| As a member of Cip/Kip family,p21Waf1/Cip1 is a cell cycle inhibition protein which is now found with the most extensive kinase activity. p21 gene is highly conserved and plays an important role in cell cycle regulation, cell differentiation and apoptosis. For the interaction with several factors in gene and protein level, p21 gene is important in complex control network and the role in tumor development cannot be ignored. In this paper, RNAi was used to silence the expression of p21 gene, in order to investigate its biological effect on cell cycle arrest, uncoupling and apoptosis. Results of the study will provide theoretical and experimental basis for clinical tumor treatment.1. Effects of X-rays on the expression of p21 in EL-4 and CHO cellsFCM was applied to detect the time-course and dose-effect changes of p21 expression in EL-4 cells and CHO cells. Results of time-course experiments showed that during 0-72 h exposed to 4.0 Gy X-rays, the expression of p21 in EL-4 cells increased significantly at 8 h, and reached peak at 24 h, then kept the increase until 72 h, compared with sham-irradiated groups (P<0.01-P<0.001). While the expression of p21 in CHO cells began to increase at 12 h, and reached peak at 48 h, then began to decrease at 72 h (P<0.01). Results of dose-effect experiments showed that, in EL-4 cells exposed to X-rays with the doses of 1.0~4.0 Gy, the expression of p21 increased significantly, compared with the sham-irradiated groups (P<0.05-P<0.01). In CHO cells exposed to X-rays with doses of 1.0-6.0 Gy, the expression of p21 increased significantly, compared with the sham-irradiated groups (P<0.05-P<0.01). For both of EL-4 cells and CHO cells, the increase of expression of p21 is most significant when exposed to X-rays with the dose of 4.0 Gy.2. Effects of X-rays on p21 mRNA level in EL-4 and CHO cellsRT-PCR assay was employed for the measurement of p21 mRNA in EL-4 and CHO cells exposed to X-rays. Results of the time-course experiments showed that, during 0-24 h exposed to 4.0 Gy X-rays, the level of p21 mRNA in EL-4 cells was higher than the control groups, especially at 4 h. During 0-48 h exposed to 4.0 Gy X-rays, the level of p21 mRNA in CHO cells was higher than the control groups, especially at 8 h. Results of dose-effect experiments showed that, when exposed to X-rays with the doses of 0.5-6.0 Gy for 4 h, the level of p21 mRNA both in EL-4 and CHO cells increased, compared with the control groups. The highest level of p21 mRNA in EL-4 cells appeared at the dose of 4.0 Gy and in CHO cells at the dose of 2.0 Gy.3. Effects of X-rays on distribution of cell cycle in EL-4 and CHO cellsFCM was applied to analyze the distribution of cell cycle in EL-4 and CHO cells exposed to X-rays. Results of time-course experiments showed that, in EL-4 cells exposed to 4.0 Gy X-rays, the percentage in G0/G1 phase increased significantly at 8 h, 24 h, 48 h and 72 h, which was significantly different with the sham-irradiated groups (P<0.05). The percentage in S phase decreased significantly during 8-48 h, and the percentage in G2/M phase increased significantly at 8 h (P<0.05-P<0.01). In CHO cells exposed to 4.0 Gy X-rays, the percentage in G0/G1 phase decreased significantly at 4 h, 8 h, 12 h, 24 h and 72 h (P<0.01-P<0.001), the percentage in S phase increased significantly at 8 h and 12 h (P<0.01-P<0.001). The percentage of G2/M phase increased significantly during 8-24 h (P<0.01-P<0.001). Results of dose-effect experiments showed that, In EL-4 cells exposed to X-rays after 24 h, the percentage in G0/G1 phase increased significantly when exposed to 1.0-6.0 Gy X-rays (P<0.01-P<0.001). The percentage in S phase decreased significantly when exposed to 2.0-6.0 Gy X-rays (P<0.01-P<0.001). The percentage in G2/M phase increased significantly