慢病毒介导Med19基因沉默对胃癌细胞生物学行为的影响
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摘要
[目的]：探讨Med19基因在胃癌组织中的表达,以及应用RNA干扰技术抑制Med19基因表达对胃癌细胞生物学行为的影响。
[方法]：检测胃癌组织中Med19的表达并分析其与胃癌临床病理的关系,利用基因重组技术构建靶向Med19的shRNA慢病毒载体,感染胃癌SGC7901和MGC803细胞,检测Med19 mRNA及蛋白抑制效率,并观察其对胃癌细胞增殖、周期和克隆形成能力的影响。
[结果]：胃癌组织中的Med19的表达显著高于癌旁组织,与肿瘤的大小、细胞分化程度及TNM分期相关。重组shRNA慢病毒载体能有效感染胃癌细胞,下调Med19基因的表达,并抑制肿瘤细胞增殖,使细胞周期阻滞于G1期,克隆形成能力显著减弱,但对肿瘤的侵袭转移没有明显的影响。
[结论]：Medl9在肿瘤组织中特异性高表达可能是胃癌形成的一个原因,慢病毒介导的RNAi能有效沉默胃癌细胞Med19基因及蛋白表达,抑制体内胃癌细胞增殖并影响细胞周期,有望成为肿瘤基因治疗的新分子靶标。
Background
Gastric cancer is the most common malignant tumor of the gastrointestinal tract, according to statistics, in the world, the mortality rate ranks second malignant tumors, about 60%of the world's new cases of gastric cancer in China, Japan and other Southeast Asian Countries. Surgical treatment of gastric cancer is still being considered the most direct and most effective means, but after 5-year survival rate is still low. According to 2009 census, although the treatment of gastric cancer has been significant progress, but the cancer patient's overall survival rate after 5 years is still less than 25%.
Tumor invasion and metastasis of tumor cells and tumor tissue micro-environment interaction. Now that the line of gastric cancer recurrence and metastasis of postoperative gastric cancer patients is the most fundamental reason of death, but on the process of gastric cancer metastasis and the molecular mechanisms of signal pathway is still not very clear, the process may involve multiple molecular mechanisms and Signal transduction pathways, such as MAPK/ERK, NF-κB and PI3K/AKT and so on.
Large number of experiments have shown that the incidence of gastric cancer process is the interaction between environmental and genetic factors in the outcome, is a cancer gene activation and inactivation of tumor suppressor gene-based multi-step, multi-stage process, and these genes the role of expression products, that is, changes in the body and disrupt cell growth, differentiation and metabolism and other important processes, which lead to cell cancer. Therefore, if you can find a way to block oncogene activation and/ or tumor suppressor gene inactivation siRNA, then there may inhibit gastric cancer. More traditional, such as gene knockout, antisense and ribozymes and other methods is often impossible to completely inhibit or reverse rumor cell growth, comparatively speaking, RNAi technology is different, because the technology can also suppress a number of different genes, and the inhibitory effect of these genes also interfere with each other. In addition, RNAi can be precisely identified to a nucleotide, for the wild-type form of cancer gene mutation, such as ras, p53, etc., are able to produce accurate and effective seal, while the wild-type gene is not affected. A large number of experimental results indicate that, RNAi technology can inhibit the cancer-specific gene mutations in cancer-related genes or gene over-expression and inhibit tumor development, and rapid identification of new tumor target goal, so this technology in cancer Gene therapy has a bright application prospect.
Objective
Of Med19 gene in gastric cancer, cancer tissue and normal tissue, as well as application of RNA interference inhibition of Medl9 expression in gastric cancer cell biological behavior.
