/Bcl-2基因表达变化。
5.采用四甲基偶氮唑盐(MTT)法和流式细胞术(FCM),检测过表达MASPIN对SGC7901细胞增殖及细胞周期的影响。
结果:
1.酶切证实成功构建真核表达质粒MASPIN/PCR2.1;转染重组质粒组MASPIN mRNA(33.62±1.2)和蛋白水平(23.36±1.6)的表达明显高于未处理组(13.72±2.0、12.01±1.3)和空质粒组(15.01±1.5、12.32±1.5, P<0.05)。
2.凝胶电泳观察到特征性凋亡梯带。
3.各组细胞均有凋亡率的增加且呈时间依赖性:转染重组质粒/TRAIL作用组最高,12h、24h、48h分别达到8.0%、16.3%、25.8%,转染重组质粒组为3.0%、8.2%、14.4%,TRAIL作用组为4.1%、9.8%、15.9%(P<0.05)。
4.48h转染重组质粒?TRAIL组Bax mRNA和蛋白表达(55.32±1.4、75.38±1.3)高于转染重组质粒组(34.32±1.2、40.67±1.8)和TRAIL作用组(43.25±1.8、36.18±1.3,P<0.05);三组Bcl-2 mRNA表达为28.33±2.5、34.31±1.2、32.84±2.1(P<0.05),蛋白表达为17.43±1.5、45.11±2.1、42.84±1.5(P<0.05)。
5.流式细胞仪检测显示和与转染空载体组相比,过表达MASPIN的SGC7901细胞G0/G1期百分比增高(70.3%vs55.6%),S期细胞减少(19.8% vs 34.9%,P<0.05)。
6.MTT实验显示,转染MASPIN/PCR2.1组细胞增殖随时间延长而减慢,第12h、24h、48h、72h的细胞增殖率分别为0.98%、6.93%、18.03%和33.5%,明显低于转染空载体组0.04%、3.01%、4.21%、4.41%(P<0.05)。
结论:
1.成功构建MASPIN/PCR2.1表达载体。转染胃癌细胞株SGC7901后上调了MASIPIN基因在mRNA和蛋白水平表达。
4.过表达MASPIN能诱导SGC7901细胞发生凋亡并显著增加胃癌细胞对凋亡刺激信号的敏感性,时间越长效应越显著。其机制可能通过上调Bax,下调Bcl-2基因的表达诱导胃癌细胞凋亡。
5.过表达MASPIN能诱导SGC7901细胞发生增殖抑制和G0/G1期阻滞。
Objective:
1. To construct the recombinant eukaryotic vector expressing MASPIN cDNA and detect its effect on MASPIN expression of SGC7901.
2. To detect the influence of cell proliferation, cell cycle and apoptosis of SGC7901 before and after transfection.
3. To detect the expression changes of Bax/Bcl-2 gene and investgate the molecular mechanism of apoptosis of SGC7901induced by MASPIN overexpresing.
Methods:
1. The fragment of MASPIN gene was amplified by polymerase chain reaction(RT-PCR).A eukaryotic expression vector MASIPIN/PCR2.1 was constructed with PCR2.1 and transfected into the SGC7901.
2. The characteristic DNA ladders of apoptosis were determined by agarose gel electrophoresis (AGE).
3.TNF-related apoptosis inducing ligand (TRAIL) was used to induce apoptosis during the different time (50ng/ml). Cell apoptosis rate was measured by FCM.
4. RT-PCR and Western blot methods were used to detect the expression changes of MASPIN、Bax/Bcl-2 gene.
5. The effects of MASPIN overexpressing on the cell proliferation, cell cycle of SGC7901 were detected by MTT and FCM.
Results:
1. Recombinant plasmid MASIPIN/PCR2.1 was successfully constructed and transfected into SGC7901 cells. The mRNA and protein level of MASPIN were significantly higher in the MASPIN/PCR2.1 group (33.62±1.2, 23.36±1.6)than in the PCR2.1 group(15.01±1.5、12.32±1.5)and the untreated group(13.72±2.0、12.01±1.3, P<0.05).
2. DNA ladders appeared by AGE. A time-dependent apoptosis inducing effect was demonstrated in each group: The apoptosis rate was the highest in the MASPIN/PCR2.1 group with TRAIL inducing and the rates at 12h、24h、48h were 8%、16.3%、25.8%; the MASPIN/PCR2.1 group’s were 3.0%、8.2%、14.4%; the TRAIL inducing group’s were 4.1%、9.8%、15.9%(P<0.05).
3.The Bax mRNA and protein expressing level at 48h of MASPIN/PCR2.1 vector group with TRAIL inducing(55.32±、75.38±1.3) were significantly higher than the PCR2.1 group (34.32±1.2、40.67±1.8)and the TRAIL inducing group(43.25±1.8、36.18±1.3,P<0.05). The Bcl-2 mRNA expression were 28.33±2.5、34.31±1.2、32.84±2.1(P<0.05)and the protein expression were 17.43±1.5、45.11±2.1、42.84±1.5(P<0.05).
4. The proliferation of SGC7901 cells was significantly lower(93.07%、81.97%、66.5%) in the MASPIN/PCR2.1 group than in the PCR2.1 group(95.98%、95.79%、95.59%,P < 0.05) at 24h、48h、72h. the G0/G1 phase proportion of the MASPIN/PCR2.1 group at 72h was significantly higher than in the PCR2.1 group(70.3%vs55.6%,P < 0.05).the S phase proportion was 19.8% and 34.9%(P<0.05)respectively .
