高尔基体α-甘露糖苷酶Ⅱ（GMⅡ）在胃癌细胞生长中的作用及机制研究
摘要
目的：成功构建GMⅡ基因过表达载体与RNAi表达载体并分别稳定转染胃癌细胞株；通过体内外实验观察过表达与沉默GMⅡ基因对胃癌细胞株生长的影响；初步探讨GMⅡ基因在胃癌细胞生长作用中可能涉及的分子机制。
方法：1、通过RT-PCR、Westernblot分别检测人永生化胃粘膜上皮GES-1、胃癌细胞株BGC-823、SGC-7901、MKN-28的GMⅡmRNA及蛋白表达水平，筛选出GMⅡ低表达与高表达的胃癌细胞株作为实验用株。2、构建GMⅡ基因过表达载体并测序验证；设计3条靶向GMⅡ的siRNA并合成GMⅡ-shRNA-1、GMⅡ-shRNA-2、GMⅡ-shRNA-3及阴性对照shRNA-NC，shRNA退火后与线性化的质粒载体相连接并转化大肠杆菌感受态细胞DH5α，重组质粒载体扩增并提取后进行酶切测序鉴定。3、通过脂质体LipofectAMINE2000介导，将GMⅡ基因过表达载体转染GMⅡ低表达的胃癌细胞株；GMⅡ-shRNA-1、GMⅡ-shRNA-2、GMⅡ-shRNA-3及GMⅡ-shRNA-NC表达载体转染GMⅡ高表达的胃癌细胞株，应用荧光倒置显微镜对转染效果进行评价；通过RT-PCR、Westernblot检测瞬时转染胃癌细胞株后GMⅡmRNA及蛋白表达水平，进一步验证GMⅡ基因过表达载体的成功构建，同时筛选出干扰效果最好的表达载体用于后续实验研究；经G418筛选以获得稳定转染GMⅡ基因过表达载体与RNAi表达载体的胃癌细胞株。4、MTT法检测GMⅡ基因过表达与沉默后胃癌细胞株增殖活性的改变，流式细胞术分析细胞周期分布的改变；Hoechst33258染色及AnnexinV/PI合染并流式细胞术检测GMⅡ基因过表达及沉默后胃癌细胞株凋亡率的改变。5、将GMⅡ基因稳定过表达及沉默的胃癌细胞株接种至裸鼠皮下，观察移植瘤的生长速度并绘制肿瘤生长曲线，将移植瘤剥除后通过免疫组织化学检测GMⅡ蛋白的表达。6、通过RealtimePCR及Westernblot检测GMⅡ基因过表达及沉默后胃癌细胞株增殖与凋亡相关基因CyclinD1、Bcl-2、c-mycmRNA及蛋白水平的改变，同时检测MAPK通路中ERK1/2、p-ERK1/2的改变。
结果：1、GMⅡ基因与蛋白在胃癌细胞株MKN-28中表达最低，而在胃癌细胞株BGC-823中表达最高，因此筛选BGC-823、MKN-28为实验用细胞株。2、酶切测序表明GMⅡ基因的过表达载体及3条GMⅡ-shRNA表达载体构建成功。3、将GMⅡ基因的过表达载体转染胃癌细胞株MKN-28，GMⅡ-shRNA-1、GMⅡ-shRNA-2、GMⅡ-shRNA-3及GMⅡ-shRNA-NC表达载体转染胃癌细胞株BGC-823，转染后细胞在荧光倒置显微镜下发出绿色荧光；与空白对照组及转染空质粒组相比，过表达载体转染后胃癌细胞株MKN-28的GMⅡmRNA及蛋白水平明显升高（P<0.05）；与空白对照组、转染空质粒组及转染GMⅡ-shRNA-1、GMⅡ-shRNA-3及GMⅡ-shRNA-NC组相比，GMⅡ-shRNA-2表达载体转染后胃癌细胞株BGC-823的GMⅡmRNA及蛋白水平明显降低（P<0.05）；经G418筛选后获得稳定表达GMⅡ基因的胃癌细胞株MKN-28及稳定表达GMⅡ-shRNA-2的胃癌细胞株BGC-823。