Methods
1. collection of our department 60 cases of human gastric cancer tissue specimens, including 36 male,24 female, mean age 58.2 years, all the specimens were pathologically confirmed gastric adenocarcinoma, all specimens were not received preoperative radiotherapy and chemotherapy. Histological examination of 20 cases of normal gastric mucosa from endoscopic biopsy specimens. Pathological types:adenocarcinoma in 13 cases, 15 cases of differentiated adenocarcinoma, poorly differentiated adenocar-cinoma in 26 cases. TNM stage:Ⅰ8 cases,ⅡPhaseⅢof the 13 cases 19 cases 18 cases ofⅣ. The group of 60 patients were clinically followed up for 48-62 months, the survival time 8-62 months, the median survival time of 37 months.
Immunohistochemistry and real-time PCR to detect Med19 gene in gastric cancer, gastric cancer and adjacent normal gastric tissues.
2. re si-Med19 lentiviral vector and packaging
The recombinant lentiviral SGC7901 gastric cancer cells, quantitative PCR analysis of RNAi on SGC7901 the Med19 mRNA Abundance.
3. application to build a good Med19 shRNA lentiviral gene and MGC803 SGC7901 cells, the experiment is divided into blank control, negative control and RNAi three groups of three duplicate wells, selected by preliminary experiments, the cells infected with reasonable parameters. Infection in more than 80%efficiency, to ensure normal cell morphology, cell outlines clear, relatively clean background. Successful expansion of infected cells cultured for subsequent functional experiments. Med19 in gene silencing by MTT method five days of continuous cell growth data, and the cell growth curve. The experimental group using PI staining of gastric cancer cells and the cell cycle by flow cytometry.
Results
1. Med19 positive expression in gastric cancer was 71.7%(43/60), significantly higher than in adjacent normal gastric tissue and gastric tissue (P <0.01). Gastric tissue and adjacent normal gastric tissues were negative and more visible expression of Medl9, a small number of weakly positive, no significant difference between the two sex (P> 0.05). Med19 expression in gastric cancer cell differentiation, tumor size, TNM staging (P<0.05), but in a different age, gender, lymph node metastasis, invasive depth of gastric cancer samples showed no significant expression (P> 0.05).
2. successfully constructed the MED19-shRNA lentiviral vectors, lentiviral transfected SGC7901, the mRNA can inhibit gene expression of Medl9 up to 81.8%, while in Western experiments also confirmed the Med19 protein was significantly inhibited, indicating that the recombinant lentiviral shRNA Med19 significantly reduced the expression of genes, the RNAi effect is effective.
3. application to build a good Med19 shRNA lentiviral gene and MGC803 SGC7901 cells, gene silencing Med19 by MTT method five days of continuous cell growth data, and the cell growth curve and found that inhibition of Med19 gene can significantly inhibit cell Proliferation, RNAi gene silencing group that Med19 group, compared with the negative control group, cell proliferation significantly decreased from the second day since. Flow cytometry showed that cells were cell cycle arrest in Gl cell cycle phase, that is, DNA synthesis phase, a large number of cells can not properly copied into the division process.
Conclusion
1. Med19 gene in gastric cancer tissues showed high expression, and its expression was significantly higher than adjacent tissues and normal tissues. Med19 expression in gastric cancer and gastric cancer tumor size, differen-tiation, TNM stage, but not with age, sex, depth of invasion, lymph node metastasis has nothing to do. Med19 expression in larger tumors, poor differentiation, TNM stage were significantly higher than the tumor is small, well differentiated and those with low TNM stage.
2. recombinant lentiviral shRNA significantly reduced Med19 gene expression.
3. shRNA slow virus infection MGC803 cells SGC7901 and Med19 gene silencing, can significantly inhibit cell proliferation, cell cycle cells were arrested in G1 phase, which concluded that Med19 gene in gastric cancer is a tumor promoting gene. We found that, Med19 gene silencing ability of colony formation of gastric cancer is weak, a single clone of cells decreased signifi-cantly. Med19 suppression gene can significantly reduce the formation of SGC7901 and MGC803 cell clones, suggesting that gene expression can be reduced Med19 abnormal proliferation of cells to be controlled.
4. Med19 highly specific expression in gastric tumor formation may be a cause of lentivirus-mediated RNAi can effectively inhibit gastric cancer cell Med19 gene and protein expression, inhibit cell proliferation in vivo and influence the cell cycle, is expected to be tumor-targeting New molecular pathway of gene therapy.
