Relationship Between B-RAF Gene And MAPK Signal Transduction Pathway And Gastric Cancer And Study Of Mechanism

Relationship between B-RAF gene and MAPK signal transduction pathway and gastric cancer and study of mechanismInvestigation 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.MethodsWe 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 samplesThere 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 tissuesImmunohistochemistry 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 mRNAThe 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 lineB-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 lineThe 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 lineInvasiveness of gastric cancer BGC823 cell line was significantly decreased after transfection with B-RAF-siRNA9. 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 analysisB-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.Conclusion1. 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...