4、MTT法及流式细胞术检测结果显示，与空白对照组及转染空质粒组细胞相比，转染GMⅡ基因过表达载体的胃癌细胞株MKN-28生长速度明显增加，并且S期细胞比例显著增加（P<0.05）；转染GMⅡ-shRNA-2表达载体的胃癌细胞株BGC-823生长速度明显减慢，并且G1期细胞比例显著增加（P<0.05）。Hoechst33258染色及AnnexinV/PI合染并流式细胞术检测结果显示，与空白对照组及转染空质粒组细胞相比，转染GMⅡ基因过表达载体的胃癌细胞株MKN-28凋亡率明显降低（P<0.05）；转染GMⅡ-shRNA-2表达载体的胃癌细胞株BGC-823凋亡率明显升高（P<0.05）。5、裸鼠体内成瘤实验结果显示，与空白对照组及转染空质粒组细胞接种裸鼠后形成的移植瘤相比，转染GMⅡ基因过表达载体的胃癌细胞株MKN-28接种裸鼠后形成的移植瘤重量及体积明显增加（P<0.05），移植瘤GMⅡ蛋白的表达明显增强（P<0.05）；转染GMⅡ-shRNA-2表达载体的胃癌细胞株BGC-823接种裸鼠后形成的移植瘤重量及体积明显降低（P<0.05），移植瘤GMⅡ蛋白的表达明显减弱（P<0.05）。6、RealtimePCR及Westernblot结果显示，与空白对照组及转染空质粒组细胞相比，转染GMⅡ基因过表达载体的胃癌细胞株MKN-28增殖与凋亡相关基因CyclinD1、c-mycmRNA与蛋白表达水平明显增加（P<0.05），而Bcl-2mRNA与蛋白表达水平三组之间无显著性差异（P>0.05），MAPK通路中p-ERK1/2蛋白表达明显增加（P<0.05）；转染GMⅡ-shRNA-2表达载体的胃癌细胞株BGC-823增殖与凋亡相关基因CyclinD1、c-mycmRNA与蛋白表达水平明显降低（P<0.05），而Bcl-2mRNA与蛋白表达水平三组之间无显著性差异（P>0.05），MAPK通路中p-ERK1/2蛋白表达明显降低（P<0.05）。
结论：GMⅡ通过促进细胞增殖、抑制细胞凋亡而促进胃癌细胞的生长；增殖与凋亡相关基因CyclinD1、c-myc及MAPK信号转导通路可能参与了GMⅡ在胃癌细胞生长中的作用；GMⅡ可能成为研究胃癌发生发展分子机理的重要靶点。
Objective:To successfully construct the overexpression and silencing plasmid vectors targeted for GM Ⅱ gene and stably transfect them to the gastric cancer cell strains; to observe the growth change of gastric cancer cell strains after GM Ⅱ gene overexpressed and silenced by the tests in vitro and in vivo; and to preliminarily explore the molecular mechanisms of GM Ⅱ gene involved in the growth of gastric cancer cells.