[方法]：检测胃癌组织中Med19的表达并分析其与胃癌临床病理的关系,利用基因重组技术构建靶向Med19的shRNA慢病毒载体,感染胃癌SGC7901和MGC803细胞,检测Med19 mRNA及蛋白抑制效率,并观察其对胃癌细胞增殖、周期和克隆形成能力的影响。
[结果]：胃癌组织中的Med19的表达显著高于癌旁组织,与肿瘤的大小、细胞分化程度及TNM分期相关。重组shRNA慢病毒载体能有效感染胃癌细胞,下调Med19基因的表达,并抑制肿瘤细胞增殖,使细胞周期阻滞于G1期,克隆形成能力显著减弱,但对肿瘤的侵袭转移没有明显的影响。
[结论]：Medl9在肿瘤组织中特异性高表达可能是胃癌形成的一个原因,慢病毒介导的RNAi能有效沉默胃癌细胞Med19基因及蛋白表达,抑制体内胃癌细胞增殖并影响细胞周期,有望成为肿瘤基因治疗的新分子靶标。
Background
Gastric cancer is the most common malignant tumor of the gastrointestinal tract, according to statistics, in the world, the mortality rate ranks second malignant tumors, about 60%of the world's new cases of gastric cancer in China, Japan and other Southeast Asian Countries. Surgical treatment of gastric cancer is still being considered the most direct and most effective means, but after 5-year survival rate is still low. According to 2009 census, although the treatment of gastric cancer has been significant progress, but the cancer patient's overall survival rate after 5 years is still less than 25%.
Tumor invasion and metastasis of tumor cells and tumor tissue micro-environment interaction. Now that the line of gastric cancer recurrence and metastasis of postoperative gastric cancer patients is the most fundamental reason of death, but on the process of gastric cancer metastasis and the molecular mechanisms of signal pathway is still not very clear, the process may involve multiple molecular mechanisms and Signal transduction pathways, such as MAPK/ERK, NF-κB and PI3K/AKT and so on.
Large number of experiments have shown that the incidence of gastric cancer process is the interaction between environmental and genetic factors in the outcome, is a cancer gene activation and inactivation of tumor suppressor gene-based multi-step, multi-stage process, and these genes the role of expression products, that is, changes in the body and disrupt cell growth, differentiation and metabolism and other important processes, which lead to cell cancer. Therefore, if you can find a way to block oncogene activation and/ or tumor suppressor gene inactivation siRNA, then there may inhibit gastric cancer. More traditional, such as gene knockout, antisense and ribozymes and other methods is often impossible to completely inhibit or reverse rumor cell growth, comparatively speaking, RNAi technology is different, because the technology can also suppress a number of different genes, and the inhibitory effect of these genes also interfere with each other. In addition, RNAi can be precisely identified to a nucleotide, for the wild-type form of cancer gene mutation, such as ras, p53, etc., are able to produce accurate and effective seal, while the wild-type gene is not affected. A large number of experimental results indicate that, RNAi technology can inhibit the cancer-specific gene mutations in cancer-related genes or gene over-expression and inhibit tumor development, and rapid identification of new tumor target goal, so this technology in cancer Gene therapy has a bright application prospect.
Objective
Of Med19 gene in gastric cancer, cancer tissue and normal tissue, as well as application of RNA interference inhibition of Medl9 expression in gastric cancer cell biological behavior.
Methods
1. collection of our department 60 cases of human gastric cancer tissue specimens, including 36 male,24 female, mean age 58.2 years, all the specimens were pathologically confirmed gastric adenocarcinoma, all specimens were not received preoperative radiotherapy and chemotherapy. Histological examination of 20 cases of normal gastric mucosa from endoscopic biopsy specimens. Pathological types:adenocarcinoma in 13 cases, 15 cases of differentiated adenocarcinoma, poorly differentiated adenocar-cinoma in 26 cases. TNM stage:Ⅰ8 cases,ⅡPhaseⅢof the 13 cases 19 cases 18 cases ofⅣ. The group of 60 patients were clinically followed up for 48-62 months, the survival time 8-62 months, the median survival time of 37 months.
