B-RAF基因及MAPK信号转导通路和胃癌相关性及其机制的研究
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摘要
B-RAF基因及MAPK信号转导通路和胃癌相关性及其机制的研究
前言
对肿瘤(癌)发生机制和防治的研究，多年来一直是人们关注和研究的热点问题。肿瘤的发生和侵袭转移是涉及许多基因参与的、多途经和多步骤的发展过程。其中，癌基因控制机体细胞的生长和分化，重要的癌基因激活可引起肿瘤的发生。
癌基因B-RAF属于RAF基因家族，编码丝氨酸/苏氨酸激酶，是有丝分裂原激活蛋白激酶(mitogen-activated protein kinase，MAPK)信号转导通路中的重要成员之一。近年来的研究发现许多肿瘤中存在B-RAF基因突变，尤其是黑色素瘤和甲状腺癌中B-RAF基因突变率高达60～80％，突变主要集中于B-RAF基因的第11和第15外显子，这说明B-RAF基因与黑色素瘤和甲状腺癌有关。到目前为止国内外有关B-RAF基因在胃癌发生、发展中的机制研究尚未见报道。
我们在前期工作中利用基因芯片技术对胃癌发病不同阶段的基因表达进行了较为全面、动态的研究。我们发现B-RAF基因在癌旁、癌及淋巴结转移癌中表达与正常胃粘膜组织相比均有明显升高，提示B-RAF基因可能是胃癌发生过程中的一个重要基因。因此我们首先对150例人胃癌组织标本中B-RAF基因突变热点第11和第15外显子进行突变筛查，结果显示阴性；但同时的免疫组化以及Western Blot结果却显示胃癌组织中B-RAF蛋白表达阳性，而癌旁组织中无B-RAF蛋白表达，这说明B-RAF基因与胃癌有相关性，但B-RAF基因在胃癌中的作用机制可能不同于黑色素瘤和甲状腺癌。为了深入探讨B-RAF基因在胃癌发生、发展中的可能作用，阐明B-RAF基因及MAPK信号转导通路和胃癌的相关性及其机制，以及B-RAF基因的上游调控基因，我们首先应用RNA干扰技术抑制胃癌BGC823细胞中B-RAF基因的表达，分析B-RAF基因对胃癌BGC823细胞生物学特性的影响；其次利用免疫沉淀(IP)技术结合蛋白质的质谱分析研究B-RAF蛋白在胃癌细胞中发挥作用时可能与之结合的未知蛋白；最后应用GGTI-298和FTI-277分别抑制RAS和RAPl蛋白活性以及利用生物信息学软件预测并用染色质免疫沉淀(CHIP)结合PCR技术研究B-RAF基因的上游调控基因。本研究对阐明B-RAF基因及MAPK信号转导通路与胃癌相关性以及对B-RAF基因在胃癌发生中调控机制研究具有重要意义。
方法
应用PCR-DHPLC结合DNA测序技术筛查B-RAF基因点突变；免疫组化和Western Blot检测胃癌组织中B-RAF蛋白表达。针对B-RAF基因和RelA基因设计siRNA，体外转录合成B-RAF-siRNA和RelA-siRNA，利用脂质体转染试剂Lipofectamine 2000将其分别转染胃癌细胞系BGC823细胞。RT-PCR和Western Blot检测胃癌BGC823细胞中B-RAF基因和RelA基因的表达；MTT、流式细胞术、Transwell和RT-PCR以及Western Blot分别检测B-RAF基因和RelA基因表达抑制后对胃癌细胞系BGC823细胞生物学特性的影响以及分子机制；免疫沉淀技术结合蛋白质的质谱分析技术研究可能与B-RAF蛋白结合的未知蛋白；利用GGTI-298和FTI-277处理胃癌细胞系BGC823细胞，Western Blot检测二种抑制剂对胃癌细胞中B-RAF基因表达的影响；利用生物信息学转录因子预测软件预测可能与B-RAF基因上游启动子区特异序列结合的转录因子，并用染色质免疫沉淀技术结合PCR技术对生物信息学转录因子预测软件预测的结果进行验证。
结果
1．人胃癌组织中B-RAF基因突变筛查结果：
在150例人胃癌组织和癌旁组织标本中未筛查到B-RAF基因第11外显子和第15外显子存在突变。
2．人胃癌组织中B-RAF蛋白表达分析：
免疫组化结果显示在20例人胃癌组织标本中，胃癌细胞中可见棕色颗粒，主要分布于细胞质，B-RAY蛋白表达阳性，而正常胃腺细胞中未见棕色颗粒，B-RAF蛋白表达阴性。
3．B-RAF-siRNA对胃癌细胞中B-RAF mRNA表达的影响：
B-RAF-siRNA转染BGC823细胞第5和第7天时B-RAF mRNA表达水平可见到明显变化，B-RAF-siRNA组较对照组B-RAF mRNA表达水平明显降低。
4．B-RAF-siRNA对胃癌细胞中B-RAF蛋白表达的影响：