Methods:1.By RT-PCR and Western blot respectively, the expression level of GM Ⅱ gene and protein in human gastric mucosal epithelium GES-1, gastric cancer cell strains BGC-823,SGC-7901and MKN-28were detected, and the cell strains with the lowest and the highest expression of GM Ⅱ gene were screenesd out as the experimental strains.2. The overexpressiom plasmid vector tageted for GM Ⅱ gene was constructed.Three siRNA segments targeted for GM Ⅱ gene were designed and the correspongding shRNA named GM Ⅱ-shRNA-1, GM Ⅱ-shRNA-2, GM Ⅱ-shRNA-3and one negative control of GM Ⅱ-shRNA-NC were synthetized by the biotech company. ShRNA after annealing were connected with linearized plasmid vector and transfected into E. coli competent cells DH5α, and the recombinant plasmid vectors were increased and the extracts were digested by restriction enzyme and identified by DNA sequencing.3. By LipofectAMINETM2000, the overexpression and silencing plamid vectors targeted for GM Ⅱ gene were transfected to the gastric cancer cell strains with the lowest and highest expression of GM Ⅱ gene respectivly. The transfection efficiency was evaluated by fluorescence inverted microscope. By RT-PCR and Western blot, the GM Ⅱ mRNA and protein of the gastric cancer cell strains after instantaneous transfection were detected, and overexpression plamid vector was further verified; at the same time, the best one of the silencing plasmid vectors used for subsequent experiments was screened out. The gastric cancer cell strains which were stably transfected with overexpression and silencing plasmid targeted for GM Ⅱ gene were selected by G418.4. After GM Ⅱ gene was overexpressed and silenced,MTT assay and flow cytometry were used to detect gastric cancer cell proliferation activity and cell cycle distribution respectively; Hoechst33258staining and Annexin V/PI co-staining and flow cytometry analysis were used to detect the rate of apoptosis.5. The gastric cancer cell strains were inoculated subcutaneously into nude mice, which were stably transfected with the overexpressed and silencing plamid vectors. The growth speed of the tumor was observed and tumor growth curve were drew, mice were sacrificed followed by HE staining of the transplanted tumors. GM Ⅱ protein expression of the transplanted tumors was detected by immunohistochemistry.6. By Real time PCR and Western blot analysis, the cell proliferation and apoptosis related genes cyclin D1, Bcl-2, c-myc mRNA and protein levels were detected, the protein level of ERK1/2, p-ERK1/2in MAPK pathway were detected by Western blot.
Results:1.GM Ⅱ gene and protein expression in gastric cancer cell strain MKN-28was the lowest while those in gastric cancer cell strain BGC-823was the highest, so the gastric cancer cell strains BGC-823and MKN-28were used for the following experiment.2. DNA sequencing showed that over expression vectors and three GM Ⅱ-shRNA expression vectors were constructed succefully.3. The overexpression vector targeted for GM Ⅱ gene was transfected to gastric cancer cell strain MKN-28, while GM Ⅱ-shRNA-1, GM Ⅱ-shRNA-2, GM Ⅱ-shRNA-3and of GM Ⅱ-shRNA-NC expression vector were transfected to gastric cancer cell strain BGC-823, the green fluorescence of the transfected