Conclusions:
1. The expression vector MASPIN/PCR2.1 is constructed successfully and can be expressed in eukaryotic cells.
2. The MASPIN overexpression can induce apoptosis and significantly enhance the sensibility to the apoptotic inducer in gastric carcinoma. The onset of apoptosis in gastric carcinoma cell lines is related to the up-regulation of Bax and down-regulation of Bcl-2 in mRNA and protein level that induced by the MASPIN overexpression.
3. MASPIN overexpressing can suppress the proliferation of gastric carcinoma cells SGC7901.
引文
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【 1】 Z ou z,Anisowicz A,Hendrix MJ,et a1.Maspin,a serpin wlth tumor suppressing activity in human mammary epithelial cells[J].Science,1994,263(5146):526-529.
【 2】 Zou Z, Cao C, Na ch AK, et a1. p53 regulates the expression on of the tumor suppressor gene Maspin[J].J Biol Chen,2000;275(9):6051-6054
【 3】 Sheng S, Carey J , Scflor EA, et a1. Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells[J]. Proe Natl Acad Sci USA, 1996, 93(21) : 11669—11674.
【 4】 B lacque OE , Worrall DM .Evidence for a direct interaction between the tumor suppressor serpin, maspin ,a nd types I and Ⅲ collagen[J]. J Pathol, 277(13):10783—10788.
【 5】 Mohsin SK, Z hang M ,C lark GM , et a1. Maspin expression in invasive breast cancer :a ssociation with other prognostic factom[J]. J Pathol, 2003,199(4):432—435.
【 6】 shi HY, Zhang W , Liang R, et a1. Modeling human breast cancer metastasis in mice ,m aspin as a paradigm[J]. Histol Histopathol ,2 003 ;l 8(1) :2 01-206.
【 7】 Sheng S, Carey J, Seftor EA, et a1. Maspin acts At the ceil Inembranet inhibitinvasion and motility of mammary and prostatic cancer cells[J]. Proc Nail Acad Sci,1996;93:1669—11674
【8 】 Khaikhali Ellis Z. Hendrix MJ. Nitric 0xide regulation of maspin expression in normal mammmry epithelial and breast cancel cell [J]. Am JPathol;2003 ;1 62(5) :1 411—14179
【9 】S heng S, P emlrton P ,S ger R .P roduction, pufcation and characterization of recombinant maspin proteins[J].J Bi0l chen,1994;269:0988—30993
【 10】 Wan g MC ,Yan g YM,Liu XH,et a1.Maspin expression and its clinic pathological significance in tumorigenesis and progression of gastric cancer[J]. World J Gastroentero1 .2 O04 .1 O(5) :6 34—637.
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【 1】 Z ou z,Anisowicz A,Hendrix MJ,et a1.Maspin,a serpin wlth tumor suppressing activity in human mammary epithelial cells[J].Science,1994,263(5146):526-529.
【 2】 Zou Z, Cao C, Na ch AK, et a1. p53 regulates the expression on of the tumor suppressor gene Maspin[J].J Biol Chen,2000;275(9):6051-6054
【 3】 Sheng S, Carey J , Scflor EA, et a1. Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells[J]. Proe Natl Acad Sci USA, 1996, 93(21) : 11669—11674.
【 4】 B lacque OE , Worrall DM .Evidence for a direct interaction between the tumor suppressor serpin, maspin ,a nd types I and Ⅲ collagen[J]. J Pathol, 277(13):10783—10788.
【 5】 Mohsin SK, Z hang M ,C lark GM , et a1. Maspin expression in invasive breast cancer :a ssociation with other prognostic factom[J]. J Pathol, 2003,199(4):432—435.
【 6】 shi HY, Zhang W , Liang R, et a1. Modeling human breast cancer metastasis in mice ,m aspin as a paradigm[J]. Histol Histopathol ,2 003 ;l 8(1) :2 01-206.
【 7】 Sheng S, Carey J, Seftor EA, et a1. Maspin acts At the ceil Inembranet inhibitinvasion and motility of mammary and prostatic cancer cells[J]. Proc Nail Acad Sci,1996;93:1669—11674
【8 】 Khaikhali Ellis Z. Hendrix MJ. Nitric 0xide regulation of maspin expression in normal mammmry epithelial and breast cancel cell [J]. Am JPathol;2003 ;1 62(5) :1 411—14179
【9 】S heng S, P emlrton P ,S ger R .P roduction, pufcation and characterization of recombinant maspin proteins[J].J Bi0l chen,1994;269:0988—30993
【 10】 Wan g MC ,Yan g YM,Liu XH,et a1.Maspin expression and its clinic pathological significance in tumorigenesis and progression of gastric cancer[J]. World J Gastroentero1 .2 O04 .1 O(5) :6 34—637.
【32】Son HJ, Sohn TS, Song SY, Lee JH, Rhee JC. et a1.Maspin expression in human gastric adenocarcinoma[J].Pathol Int. 2002 Aug;52(8):508-13.
【33】Terashima M, Maesawa C, Oyama K, et a1. Gene expression profiles in human gastric cancer: expression of maspin correlates with lymph node metastasis[J].Br J Cancer. 2005 Mar 28;92(6):1130-6.
【34 】S ong SY, Son HJ, Kim MH, et a1 . Prognostic significance of maspin expressionin human gastric adenocarcinoma. Hepatogastroenterology. [J]2007 Apr-May;54(75):973-6.
【35】Yu M, Zheng H, Tsuneyama K, Takahashi H, et a1.Paradoxical expression of maspin in gastric carcinomas: correlation with carcinogenesis and progression[J].Hum Pathol. 2007 Aug;38(8):1248-55. Epub 2007 May 8.
【36】Mori K, Suzuki T, Uozaki H, et a1.Detection of minimal gastric cancer cells in peritoneal washings by focused microarray analysis with multiple markers: clinical implications.Ann Surg Oncol[J]. 2007 May;14(5):1694-702. Epub 2007 Feb 9.