B-RAF-siRNA转染BGC823细胞第9天时B-RAF蛋白表达水平可见到明显变化，B-RAF-siRNA组较对照组B-RAF蛋白表达水平明显降低，表明B-RAF蛋白表达受到抑制。
5．B-RAF-siRNA对人胃癌BGC823细胞生长的影响
与细胞对照组相比，B-RAF-siRNA对体外培养的胃癌BGC823细胞的生长有明显抑制作用。其中，转染后72h抑制率达20％。
6．B-RAF-siRNA对胃癌细胞凋亡的影响
B-RAF-siRNA组胃癌BGC823细胞凋亡率为19.58％，B-RAF-siRNA组较对照组胃癌BGC823细胞凋亡率明显升高，说明B-RAF基因抑制能够引起胃癌BGC823细胞凋亡。
7．B-RAF-siRNA抑制人胃癌BGC823细胞中BCL-2 mRNA的表达。
8．B-RAF-siRNA对细胞侵袭力的影响
B-RAF-siRNA转染后第9天，细胞侵袭力明显降低。
9．RAP1抑制剂GGTI-298和RAS抑制剂FTI-277均明显抑制胃癌BGC823细胞中B-RAF mRNA和蛋白的表达。
10．RelA-siRNA对胃癌BGC823细胞中B-RAF蛋白表达的影响
RelA-siRNA有效地抑制RelA基因表达后，胃癌BGC823细胞中B-RAF蛋白表达水平也明显降低。
11．免疫沉淀技术结合质谱分析结果
胃癌BGC823细胞中B-RAF蛋白可能与PR02619蛋白结合。
12．染色质免疫沉淀技术结合PCR技术检测结果
在NF-κB抗体免疫沉淀的DNA片断中含有B-RAF基因的启动子区NF-κB蛋白的结合序列。
结论
1．B-RAF基因点突变可能不是B-RAF基因参与胃癌发生的途径。
2．B-RAF基因表达增高可能与胃癌发生密切相关。
3．B-RAF基因是与胃癌相关的重要癌基因，促进胃癌细胞的生长增殖、抑制细胞凋亡和增强胃癌细胞的侵袭力。
4．BCL-2基因在胃癌细胞中可能位于B-RAF基因的下游并受其调控。
5．B-RAF基因在其信号转导通路中受RAPI和RAS上游基因的双重调控；
6．RAP1和RAS的抑制剂GGTI-298与FTI-277可抑制胃癌细胞的增殖，促进胃癌细胞的凋亡；
7．NF-κB可能是调控B-RAF基因的重要转录因子
8．B-RAF蛋白在胃癌BGC823细胞中可能与PR02619蛋白结合。
Relationship between B-RAF gene and MAPK signal transduction pathway and gastric cancer and study of mechanism
Investigation of tumorigenesis mechanism, prevention, and therapy have already been hot point for many years. The tumorigenesis and metastasis are involved in developing process of many pathways and steps in which many genes participate, especially oncogenes control growth and differentiation of somatic cells, and activation of critical oncogenes can cause tumorigenesis.
Oncogene B-RAF which belongs to RAF gene family, encoding sefine/threonine kinase, is one of the important members of MAPK (mitogen-activated protein kinase) signal transduction pathway. Recent research found that the mutation of B-RAF gene exists in many tumors, especially melanoma and thyroid cancer, in which the rate of mutation in B-RAF gene is up to 60-80%, It indicates that B-RAF gene is related to tumors. Until now, there is no report about the mechanism of B-RAF gene in gastric carcinogenesis and development in the world.
