| ObjectivePrevious work demonstratesd that overexpression of Rap2 B was generated in lung cacer tissues.Its overexpression can active NF-κB pathway as well as p38 mitogen-activated protein kinase. The purpose of this study was to detect the expression of NF-κB and p38 in the level of m RNA and protein, the colony forming efficiency, the ability of proliferation and migtation, as well as the assay of cell cycle and apoptosis to explore the role of Rap2 B in lung cancer by RNA interference. This study aims to confirm whether Rap2 B may serve as an efficient and specific biomarker for lung cancer. Methods1. B(a)P was used as a test substance to induce the malignant transformation model of BEAS-2B cells in vitro.Three groups were divided as control group, Dimethyl Sulfoxide(DMSO) group, and Benzo(a) pyrene, B(a)P group. The transcription and protein level were measured by RT-PCR and Western blot respectively.2. First: si RNA transfects the malignant transformed cell of BEAS-2B. Second:to screen the most appropriate sequence of one silent Rap2 B gene from three si RNA sequence. Last: Using RT-PCR to test the consequence.Taking an observation of the expression of p65 and p38 in the m RNA and protein level and the cell colony forming efficiency, proliferation ability, migration ability, the change of apoptosis and cell cycle after the interference of the Rap2 B. Results1. The establishment of malignant transformation model of BEAS-2B cellsThe results of cell morphological observation and colony formation experiment indicate the malignant transformation of BEAS-2B cells.2. The expression of Rap2 B in malignant transformated cell BEAS-2B.The m RNA expression of Rap2 B gene in B(a)P group was higher than control group and DMSO group, the difference was statistically significant(P<0.001); the protein expression of Rap2 B in B(a)P group was higher than control group and DMSO group, the difference was statistically significant(P<0.001).3. The test on the efficiency of cell transfection and the interference of Rap2 B gene. 6 hours after cell transfection, fluorescence microscope and flow cytometry showed that the transfection efficiency was 90.39%; the optimal si RNA of interfering Rap2 B gene was Rap2b-homo-814, the interfering efficiency was 82.3%.4. After Rap2 B gene interference, the expression of p38 lightning and p65 gene and protein. Compared RNAi- group with NC group, the differences of m RNA expression level of p38 and p65 gene had no statistical significance.(P=0.612,P=0.408).However,the m RNA expression quantity of p38 and p65 gene in RNAi+ group were significantly lower than the RNAi- and NC groups, the differences were statistically significant.(P﹤0.001); Compared RNAi- group with NC group,the differences of protein expression level of p38 and p65 gene had no statistical significance(P=0.158,P=0.111).However, the protein expression quantity of p38 and p65 gene in RNAi+ group were significantly lower than the RNAi- and NC groups, the differences were statistically significant(P﹤0.001).5. The cells’ proliferation and migration ability change after interfered by Rap2 B gene.MTT method was to observe the growth of groups of cells after transfection, and interfering Rap2 B gene can significantly inhibit the growth of malignant transformation of BEAS- 2B cells,then compared with RNAi- group and NC group, the differences was statistically significant(P ﹤ 0.001).Scratch experiment tested Rap2 B gene which was interferenced after malignant transformation BEAS- 2B cell migration ability, and there was no statistically significant difference on average gap between RNAi- group and NC group(P=0.739), and average gap of NAi + group compared with RNAi- group,and the NC group the differences were statistically significant(P﹤0.001).6. The changing of Cell apoptosis and cell cycle after Rap2 B gene interfered Compared with RNAi– group and NC group, cells apoptosis rate significantly increased within RNAi + group, then the difference has statistical significance(P﹤0.001), while apoptosis rate differences of RNAi- group and NC group has no statistical significance(P=0.226);RNAi+ group G1 phase cells, percentage increase significantly contrasting RNAi– group and NC group, then the difference has statistical significance(P﹤0.001). the percentage of S phase cells significantly decreased compared with RNAi- group, the NC group, the difference has statistical significance(P﹤0.001), G2 / M phase cells percentage compared with RNAi-group, the NC group significantly decreased, the difference has statistical significance(P﹤0.001), and RNAi- and NC groups from one period to the cell percentage than there was no statistically significant difference(P=0.820, P=0.923, P=0.791). ConclusionIt can make relative expression of p38, p65 gene and protein decrease as Rap2 B gene interfered, thus inhibiting the activity of the NF-κB signaling pathway and MAPK pathway, and then reducing cells proliferation and migration ability, promoting apoptosis. This block the cells in G0 / G1 phase, making the cell division mitigate significantly. |
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