Immunohistochemistry and real-time PCR to detect Med19 gene in gastric cancer, gastric cancer and adjacent normal gastric tissues.
2. re si-Med19 lentiviral vector and packaging
The recombinant lentiviral SGC7901 gastric cancer cells, quantitative PCR analysis of RNAi on SGC7901 the Med19 mRNA Abundance.
3. application to build a good Med19 shRNA lentiviral gene and MGC803 SGC7901 cells, the experiment is divided into blank control, negative control and RNAi three groups of three duplicate wells, selected by preliminary experiments, the cells infected with reasonable parameters. Infection in more than 80%efficiency, to ensure normal cell morphology, cell outlines clear, relatively clean background. Successful expansion of infected cells cultured for subsequent functional experiments. Med19 in gene silencing by MTT method five days of continuous cell growth data, and the cell growth curve. The experimental group using PI staining of gastric cancer cells and the cell cycle by flow cytometry.
Results
1. Med19 positive expression in gastric cancer was 71.7%(43/60), significantly higher than in adjacent normal gastric tissue and gastric tissue (P <0.01). Gastric tissue and adjacent normal gastric tissues were negative and more visible expression of Medl9, a small number of weakly positive, no significant difference between the two sex (P> 0.05). Med19 expression in gastric cancer cell differentiation, tumor size, TNM staging (P<0.05), but in a different age, gender, lymph node metastasis, invasive depth of gastric cancer samples showed no significant expression (P> 0.05).
2. successfully constructed the MED19-shRNA lentiviral vectors, lentiviral transfected SGC7901, the mRNA can inhibit gene expression of Medl9 up to 81.8%, while in Western experiments also confirmed the Med19 protein was significantly inhibited, indicating that the recombinant lentiviral shRNA Med19 significantly reduced the expression of genes, the RNAi effect is effective.
3. application to build a good Med19 shRNA lentiviral gene and MGC803 SGC7901 cells, gene silencing Med19 by MTT method five days of continuous cell growth data, and the cell growth curve and found that inhibition of Med19 gene can significantly inhibit cell Proliferation, RNAi gene silencing group that Med19 group, compared with the negative control group, cell proliferation significantly decreased from the second day since. Flow cytometry showed that cells were cell cycle arrest in Gl cell cycle phase, that is, DNA synthesis phase, a large number of cells can not properly copied into the division process.
Conclusion
1. Med19 gene in gastric cancer tissues showed high expression, and its expression was significantly higher than adjacent tissues and normal tissues. Med19 expression in gastric cancer and gastric cancer tumor size, differen-tiation, TNM stage, but not with age, sex, depth of invasion, lymph node metastasis has nothing to do. Med19 expression in larger tumors, poor differentiation, TNM stage were significantly higher than the tumor is small, well differentiated and those with low TNM stage.
2. recombinant lentiviral shRNA significantly reduced Med19 gene expression.
3. shRNA slow virus infection MGC803 cells SGC7901 and Med19 gene silencing, can significantly inhibit cell proliferation, cell cycle cells were arrested in G1 phase, which concluded that Med19 gene in gastric cancer is a tumor promoting gene. We found that, Med19 gene silencing ability of colony formation of gastric cancer is weak, a single clone of cells decreased signifi-cantly. Med19 suppression gene can significantly reduce the formation of SGC7901 and MGC803 cell clones, suggesting that gene expression can be reduced Med19 abnormal proliferation of cells to be controlled.
4. Med19 highly specific expression in gastric tumor formation may be a cause of lentivirus-mediated RNAi can effectively inhibit gastric cancer cell Med19 gene and protein expression, inhibit cell proliferation in vivo and influence the cell cycle, is expected to be tumor-targeting New molecular pathway of gene therapy.
引文
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