Our previous work studied the expression of genes in different satges of gastric cancer by using gene chip. We found that the expression of B-RAF gene is significantly elevated in paracancerous tissue, cancer tissue and metastatic lymphode. Subsequently, we screened the mutation of exon11 and exon 15 of B-RAF gene in 150 samples of human gastric cancer tissue and result showed negative, but the result of simultaneous immunohistochemistry and Western Blot indicated that the expression of B-RAF protein was positive and suggest that B-RAF gene is related to gastric cancel but the functional mechanism of B-RAF in gatric cancer may be different in melanoma and thyroid cancer. To further research the possible function of B-RAF gene in gastric carcinogenesis and development, clarify the correlation between B-RAF gene and MAPK signal transduction pathway and study of mechanism and upstream regulating gene of B-RAF gene, we first utilized RNA interference to inhibit the expression of B-RAF gene in gastric cancer BGC823 cell line, analyzed the biological effect of B-RAF gene on BGC823 cell line before and after RNA interference; Secondly, we used IP(immunoprecipitation, IP) with mass spectrometry analysis technique to study unknown protein which may bind to B-RAF protein in gastric cancer cellsl; Finally, we employed GGTI-298 and FTI-277 to inhibit the activation of RAS and RAP1 protein, and biological information software to predict the transcriptional factors of regulating B-RAF gene and CHIP with PCR to investigate the upstream regulating genes of B-RAF gene. Our research is of significance to elucidate the correlation between B-RAF gene and gastric cancer, and explore its functional mechanism.
Methods
We used PCR-DHPLC with DNA sequencing to screen point mutation of B-RAF gene; Immunohistochemistry and Western Blot for the expression of B-RAF protein in gastric cancer. We designed siRNA specific for B-RAF gene and RelA gene and in vitro synthesize B-RAF-siRNA and RelA-siRNA, then was transfected into gastric cancer BGC823 cell line by Lipofectamin2000 reagent. The inhibitory effect of B-RAF-siRNA and RelA-siRNA on B-RAF gene and RelA gene of gastric cancer BGC823 cell line was analyzed by RT-PCR and Western Blot; The influence of inhibition of B-RAF gene and RelA gene expression on the biological characteristics of gastric cancer BGC823 cell line was investigated with MTT, flow cytometry, RT-PCR, and Western Blot; IP with mass spectrometry analysis technique was used to study the unknown protein which binds to B-RAF protein; Secondly, we used GGTI-298 and FTI-277 to treat gastric cancer BGC823 cell line in order to detect the effect of both reagents on the expression of B-RAF gene by "Western Blot; Finally, we made use of biological information soft-ware to predict and verify the possible regulating sequence ofB-RAF gene by using CHIP with PCR technique.
Results
1. Screening the mutation ofB-RAF gene in gastric cancer samples
There is no mutation in exon 11 and exon 15 of B-RAF gene in 150 cases of samples of human gastric cancer tissues and paracancerous tissues.
2. Expression of B-RAF protein in gastric cancer tissues
Immunohistochemistry has shown that there are brown particles in gastric cell, mainly distributed in cytoplasm; the expression of B-RAF protein is positive, while there is no brown particle in normal gastric gland and B-RAF protein negative.
3. Influence of B-RAF-siRNA on the expression of B-RAF mRNA
The expression of B-RAF mRNA is significantly changed on day 5th and 7th after transfection of B-RAF-siRNA into gastric cancer BGC823 cell line. The expression of B-RAF mRNA is significantly decreased, compared to control group.
4. Influence of B-RAF-siRNA on the expression of B-RAF protein in gastric cancer BGC823 cell line.
day 9th after RNAi, the expression of B-RAF protein was clearly decreased, compared to control group.
5. Influence of B-RAF-siRNA on growth of gastric cancer BGC823 cell line
B-RAF-siRNA significantly inhibited proliferation of gastric cancer BGC823 cell line, especially 72 h after transfection, up to20%.
6. Influence of B-RAF-siRNA on cell apoptosis of gastric cancer BGC823 cell line
The rate of cell apoptosis of gastric cancer BGC823 cell line in B-RAF-siRNA group is 19.5%, clearly higher than control group. It means the inhibition of B-RAF gene could promote cell apoptosis.