cells could be observed under the fluorescence microscope.4. Compared to those of the black control group and the group transfected with empty plasmid, GM Ⅱ mRNA and protein levels of gastric cancer cell strain MKN-28transfected with overexpression vector increased significantly (P<0.05); Compared with those of the black control group and the group transfected with empty plasmid, the group transfected with GM Ⅱ-shRNA-1, GM Ⅱ-shRNA-3and GM Ⅱ-shRNA-NC, the GM Ⅱ mRNA and protein levels of the gastric cancer cell strain BGC-823transfected with GM Ⅱ-shRNA-2expression vector was decreased significantly (P<0.05); after G418selection, the gastric cancer cell MKN-28with stably overexpressed GM Ⅱ gene and the gastric cancer cell strain BGC-823with stably expressed GM Ⅱ-shRNA-2were obtained. Compared to those of the bland control group and the group transfected with the empty plasmid, MTT assay and flow cytometry results showed the growth rate of gastric cancer cell strain MKN-28transfected with overexpression vector increased significantly, and the S-phase cells increased significantly (P<0.05); the growth rate of gastric cancer cell strain BGC-823transfected with GM Ⅱ-shRNA-2expression vector was decreased significantly, and the rate of cells in G1phase was increased significantly (P<0.05). Hoechst33258staining and Annexin V/PI co-staining and flow cytometry results showed that the apoptosis rate of the gastric cancer cell strain MKN-28transfected with overexpression vector was decreased significantly (P<0.05); the apoptosis rate of the gastric cancer cell strain BGC-823transfected with the GM Ⅱ-shRNA-2expression vector was increased significantly (P<0.05).5. After the gastric cancer cell strain MKN-28transfected with overexpression vector were inoculated to the nude mouse, the weight and volume of transplanted tumor were significantly increased compare to those of transplanted tumor arising from inoculation of the blank control and the gastric cancer cell strain MKN-28transfected with the empty plamid(P<0.05); After the gastric cancer cell strain BGC-823transfected with GM Ⅱ-shRNA-2expression vector were inoculated to the nude mouse, the weight and volume of transplanted tumor were significantly decreased compare to those of transplanted tumor arising from inoculation of the blank control and the gastric cancer cell strain BGC-823transfected with the empty plamid(P<0.05).6. Real time PCR and Western blot results showed that compare to those of the control group and the group transfected with the empty plasmid, the proliferation and apoptosis-related genes Cyclin D1, c-myc mRNA and protein expression levels of gastric cancer cell strain MKN-28transfected with overexpressin vector were increased significantly (P<0.05), and p-ERK1/2protein expression the of MAPK pathway was increased significantly (P<0.05), but Bcl-2mRNA and protein expression levels among the three groups showed no significant difference (P>0.05); the proliferation and apoptosis-related genes Cyclin D1, c-myc mRNA and protein expression levels of gastric cancer cell strain BGC-823transfected with GM Ⅱ-shRNA-2expression vector were decreased significantly (P<0.05), and p-ERKl/2protein expression the of MAPK pathway was decreased significantly (P<0.05), but Bcl-2mRNA and protein expression levels among the three groups showed no significant difference (P>0.05).
