RNAi抑制COX-2活性对人胃腺癌细胞系SGC-7901生长抑制的体内外研究
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摘要
目的研究COX-2在胃腺癌组织中的表达,探讨COX-2与PCNA,PGE_2,VEGF,CD31,cyclin D1在胃腺癌发生发展中作用的相互关系。构建COX-2的siRNA,检测其在胃腺癌细胞株SGC-7901中对COX-2基因表达的抑制作用,以及敲低COX-2的表达后,观察PGE_2,PCNA,VEGF,CD31,cyclin D1的表达变化,以及细胞周期,增殖活性,凋亡,侵袭活性的变化,并进一步探讨COX-2在胃腺癌细胞系中的作用机制。应用COX-2 siRNA导入裸鼠胃腺癌皮下动物模型后,验证在体内COX-2 siRNA对SGC-7901胃腺癌的抑制作用。
方法本课题分三部分进行:
第一部分构造胃腺癌组织芯片,利用免疫组化检测COX-2,PCNA,PGE_2与VEGF的蛋白表达。
第二部分在体外应用RNA干涉技术以Oligofectamine介导siRNA靶向COX-2转染人胃腺癌细胞系SGC-7901后,应用realtime PCR检测目的基因的敲低情况,并用Western blot和免疫荧光技术检测了RNAi敲低COX-2的表达后,COX-2通路相关基因PCNA,PGE_2,Bcl-2,Cyclin D1与VEGF的表达变化。并用MTT、annexin V标记、流式细胞术及Matrigel 3D生长实验等方法分析了转染前后细胞增殖、凋亡、细胞周期分布和侵袭能力的变化。
第三部分中则通过建立裸鼠胃腺癌皮下动物模型,瘤内注射Oligofectamine和siRNA的混合物,动态观察肿瘤生长情况,检测RNA干涉技术敲低SGC-7901裸鼠胃腺癌COX-2的表达后PCNA,VEGF,CD31的表达变化;并应用TUNEL法检测细胞凋亡,对体外实验的结果进一步验证。
结果1.COX-2在胃腺癌组织中过度表达(68.9%),且随组织的恶性分化程度升高而表达增强(x~2=31.855,P＜0.001)。COX-2在胃腺癌组织中的表达与癌旁组织及正常组织相比较具有差异性(x~2=14.231,P=0.027)。PGE_2,PCNA,VEGF(80.0%,84.4%和73.3%)也在胃腺癌组织中呈现过表达的现象,同时,COX-2,PCNA,PGE_2与VEGF表达之间存在正相关性。
2.realtime PCR结果提示,转染COX-2 siRNA后可明显敲低COX-2 mRNA的表达。MTT结果显示转染COX-2 siRNA后细胞增殖率与转染nonsense siRNA和对照组相比明显受抑制(P＜0.05),通过Western blot和免疫荧光检测发现除COX-2的蛋白明显敲低外,COX-2通路相关基因PCNA,PGE_2,Bcl-2,CyclinD1与VEGF的表达水平均明显降低。应用Annexin V检测发现转染COX-2siRNA组细胞凋亡率明显升高,进一步应用流式细胞术检测细胞周期分布发现S期细胞比例降低,细胞阻滞于G_0/G_1期。侵袭试验结果显示COX-2 siRNA可明显抑制SGC-7901细胞的侵袭。
3.成功建立胃腺癌SGC-7901细胞系裸鼠皮下肿瘤模型后,每4天测量一次肿瘤体积,并在肿瘤局部多点注射siRNA和Oligofectamine混合物,发现和对照组相比,COX-2 siRNA治疗组肿瘤生长缓慢,从治疗后的第15天起,肿瘤体积出现明显差别,这种差别在观察末期(30天)最显著。应用免疫组化法检测肿瘤标本,发现COX-2的蛋白明显敲低的同时,PCNA,CD31,VEGF表达水平均明显降低。应用TUNEL法检测原位细胞凋亡发现COX-2 siRNA治疗组凋亡细胞明显增加。体内和体外实验的结果一致。
结论1.COX-2,PCNA,PGE_2,VEGF的表达上调与胃腺癌的分化的严重程度、发生与进展呈正相关。
2.应用以Oligofectamine介导的RNA干涉技术转染siRNA靶向COX-2可有效敲低COX-2在胃腺癌SGC-7901细胞内的表达,同时抑制PCNA,PGE_2,Bcl-2,Cyclin D1与VEGF的表达活性,从而抑制了细胞的增殖和侵袭,出现G_0/G_1期阻滞,并诱发细胞凋亡。
3.裸鼠皮下胃腺癌模型应用Oligofectamine介导的COX-2 siRNA治疗,同样可有效敲低COX-2的表达,抑制肿瘤的生长,诱导细胞凋亡,与体外试验结果一致。
AIM:The aim of this study was to investigate the overexpression and co-expression of COX-2 and its related signaling proteins in gastric adenocarcinoma patients,and to find their relationship to gastric carcinogenesis. Cyclooxygenases-2(COX-2)- short interfering RNA is constructed so as to suppress the expression of COX-2 in gastric carcinoma cell SGC-7901.We examined the effects of COX-2 siRNA on growth,apoptosis,cell cycle and invasion of gastric cancer,and the effect of the down-regulation of COX-2 on expression of Prostaglandin E_2(PGE_2)and cancer related genes.Subcutaneous SGC-7901 gastric carcinoma model was established in nude mice.The mechanism of RNAi applying for gene therapy in gastric carcinoma by inhibiting COX-2 in nude mice is investigated.