7. B-RAF-siRNA inhibited the expression of BCL-2 mRNA in gastric cancer BGC823 cell line.
8. Influence of B-RAF-siRNA on invasiveness of gastric cancer BGC823 cell line
Invasiveness of gastric cancer BGC823 cell line was significantly decreased after transfection with B-RAF-siRNA
9. GGTI-298 and FTI-277 both clearly inhibited the expression of B-RAF protein of gastric cancer BGC823 cell line.
10. Influence of RelA-siRNA on expression of B-RAF protein in gastric cancer BGC823 cell line:
When RelA-siRNA effectively inhibited the expression of RelA gene, the expression of B-RAF protein was also significantly decreased.
11. The result of IP with mass spectrometry analysis
B-RAF protein in gastric cancer BGC823 cell may bind to PRO2619 protein.
12. Result of CHIP with PCR:
DNA fragment which was immunoprecipitated with NF-κB antibody contains NF-κB protein binding sequence of B-RAF promoter region.
Conclusion
1. The point mutation of B-RAF gene may not be the pathway of gastric carcinogenesis in which B-RAF gene participate.
2. The elevation of the expression of B-RAF gene may be closely related to gastric carcinogenesis.
3. B-RAF gene is an important oncogene related to gastric cancer, promote growth, proliferation and invasiveness and inhibit cell apoptosis of gastric cancer cells.
4. BCL-2 may be located in the down-stream of B-RAF gene and be regulated by it.
5. B-RAF gene is doublely regulated by upstream gene, RAP1 and RAS, in its signal transduction pathway.
6. GGTI-298 and FTI-277 could inhibit proliferation and promote cell apoptosis of gastric cancer cells.
7. NF-κB may be an important transcriptional factor which regulates B-RAF gene.
8. B-RAF protein may bind to PRO2619 protein in gastric cancer BGC823 cell
前言
对肿瘤(癌)发生机制和防治的研究，多年来一直是人们关注和研究的热点问题。肿瘤的发生和侵袭转移是涉及许多基因参与的、多途经和多步骤的发展过程。其中，癌基因控制机体细胞的生长和分化，重要的癌基因激活可引起肿瘤的发生。
癌基因B-RAF属于RAF基因家族，编码丝氨酸/苏氨酸激酶，是有丝分裂原激活蛋白激酶(mitogen-activated protein kinase，MAPK)信号转导通路中的重要成员之一。近年来的研究发现许多肿瘤中存在B-RAF基因突变，尤其是黑色素瘤和甲状腺癌中B-RAF基因突变率高达60～80％，突变主要集中于B-RAF基因的第11和第15外显子，这说明B-RAF基因与黑色素瘤和甲状腺癌有关。到目前为止国内外有关B-RAF基因在胃癌发生、发展中的机制研究尚未见报道。
我们在前期工作中利用基因芯片技术对胃癌发病不同阶段的基因表达进行了较为全面、动态的研究。我们发现B-RAF基因在癌旁、癌及淋巴结转移癌中表达与正常胃粘膜组织相比均有明显升高，提示B-RAF基因可能是胃癌发生过程中的一个重要基因。因此我们首先对150例人胃癌组织标本中B-RAF基因突变热点第11和第15外显子进行突变筛查，结果显示阴性；但同时的免疫组化以及Western Blot结果却显示胃癌组织中B-RAF蛋白表达阳性，而癌旁组织中无B-RAF蛋白表达，这说明B-RAF基因与胃癌有相关性，但B-RAF基因在胃癌中的作用机制可能不同于黑色素瘤和甲状腺癌。为了深入探讨B-RAF基因在胃癌发生、发展中的可能作用，阐明B-RAF基因及MAPK信号转导通路和胃癌的相关性及其机制，以及B-RAF基因的上游调控基因，我们首先应用RNA干扰技术抑制胃癌BGC823细胞中B-RAF基因的表达，分析B-RAF基因对胃癌BGC823细胞生物学特性的影响；其次利用免疫沉淀(IP)技术结合蛋白质的质谱分析研究B-RAF蛋白在胃癌细胞中发挥作用时可能与之结合的未知蛋白；最后应用GGTI-298和FTI-277分别抑制RAS和RAPl蛋白活性以及利用生物信息学软件预测并用染色质免疫沉淀(CHIP)结合PCR技术研究B-RAF基因的上游调控基因。本研究对阐明B-RAF基因及MAPK信号转导通路与胃癌相关性以及对B-RAF基因在胃癌发生中调控机制研究具有重要意义。