Conclusion:GM Ⅱ gene could promote the growth of gastric cancer by promoting the proliferation and inhibting the apoptosis; the proliferation and apoptosis related gene such as Cyclin D1, c-myc and MAPK signal transduction pathway may be involved in the role of GM Ⅱ gene in the growth of gastric cancer; and GM II can be become an important target used to explore the molecular mechanism in gastric cancer occurrence and development.
方法：1、通过RT-PCR、Westernblot分别检测人永生化胃粘膜上皮GES-1、胃癌细胞株BGC-823、SGC-7901、MKN-28的GMⅡmRNA及蛋白表达水平，筛选出GMⅡ低表达与高表达的胃癌细胞株作为实验用株。2、构建GMⅡ基因过表达载体并测序验证；设计3条靶向GMⅡ的siRNA并合成GMⅡ-shRNA-1、GMⅡ-shRNA-2、GMⅡ-shRNA-3及阴性对照shRNA-NC，shRNA退火后与线性化的质粒载体相连接并转化大肠杆菌感受态细胞DH5α，重组质粒载体扩增并提取后进行酶切测序鉴定。3、通过脂质体LipofectAMINE2000介导，将GMⅡ基因过表达载体转染GMⅡ低表达的胃癌细胞株；GMⅡ-shRNA-1、GMⅡ-shRNA-2、GMⅡ-shRNA-3及GMⅡ-shRNA-NC表达载体转染GMⅡ高表达的胃癌细胞株，应用荧光倒置显微镜对转染效果进行评价；通过RT-PCR、Westernblot检测瞬时转染胃癌细胞株后GMⅡmRNA及蛋白表达水平，进一步验证GMⅡ基因过表达载体的成功构建，同时筛选出干扰效果最好的表达载体用于后续实验研究；经G418筛选以获得稳定转染GMⅡ基因过表达载体与RNAi表达载体的胃癌细胞株。4、MTT法检测GMⅡ基因过表达与沉默后胃癌细胞株增殖活性的改变，流式细胞术分析细胞周期分布的改变；Hoechst33258染色及AnnexinV/PI合染并流式细胞术检测GMⅡ基因过表达及沉默后胃癌细胞株凋亡率的改变。5、将GMⅡ基因稳定过表达及沉默的胃癌细胞株接种至裸鼠皮下，观察移植瘤的生长速度并绘制肿瘤生长曲线，将移植瘤剥除后通过免疫组织化学检测GMⅡ蛋白的表达。6、通过RealtimePCR及Westernblot检测GMⅡ基因过表达及沉默后胃癌细胞株增殖与凋亡相关基因CyclinD1、Bcl-2、c-mycmRNA及蛋白水平的改变，同时检测MAPK通路中ERK1/2、p-ERK1/2的改变。
结果：1、GMⅡ基因与蛋白在胃癌细胞株MKN-28中表达最低，而在胃癌细胞株BGC-823中表达最高，因此筛选BGC-823、MKN-28为实验用细胞株。2、酶切测序表明GMⅡ基因的过表达载体及3条GMⅡ-shRNA表达载体构建成功。3、将GMⅡ基因的过表达载体转染胃癌细胞株MKN-28，GMⅡ-shRNA-1、GMⅡ-shRNA-2、GMⅡ-shRNA-3及GMⅡ-shRNA-NC表达载体转染胃癌细胞株BGC-823，转染后细胞在荧光倒置显微镜下发出绿色荧光；与空白对照组及转染空质粒组相比，过表达载体转染后胃癌细胞株MKN-28的GMⅡmRNA及蛋白水平明显升高（P<0.05）；与空白对照组、转染空质粒组及转染GMⅡ-shRNA-1、GMⅡ-shRNA-3及GMⅡ-shRNA-NC组相比，GMⅡ-shRNA-2表达载体转染后胃癌细胞株BGC-823的GMⅡmRNA及蛋白水平明显降低（P<0.05）；经G418筛选后获得稳定表达GMⅡ基因的胃癌细胞株MKN-28及稳定表达GMⅡ-shRNA-2的胃癌细胞株BGC-823。4、MTT法及流式细胞术检测结果显示，与空白对照组及转染空质粒组细胞相比，转染GMⅡ基因过表达载体的胃癌细胞株MKN-28生长速度明显增加，并且S期细胞比例显著增加（P<0.05）；转染GMⅡ-shRNA-2表达载体的胃癌细胞株BGC-823生长速度明显减慢，并且G1期细胞比例显著增加（P<0.05）。Hoechst33258染色及AnnexinV/PI合染并流式细胞术检测结果显示，与空白对照组及转染空质粒组细胞相比，转染GMⅡ基因过表达载体的胃癌细胞株MKN-28凋亡率明显降低（P<0.05）；转染GMⅡ-shRNA-2表达载体的胃癌细胞株BGC-823凋亡率明显升高（P<0.05）。5、裸鼠体内成瘤实验结果显示，与空白对照组及转染空质粒组细胞接种裸鼠后形成的移植瘤相比，转染GMⅡ基因过表达载体的胃癌细胞株MKN-28接种裸鼠后形成的移植瘤重量及体积明显增加（P<0.05），移植瘤GMⅡ蛋白的表达明显增强（P<0.05）；转染GMⅡ-shRNA-2表达载体的胃癌细胞株BGC-823接种裸鼠后形成的移植瘤重量及体积明显降低（P<0.05），移植瘤GMⅡ蛋白的表达明显减弱（P<0.05）。6、RealtimePCR及Westernblot结果显示，与空白对照组及转染空质粒组细胞相比，转染GMⅡ基因过表达载体的胃癌细胞株MKN-28增殖与凋亡相关基因CyclinD1、c-mycmRNA与蛋白表达水平明显增加（P<0.05），而Bcl-2mRNA与蛋白表达水平三组之间无显著性差异（P>0.05），MAPK通路中p-ERK1/2蛋白表达明显增加（P<0.05）；转染GMⅡ-shRNA-2表达载体的胃癌细胞株BGC-823增殖与凋亡相关基因CyclinD1、c-mycmRNA与蛋白表达水平明显降低（P<0.05），而Bcl-2mRNA与蛋白表达水平三组之间无显著性差异（P>0.05），MAPK通路中p-ERK1/2蛋白表达明显降低（P<0.05）。
结论：GMⅡ通过促进细胞增殖、抑制细胞凋亡而促进胃癌细胞的生长；增殖与凋亡相关基因CyclinD1、c-myc及MAPK信号转导通路可能参与了GMⅡ在胃癌细胞生长中的作用；GMⅡ可能成为研究胃癌发生发展分子机理的重要靶点。
Objective:To successfully construct the overexpression and silencing plasmid vectors targeted for GM Ⅱ gene and stably transfect them to the gastric cancer cell strains; to observe the growth change of gastric cancer cell strains after GM Ⅱ gene overexpressed and silenced by the tests in vitro and in vivo; and to preliminarily explore the molecular mechanisms of GM Ⅱ gene involved in the growth of gastric cancer cells.
