| ObjectiveGastric cancer is one of the most frequent malignancies in china. Its occurrence, development, invasion and metastasis are composed of multi-steps, involving multiple genes and factors. Although a large number of investigation, their molecular mechanisms are still not clear. S100A4, a small calcium binding protein, plays a key role in the process of metastasis via decreasing the adhesive ability of tumor cells promoting the hydrolysis of extra-cellular matrix, the remodeling of the matrix, mobility of tumor cells, angiogenesis of the tumors etc. Our previous works and other reports have demonstrated the close correlation between S100A4 gene and gastric cancer infiltration, lymph node metastasis and in vitro invasiveness of gastric cancer cells. But the function of S100A4 gene and its regulation in gastric cancer are still to be disclosed. We apply RNA interference technology to inhibit the expression of S100A4 gene in gastric cancer cell line BGC823 cell, analyzing the change of cell apoptosis, proliferation and so on as well as its molecular mechanism. Recently it is widely concerned for the correlation of tumor's microenviroment and its invasiveness and metastasis, especially microenviromental hypoxia of tumor, because it could increase genomic instability and heterogeneity and regulate gene expression via epigenetics. Our previous research has predicted the core sequence of HIF response element in the first intron of S100A4 gene by using bioinformatic software. So we speculate that microenviromental hypoxia may up-regulate the expression of S100A4. Therefore, we treat gastric cancer cell line BGC823 cell with hypoxia and observe its influence on expression of S100A4 gene. Our research will be not only useful for clarifying the molecular mechanism of gastric carcinogenesis and development, also of significance for diagnosis and therapy of gastric cancer.Materials and methods1. Materials: Gastric cancer cell line BGC823, TRIZOL Reagent, antibody of S100A4/NF-κB,Taq DNA polymerase, Reverse Transcription System, pSilencerTM4.1-CMV neo vector was brought from Ambion.2. Methods: derived from low differential adenoma was cultured in RPMI 1640 medium containing 10% fetal boving serum, 100U/ml of penicillin, and 100μg of streptomycin in a humidified atmosphere of 5% CO2 at 37℃S100A4 -specific siRNA was designed according to software of Ambion.iS100A4-specific shRNA expression vector was constructed by using recombinant DNA technology and transfected into gastric cancer cell line BGC823 cell. The expression of S100A4 and related genes were analyzed by RT-PCR and Western Blot; Cell cycle and apoptosis were detected by flow cytometry; Cell proliferation was detected by MTT.Results1. The influence of RNAi on expression of S100A4 gene in gastric cancer cell BGC823 cell: After transfection of S100A4-shRNA into BGC823 cell, the expression of S100A4 gene was significantly inhibited as detected by RT-PCR and Western Blot;2. The influence of RNAi on gastric cancer cell apoptosis and proliferation and possible molecular mechanism: The inhibition of expression of S100A4 gene promotes apoptosis, induces the arrest of cell cycle in G1 phase, decrease proliferation ability of gastric cancer cell BGC823 and simultaneously down-regulate the expression of NF-κB, BCL-2 and so on.3. The influence of hypoxia inducer CoCl2 on the expression of S100A4 gene in gastric cancer cell BGC823 cell: After treatment with CoCl2, the expression of S100A4 mRNA and protein in BGC823 cell was clearly increased as detected by RT-PCR and Western Blot.Conclusion1. S100A4-specific shRNA expression vector may efficiently inhibit the expression after transfected into gastric cancer.2. S100A4-specific shRNA increase apoptosis, decrease proliferation ability, induces the arrest of cell cycle in G1 phase, and simultaneously down-regulate the expression of NF-κB, BCL-2 gene and so on in gastric cancer cell.3. Microenvironmental hypoxia can up regulate the expression of S100A4.
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