方法
应用PCR-DHPLC结合DNA测序技术筛查B-RAF基因点突变；免疫组化和Western Blot检测胃癌组织中B-RAF蛋白表达。针对B-RAF基因和RelA基因设计siRNA，体外转录合成B-RAF-siRNA和RelA-siRNA，利用脂质体转染试剂Lipofectamine 2000将其分别转染胃癌细胞系BGC823细胞。RT-PCR和Western Blot检测胃癌BGC823细胞中B-RAF基因和RelA基因的表达；MTT、流式细胞术、Transwell和RT-PCR以及Western Blot分别检测B-RAF基因和RelA基因表达抑制后对胃癌细胞系BGC823细胞生物学特性的影响以及分子机制；免疫沉淀技术结合蛋白质的质谱分析技术研究可能与B-RAF蛋白结合的未知蛋白；利用GGTI-298和FTI-277处理胃癌细胞系BGC823细胞，Western Blot检测二种抑制剂对胃癌细胞中B-RAF基因表达的影响；利用生物信息学转录因子预测软件预测可能与B-RAF基因上游启动子区特异序列结合的转录因子，并用染色质免疫沉淀技术结合PCR技术对生物信息学转录因子预测软件预测的结果进行验证。
结果
1．人胃癌组织中B-RAF基因突变筛查结果：
在150例人胃癌组织和癌旁组织标本中未筛查到B-RAF基因第11外显子和第15外显子存在突变。
2．人胃癌组织中B-RAF蛋白表达分析：
免疫组化结果显示在20例人胃癌组织标本中，胃癌细胞中可见棕色颗粒，主要分布于细胞质，B-RAY蛋白表达阳性，而正常胃腺细胞中未见棕色颗粒，B-RAF蛋白表达阴性。
3．B-RAF-siRNA对胃癌细胞中B-RAF mRNA表达的影响：
B-RAF-siRNA转染BGC823细胞第5和第7天时B-RAF mRNA表达水平可见到明显变化，B-RAF-siRNA组较对照组B-RAF mRNA表达水平明显降低。
4．B-RAF-siRNA对胃癌细胞中B-RAF蛋白表达的影响：
B-RAF-siRNA转染BGC823细胞第9天时B-RAF蛋白表达水平可见到明显变化，B-RAF-siRNA组较对照组B-RAF蛋白表达水平明显降低，表明B-RAF蛋白表达受到抑制。
5．B-RAF-siRNA对人胃癌BGC823细胞生长的影响
与细胞对照组相比，B-RAF-siRNA对体外培养的胃癌BGC823细胞的生长有明显抑制作用。其中，转染后72h抑制率达20％。
6．B-RAF-siRNA对胃癌细胞凋亡的影响
B-RAF-siRNA组胃癌BGC823细胞凋亡率为19.58％，B-RAF-siRNA组较对照组胃癌BGC823细胞凋亡率明显升高，说明B-RAF基因抑制能够引起胃癌BGC823细胞凋亡。
7．B-RAF-siRNA抑制人胃癌BGC823细胞中BCL-2 mRNA的表达。
8．B-RAF-siRNA对细胞侵袭力的影响
B-RAF-siRNA转染后第9天，细胞侵袭力明显降低。
9．RAP1抑制剂GGTI-298和RAS抑制剂FTI-277均明显抑制胃癌BGC823细胞中B-RAF mRNA和蛋白的表达。
10．RelA-siRNA对胃癌BGC823细胞中B-RAF蛋白表达的影响
RelA-siRNA有效地抑制RelA基因表达后，胃癌BGC823细胞中B-RAF蛋白表达水平也明显降低。
11．免疫沉淀技术结合质谱分析结果
胃癌BGC823细胞中B-RAF蛋白可能与PR02619蛋白结合。
12．染色质免疫沉淀技术结合PCR技术检测结果
在NF-κB抗体免疫沉淀的DNA片断中含有B-RAF基因的启动子区NF-κB蛋白的结合序列。
结论
1．B-RAF基因点突变可能不是B-RAF基因参与胃癌发生的途径。
2．B-RAF基因表达增高可能与胃癌发生密切相关。
3．B-RAF基因是与胃癌相关的重要癌基因，促进胃癌细胞的生长增殖、抑制细胞凋亡和增强胃癌细胞的侵袭力。
4．BCL-2基因在胃癌细胞中可能位于B-RAF基因的下游并受其调控。
5．B-RAF基因在其信号转导通路中受RAPI和RAS上游基因的双重调控；
6．RAP1和RAS的抑制剂GGTI-298与FTI-277可抑制胃癌细胞的增殖，促进胃癌细胞的凋亡；
7．NF-κB可能是调控B-RAF基因的重要转录因子
8．B-RAF蛋白在胃癌BGC823细胞中可能与PR02619蛋白结合。
Relationship between B-RAF gene and MAPK signal transduction pathway and gastric cancer and study of mechanism
Investigation of tumorigenesis mechanism, prevention, and therapy have already been hot point for many years. The tumorigenesis and metastasis are involved in developing process of many pathways and steps in which many genes participate, especially oncogenes control growth and differentiation of somatic cells, and activation of critical oncogenes can cause tumorigenesis.