Methods:1.By RT-PCR and Western blot respectively, the expression level of GM Ⅱ gene and protein in human gastric mucosal epithelium GES-1, gastric cancer cell strains BGC-823,SGC-7901and MKN-28were detected, and the cell strains with the lowest and the highest expression of GM Ⅱ gene were screenesd out as the experimental strains.2. The overexpressiom plasmid vector tageted for GM Ⅱ gene was constructed.Three siRNA segments targeted for GM Ⅱ gene were designed and the correspongding shRNA named GM Ⅱ-shRNA-1, GM Ⅱ-shRNA-2, GM Ⅱ-shRNA-3and one negative control of GM Ⅱ-shRNA-NC were synthetized by the biotech company. ShRNA after annealing were connected with linearized plasmid vector and transfected into E. coli competent cells DH5α, and the recombinant plasmid vectors were increased and the extracts were digested by restriction enzyme and identified by DNA sequencing.3. By LipofectAMINETM2000, the overexpression and silencing plamid vectors targeted for GM Ⅱ gene were transfected to the gastric cancer cell strains with the lowest and highest expression of GM Ⅱ gene respectivly. The transfection efficiency was evaluated by fluorescence inverted microscope. By RT-PCR and Western blot, the GM Ⅱ mRNA and protein of the gastric cancer cell strains after instantaneous transfection were detected, and overexpression plamid vector was further verified; at the same time, the best one of the silencing plasmid vectors used for subsequent experiments was screened out. The gastric cancer cell strains which were stably transfected with overexpression and silencing plasmid targeted for GM Ⅱ gene were selected by G418.4. After GM Ⅱ gene was overexpressed and silenced,MTT assay and flow cytometry were used to detect gastric cancer cell proliferation activity and cell cycle distribution respectively; Hoechst33258staining and Annexin V/PI co-staining and flow cytometry analysis were used to detect the rate of apoptosis.5. The gastric cancer cell strains were inoculated subcutaneously into nude mice, which were stably transfected with the overexpressed and silencing plamid vectors. The growth speed of the tumor was observed and tumor growth curve were drew, mice were sacrificed followed by HE staining of the transplanted tumors. GM Ⅱ protein expression of the transplanted tumors was detected by immunohistochemistry.6. By Real time PCR and Western blot analysis, the cell proliferation and apoptosis related genes cyclin D1, Bcl-2, c-myc mRNA and protein levels were detected, the protein level of ERK1/2, p-ERK1/2in MAPK pathway were detected by Western blot.