METHODS:The study was divided into 3 parts.
1.Immunohistochemical staining was applied to tissue microarray,we examined COX-2,PGE_2,PCNA,VEGF expressions in gastric adenocarcinoma tissue array.
2.siRNA targeting COX-2 was transfected into SGC-7901 cells mediated by Oligofectamine.COX-2 mRNA expression were detected by realtime PCR after transfection.The expression of COX-2 and other cancer related genes PCNA, PGE_2,Bcl-2,Cyclin D1 and VEGF was also studied by Western blotting and immunofiuorescence staining after transfection.The phenotypic change of SGC-7901 cells including proliferation,apoptosis and invasive ability after RNAi trasfection was studied by MTT assay,annexinⅤstaining,flow cytometry, Matrigel 3D growth experiment and Transwell.
3.Subcutaneous SGC-7901 gastric carcinoma model model was established in nude mice.Every 4 day the mixture of Oligofectamine and siRNA was injected into the tumors and the tumor volumes were measured.On 30~(nd)day after the first injection,tumors were resected.The expression of COX-2 and PCNA,CD31, VEGF were studied by immunohistochemistry.Apoptosis in tumors were detected by TUNEL method.
RESULTS:1.COX-2 was commonly expressed in gastric adenocarcinoma (68.9%).PGE_2,PCNA,and VEGF(80.0%,84.4%and 73.3%of malignant cells, respectively)overexpression was also detected.The COX-2 expression was elevated with the rising gastric adenocarcinoma stages(χ~2=31.855,P＜0.001).The expression of COX-2 in gastric adenocarcinoma was higher than that of the gastric mucosa surrounding carcinomas and normal stomach tissue(χ~2=14.231, P=0.027);The result of Spearman rank correlation analysis indicated that the relevance of COX-2,PGE_2,VEGF,PCNA expression was considered to be statistically significant.
2.SGC-7901 cells were transfected with siRNA targeting COX-2 mediated by Oligofectamine in vitro.The transfection efficiency was up to over 70%.COX-2 mRNA expression were obviously knocked down after transfection with siRNA. COX-2,PCNA,PGE_2,Bcl-2,Cyclin D1 and VEGF protein expression were also decreased.SGC-7901 cells transfected with siRNA targeting COX-2 showed lowering proliferation activity,decrease of SPF and arresting cell cycle in the G_0/G_1phase.The cell invasive ability was attenuated and cell apoptosis was induced.
3.As compared with the control group,the tumors in mice treated with siRNA targeting COX-2 grew slowly and the difference of tumor volumes became significant since the 15th day after the first time of siRNA therapy until the 30~(nd) day tumors were resected.Meanwhile,cell apoptosis was increased.The expressions of PCNA,CD31 and VEGF were downregulated as shown by immunohistochemistry.
CONCLUSION:
1.The COX-2,PGE_2,PCNA,VEGF expressions increased with gastric adenocarcinoma histodifferentiation grade rise,which are useful for predicting the degree of malignancy for gastric cancer cells.COX-2 overexpression,via inducing angiogenesis,promoting proliferation,inducting immunosuppression, inducing angiogenesis,may play an important role in gastric carcinogenesis.