Oncogene B-RAF which belongs to RAF gene family, encoding sefine/threonine kinase, is one of the important members of MAPK (mitogen-activated protein kinase) signal transduction pathway. Recent research found that the mutation of B-RAF gene exists in many tumors, especially melanoma and thyroid cancer, in which the rate of mutation in B-RAF gene is up to 60-80%, It indicates that B-RAF gene is related to tumors. Until now, there is no report about the mechanism of B-RAF gene in gastric carcinogenesis and development in the world.
Our previous work studied the expression of genes in different satges of gastric cancer by using gene chip. We found that the expression of B-RAF gene is significantly elevated in paracancerous tissue, cancer tissue and metastatic lymphode. Subsequently, we screened the mutation of exon11 and exon 15 of B-RAF gene in 150 samples of human gastric cancer tissue and result showed negative, but the result of simultaneous immunohistochemistry and Western Blot indicated that the expression of B-RAF protein was positive and suggest that B-RAF gene is related to gastric cancel but the functional mechanism of B-RAF in gatric cancer may be different in melanoma and thyroid cancer. To further research the possible function of B-RAF gene in gastric carcinogenesis and development, clarify the correlation between B-RAF gene and MAPK signal transduction pathway and study of mechanism and upstream regulating gene of B-RAF gene, we first utilized RNA interference to inhibit the expression of B-RAF gene in gastric cancer BGC823 cell line, analyzed the biological effect of B-RAF gene on BGC823 cell line before and after RNA interference; Secondly, we used IP(immunoprecipitation, IP) with mass spectrometry analysis technique to study unknown protein which may bind to B-RAF protein in gastric cancer cellsl; Finally, we employed GGTI-298 and FTI-277 to inhibit the activation of RAS and RAP1 protein, and biological information software to predict the transcriptional factors of regulating B-RAF gene and CHIP with PCR to investigate the upstream regulating genes of B-RAF gene. Our research is of significance to elucidate the correlation between B-RAF gene and gastric cancer, and explore its functional mechanism.
Methods
We used PCR-DHPLC with DNA sequencing to screen point mutation of B-RAF gene; Immunohistochemistry and Western Blot for the expression of B-RAF protein in gastric cancer. We designed siRNA specific for B-RAF gene and RelA gene and in vitro synthesize B-RAF-siRNA and RelA-siRNA, then was transfected into gastric cancer BGC823 cell line by Lipofectamin2000 reagent. The inhibitory effect of B-RAF-siRNA and RelA-siRNA on B-RAF gene and RelA gene of gastric cancer BGC823 cell line was analyzed by RT-PCR and Western Blot; The influence of inhibition of B-RAF gene and RelA gene expression on the biological characteristics of gastric cancer BGC823 cell line was investigated with MTT, flow cytometry, RT-PCR, and Western Blot; IP with mass spectrometry analysis technique was used to study the unknown protein which binds to B-RAF protein; Secondly, we used GGTI-298 and FTI-277 to treat gastric cancer BGC823 cell line in order to detect the effect of both reagents on the expression of B-RAF gene by "Western Blot; Finally, we made use of biological information soft-ware to predict and verify the possible regulating sequence ofB-RAF gene by using CHIP with PCR technique.