Results:1.GM Ⅱ gene and protein expression in gastric cancer cell strain MKN-28was the lowest while those in gastric cancer cell strain BGC-823was the highest, so the gastric cancer cell strains BGC-823and MKN-28were used for the following experiment.2. DNA sequencing showed that over expression vectors and three GM Ⅱ-shRNA expression vectors were constructed succefully.3. The overexpression vector targeted for GM Ⅱ gene was transfected to gastric cancer cell strain MKN-28, while GM Ⅱ-shRNA-1, GM Ⅱ-shRNA-2, GM Ⅱ-shRNA-3and of GM Ⅱ-shRNA-NC expression vector were transfected to gastric cancer cell strain BGC-823, the green fluorescence of the transfected cells could be observed under the fluorescence microscope.4. Compared to those of the black control group and the group transfected with empty plasmid, GM Ⅱ mRNA and protein levels of gastric cancer cell strain MKN-28transfected with overexpression vector increased significantly (P<0.05); Compared with those of the black control group and the group transfected with empty plasmid, the group transfected with GM Ⅱ-shRNA-1, GM Ⅱ-shRNA-3and GM Ⅱ-shRNA-NC, the GM Ⅱ mRNA and protein levels of the gastric cancer cell strain BGC-823transfected with GM Ⅱ-shRNA-2expression vector was decreased significantly (P<0.05); after G418selection, the gastric cancer cell MKN-28with stably overexpressed GM Ⅱ gene and the gastric cancer cell strain BGC-823with stably expressed GM Ⅱ-shRNA-2were obtained. Compared to those of the bland control group and the group transfected with the empty plasmid, MTT assay and flow cytometry results showed the growth rate of gastric cancer cell strain MKN-28transfected with overexpression vector increased significantly, and the S-phase cells increased significantly (P<0.05); the growth rate of gastric cancer cell strain BGC-823transfected with GM Ⅱ-shRNA-2expression vector was decreased significantly, and the rate of cells in G1phase was increased significantly (P<0.05). Hoechst33258staining and Annexin V/PI co-staining and flow cytometry results showed that the apoptosis rate of the gastric cancer cell strain MKN-28transfected with overexpression vector was decreased significantly (P<0.05); the apoptosis rate of the gastric cancer cell strain BGC-823transfected with the GM Ⅱ-shRNA-2expression vector was increased significantly (P<0.05).5. After the gastric cancer cell strain MKN-28transfected with overexpression vector were inoculated to the nude mouse, the weight and volume of transplanted tumor were significantly increased compare to those of transplanted tumor arising from inoculation of the blank control and the gastric cancer cell strain MKN-28transfected with the empty plamid(P<0.05); After the gastric cancer cell strain BGC-823transfected with GM Ⅱ-shRNA-2expression vector were inoculated to the nude mouse, the weight and volume of transplanted tumor were significantly decreased compare to those of transplanted tumor arising from inoculation of the blank control and the gastric cancer cell strain BGC-823transfected with the empty plamid(P<0.05).6. Real time PCR and Western blot results showed that compare to those of the control group and the group transfected with the empty plasmid, the proliferation and apoptosis-related genes Cyclin D1, c-myc mRNA and protein expression levels of gastric cancer cell strain MKN-28transfected with overexpressin vector were increased significantly (P<0.05), and p-ERK1/2protein expression the of MAPK pathway was increased significantly (P<0.05), but Bcl-2mRNA and protein expression levels among the three groups showed no significant difference (P>0.05); the proliferation and apoptosis-related genes Cyclin D1, c-myc mRNA and protein expression levels of gastric cancer cell strain BGC-823transfected with GM Ⅱ-shRNA-2expression vector were decreased significantly (P<0.05), and p-ERKl/2protein expression the of MAPK pathway was decreased significantly (P<0.05), but Bcl-2mRNA and protein expression levels among the three groups showed no significant difference (P>0.05).
Conclusion:GM Ⅱ gene could promote the growth of gastric cancer by promoting the proliferation and inhibting the apoptosis; the proliferation and apoptosis related gene such as Cyclin D1, c-myc and MAPK signal transduction pathway may be involved in the role of GM Ⅱ gene in the growth of gastric cancer; and GM II can be become an important target used to explore the molecular mechanism in gastric cancer occurrence and development.
引文
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