2.Using RNAi technology,siRNA targeting COX-2 mediated by Oligofectamine efficiently knocks down the expression of COX-2 in human gastric carcinoma cell SGC-7901 cells,inhibits activation of PCNA,PGE_2,Bcl-2,Cyclin D1 and VEGF, results in decrease of cell proliferation activity and invasive ability,arrests cell cycle and induces apoptosis.
3.The established subcutaneous SGC-7901 gastric carcinoma models in nude mice are treated with siRNA targeting COX-2.the tumor growth is inhibited and cell apoptosis is induced.The findings of in vivo study is in accordance with those in vitro study.
方法本课题分三部分进行:
第一部分构造胃腺癌组织芯片,利用免疫组化检测COX-2,PCNA,PGE_2与VEGF的蛋白表达。
第二部分在体外应用RNA干涉技术以Oligofectamine介导siRNA靶向COX-2转染人胃腺癌细胞系SGC-7901后,应用realtime PCR检测目的基因的敲低情况,并用Western blot和免疫荧光技术检测了RNAi敲低COX-2的表达后,COX-2通路相关基因PCNA,PGE_2,Bcl-2,Cyclin D1与VEGF的表达变化。并用MTT、annexin V标记、流式细胞术及Matrigel 3D生长实验等方法分析了转染前后细胞增殖、凋亡、细胞周期分布和侵袭能力的变化。
第三部分中则通过建立裸鼠胃腺癌皮下动物模型,瘤内注射Oligofectamine和siRNA的混合物,动态观察肿瘤生长情况,检测RNA干涉技术敲低SGC-7901裸鼠胃腺癌COX-2的表达后PCNA,VEGF,CD31的表达变化;并应用TUNEL法检测细胞凋亡,对体外实验的结果进一步验证。
结果1.COX-2在胃腺癌组织中过度表达(68.9%),且随组织的恶性分化程度升高而表达增强(x~2=31.855,P＜0.001)。COX-2在胃腺癌组织中的表达与癌旁组织及正常组织相比较具有差异性(x~2=14.231,P=0.027)。PGE_2,PCNA,VEGF(80.0%,84.4%和73.3%)也在胃腺癌组织中呈现过表达的现象,同时,COX-2,PCNA,PGE_2与VEGF表达之间存在正相关性。
2.realtime PCR结果提示,转染COX-2 siRNA后可明显敲低COX-2 mRNA的表达。MTT结果显示转染COX-2 siRNA后细胞增殖率与转染nonsense siRNA和对照组相比明显受抑制(P＜0.05),通过Western blot和免疫荧光检测发现除COX-2的蛋白明显敲低外,COX-2通路相关基因PCNA,PGE_2,Bcl-2,CyclinD1与VEGF的表达水平均明显降低。应用Annexin V检测发现转染COX-2siRNA组细胞凋亡率明显升高,进一步应用流式细胞术检测细胞周期分布发现S期细胞比例降低,细胞阻滞于G_0/G_1期。侵袭试验结果显示COX-2 siRNA可明显抑制SGC-7901细胞的侵袭。
3.成功建立胃腺癌SGC-7901细胞系裸鼠皮下肿瘤模型后,每4天测量一次肿瘤体积,并在肿瘤局部多点注射siRNA和Oligofectamine混合物,发现和对照组相比,COX-2 siRNA治疗组肿瘤生长缓慢,从治疗后的第15天起,肿瘤体积出现明显差别,这种差别在观察末期(30天)最显著。应用免疫组化法检测肿瘤标本,发现COX-2的蛋白明显敲低的同时,PCNA,CD31,VEGF表达水平均明显降低。应用TUNEL法检测原位细胞凋亡发现COX-2 siRNA治疗组凋亡细胞明显增加。体内和体外实验的结果一致。
结论1.COX-2,PCNA,PGE_2,VEGF的表达上调与胃腺癌的分化的严重程度、发生与进展呈正相关。
2.应用以Oligofectamine介导的RNA干涉技术转染siRNA靶向COX-2可有效敲低COX-2在胃腺癌SGC-7901细胞内的表达,同时抑制PCNA,PGE_2,Bcl-2,Cyclin D1与VEGF的表达活性,从而抑制了细胞的增殖和侵袭,出现G_0/G_1期阻滞,并诱发细胞凋亡。
3.裸鼠皮下胃腺癌模型应用Oligofectamine介导的COX-2 siRNA治疗,同样可有效敲低COX-2的表达,抑制肿瘤的生长,诱导细胞凋亡,与体外试验结果一致。
AIM:The aim of this study was to investigate the overexpression and co-expression of COX-2 and its related signaling proteins in gastric adenocarcinoma patients,and to find their relationship to gastric carcinogenesis. Cyclooxygenases-2(COX-2)- short interfering RNA is constructed so as to suppress the expression of COX-2 in gastric carcinoma cell SGC-7901.We examined the effects of COX-2 siRNA on growth,apoptosis,cell cycle and invasion of gastric cancer,and the effect of the down-regulation of COX-2 on expression of Prostaglandin E_2(PGE_2)and cancer related genes.Subcutaneous SGC-7901 gastric carcinoma model was established in nude mice.The mechanism of RNAi applying for gene therapy in gastric carcinoma by inhibiting COX-2 in nude mice is investigated.