Results
1. Screening the mutation ofB-RAF gene in gastric cancer samples
There is no mutation in exon 11 and exon 15 of B-RAF gene in 150 cases of samples of human gastric cancer tissues and paracancerous tissues.
2. Expression of B-RAF protein in gastric cancer tissues
Immunohistochemistry has shown that there are brown particles in gastric cell, mainly distributed in cytoplasm; the expression of B-RAF protein is positive, while there is no brown particle in normal gastric gland and B-RAF protein negative.
3. Influence of B-RAF-siRNA on the expression of B-RAF mRNA
The expression of B-RAF mRNA is significantly changed on day 5th and 7th after transfection of B-RAF-siRNA into gastric cancer BGC823 cell line. The expression of B-RAF mRNA is significantly decreased, compared to control group.
4. Influence of B-RAF-siRNA on the expression of B-RAF protein in gastric cancer BGC823 cell line.
day 9th after RNAi, the expression of B-RAF protein was clearly decreased, compared to control group.
5. Influence of B-RAF-siRNA on growth of gastric cancer BGC823 cell line
B-RAF-siRNA significantly inhibited proliferation of gastric cancer BGC823 cell line, especially 72 h after transfection, up to20%.
6. Influence of B-RAF-siRNA on cell apoptosis of gastric cancer BGC823 cell line
The rate of cell apoptosis of gastric cancer BGC823 cell line in B-RAF-siRNA group is 19.5%, clearly higher than control group. It means the inhibition of B-RAF gene could promote cell apoptosis.
7. B-RAF-siRNA inhibited the expression of BCL-2 mRNA in gastric cancer BGC823 cell line.
8. Influence of B-RAF-siRNA on invasiveness of gastric cancer BGC823 cell line
Invasiveness of gastric cancer BGC823 cell line was significantly decreased after transfection with B-RAF-siRNA
9. GGTI-298 and FTI-277 both clearly inhibited the expression of B-RAF protein of gastric cancer BGC823 cell line.
10. Influence of RelA-siRNA on expression of B-RAF protein in gastric cancer BGC823 cell line:
When RelA-siRNA effectively inhibited the expression of RelA gene, the expression of B-RAF protein was also significantly decreased.
11. The result of IP with mass spectrometry analysis
B-RAF protein in gastric cancer BGC823 cell may bind to PRO2619 protein.
12. Result of CHIP with PCR:
DNA fragment which was immunoprecipitated with NF-κB antibody contains NF-κB protein binding sequence of B-RAF promoter region.
Conclusion
1. The point mutation of B-RAF gene may not be the pathway of gastric carcinogenesis in which B-RAF gene participate.
2. The elevation of the expression of B-RAF gene may be closely related to gastric carcinogenesis.
3. B-RAF gene is an important oncogene related to gastric cancer, promote growth, proliferation and invasiveness and inhibit cell apoptosis of gastric cancer cells.
4. BCL-2 may be located in the down-stream of B-RAF gene and be regulated by it.
5. B-RAF gene is doublely regulated by upstream gene, RAP1 and RAS, in its signal transduction pathway.
6. GGTI-298 and FTI-277 could inhibit proliferation and promote cell apoptosis of gastric cancer cells.
7. NF-κB may be an important transcriptional factor which regulates B-RAF gene.
8. B-RAF protein may bind to PRO2619 protein in gastric cancer BGC823 cell
引文
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26. Kim J, Giuliano AE, Turner RR, Lymphatic mapping establishes the role of BRAF gene mutation in papillary thyroid carcinoma. Ann Surg. 2006, 244(5):799-804.
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29. Elbashir SM, Harborth J, Weber K, Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 2002, 26:199-213.
30. Mitra RS, Zhang Z, Henson BS Rap1A and rap1B ras-family proteins are prominently expressed in the nucleus of squamous carcinomas: nuclear translocation of GTP-bound active form. Oncogene. 2003, 22(40):6243-56.
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35. Scheidereit C IkappaB kinase complexes: gateways to NF-kappaB activation and transcription.Oncogene. 2006, 25(51):6685-705.
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29. Elbashir SM, Harborth J, Weber K, Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 2002, 26:199-213.
30. Mitra RS, Zhang Z, Henson BS Rap1A and rap1B ras-family proteins are prominently expressed in the nucleus of squamous carcinomas: nuclear translocation of GTP-bound active form. Oncogene. 2003, 22(40):6243-56.
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