METHODS:The study was divided into 3 parts.
1.Immunohistochemical staining was applied to tissue microarray,we examined COX-2,PGE_2,PCNA,VEGF expressions in gastric adenocarcinoma tissue array.
2.siRNA targeting COX-2 was transfected into SGC-7901 cells mediated by Oligofectamine.COX-2 mRNA expression were detected by realtime PCR after transfection.The expression of COX-2 and other cancer related genes PCNA, PGE_2,Bcl-2,Cyclin D1 and VEGF was also studied by Western blotting and immunofiuorescence staining after transfection.The phenotypic change of SGC-7901 cells including proliferation,apoptosis and invasive ability after RNAi trasfection was studied by MTT assay,annexinⅤstaining,flow cytometry, Matrigel 3D growth experiment and Transwell.
3.Subcutaneous SGC-7901 gastric carcinoma model model was established in nude mice.Every 4 day the mixture of Oligofectamine and siRNA was injected into the tumors and the tumor volumes were measured.On 30~(nd)day after the first injection,tumors were resected.The expression of COX-2 and PCNA,CD31, VEGF were studied by immunohistochemistry.Apoptosis in tumors were detected by TUNEL method.
RESULTS:1.COX-2 was commonly expressed in gastric adenocarcinoma (68.9%).PGE_2,PCNA,and VEGF(80.0%,84.4%and 73.3%of malignant cells, respectively)overexpression was also detected.The COX-2 expression was elevated with the rising gastric adenocarcinoma stages(χ~2=31.855,P＜0.001).The expression of COX-2 in gastric adenocarcinoma was higher than that of the gastric mucosa surrounding carcinomas and normal stomach tissue(χ~2=14.231, P=0.027);The result of Spearman rank correlation analysis indicated that the relevance of COX-2,PGE_2,VEGF,PCNA expression was considered to be statistically significant.
2.SGC-7901 cells were transfected with siRNA targeting COX-2 mediated by Oligofectamine in vitro.The transfection efficiency was up to over 70%.COX-2 mRNA expression were obviously knocked down after transfection with siRNA. COX-2,PCNA,PGE_2,Bcl-2,Cyclin D1 and VEGF protein expression were also decreased.SGC-7901 cells transfected with siRNA targeting COX-2 showed lowering proliferation activity,decrease of SPF and arresting cell cycle in the G_0/G_1phase.The cell invasive ability was attenuated and cell apoptosis was induced.
3.As compared with the control group,the tumors in mice treated with siRNA targeting COX-2 grew slowly and the difference of tumor volumes became significant since the 15th day after the first time of siRNA therapy until the 30~(nd) day tumors were resected.Meanwhile,cell apoptosis was increased.The expressions of PCNA,CD31 and VEGF were downregulated as shown by immunohistochemistry.
CONCLUSION:
1.The COX-2,PGE_2,PCNA,VEGF expressions increased with gastric adenocarcinoma histodifferentiation grade rise,which are useful for predicting the degree of malignancy for gastric cancer cells.COX-2 overexpression,via inducing angiogenesis,promoting proliferation,inducting immunosuppression, inducing angiogenesis,may play an important role in gastric carcinogenesis.
2.Using RNAi technology,siRNA targeting COX-2 mediated by Oligofectamine efficiently knocks down the expression of COX-2 in human gastric carcinoma cell SGC-7901 cells,inhibits activation of PCNA,PGE_2,Bcl-2,Cyclin D1 and VEGF, results in decrease of cell proliferation activity and invasive ability,arrests cell cycle and induces apoptosis.
3.The established subcutaneous SGC-7901 gastric carcinoma models in nude mice are treated with siRNA targeting COX-2.the tumor growth is inhibited and cell apoptosis is induced.The findings of in vivo study is in accordance with those in vitro study.
引文
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