when exposed to 6.0 Gy X-rays (P<0.01-P<0.001). In CHO cells exposed to 4.0 and 6.0 Gy X-rays after 24 h, the percentage in S phase decreased significantly (P<0.01-P<0.001) and that in G2/M phase increased significantly (P<0.01-P<0.001).4. Effects of X-rays on the number of chromosome in EL-4 and CHO cellsFCM was applied to detecte the number of chromosome in EL-4 and CHO cells exposed to X-rays. Results of time-course experiments showed that, in EL-4 cells, the percentage of diploid cells increased significantly during 8-48 h (P<0.01) and the percentage of tetraploid cells decreased significantly at 2 h and 8 h (P<0.05). The percentage of octoploid cells decreased significantly at 4 h and 24 h (P<0.05-P<0.01). In CHO cells, the percentage of diploid cells decreased significantly at 8 h, 12 h and 72 h (P<0.05-P<0.001) and the percentage of tetraploid cells increased significantly at 4 h, 8 h, 12 h and 48 h (P<0.05-P<0.001). The percentage of octoploid cells increased significantly at 12 h (P<0.001). Results of dose-effect experiments showed that, in EL-4 cells exposed to 0.5 Gy, 2.0 Gy and 4.0 Gy X-rays, the percentage of diploid cells increased significantly (P<0.05-P<0.01), and the percentage of tetraploid cells decreased significantly (P<0.05-P<0.001). In CHO cells exposed to 2.0-6.0 Gy X-rays, the percentage of diploid cells decreased significantly (P<0.05-P<0.001), and the percentage of tetraploid cells increased significantly (P<0.01-P<0.001).5. Effects of X-rays on apoptosis in EL-4 and CHO cellsFCM was applied to detect the percentage of apoptosis in EL-4 and CHO cells exposed to X-rays. Results of time-course experiments showed that, exposed to 4.0 Gy X-ray, the percentage of apoptosis in EL-4 cells during 2-72 h and in CHO cells during 24-72 h both increased significantly (P<0.05-P<0.01). Results of dose-effect experiments showed that, in EL-4 cells exposed to 1.0 Gy and 4.0 Gy X-rays after 24 h, the percentage of apoptosis increased significantly (P<0.01). In CHO cells exposed to 4.0 Gy and 6.0 Gy X-rays after 24 h, the percentage of apoptosis increased significantly (P<0.05-P<0.01).6. Construction and identification of RNA interference plasmid pSilencer3.1-H1 neo-p21An oligonuclear hairpin sequence targeting p21 gene was designed by internet tool. The fragments were chemically synthesized and inserted into the plasmid pSilencer3.1-H1 neo forming recombinant plasmid pSilencer3.1-H1 neo-p21. E.colicells which was transformed with the recombinant plasmid were screened by ampicillin resistance. The recombinant plasmid pSilencer3.1-H1 neo-p21 prepared from positive clones was identified as correct by sequencing.7. Effects of siRNA on expression of p21 in EL-4 and CHO cellsEL-4 and CHO cells were transfected with the interference plasmid pSilencer3.1-H1 neo-p21. At 48 h after transfection, the cells were exposed to 4.0 Gy X-rays. FCM was applied to detect the changes of p21 expression at 24 h and 48 h after X-irradiation. It was showed that, compared with the control, the expression of p21 in cells transfected with pSilencer3.1-H1 neo-p21 was inhibited significantly (P<0.01-P<0.001). The results demonstrated that the siRNA selected was able to specifically inhibit the p21 expression effectively.8. Effects of siRNA interference on the transcription of p21 mRNA in EL-4 and CHO cellsEL-4 and CHO cells were transfected with pSilencer3.1-H1 neo-p21. At 48 h after transfection, the cells were exposed to 4.0 Gy X-rays. FCM was applied to detect the level of p21 mRNA at 4 h after X-irradiation. It was showed that, compared with the control, the level of p21 mRNA in cells of the transfection group decreased significantly. It demonstrated that siRNA selected was able to inhibit the transcription of p21 gene effectively.9. Effects of siRNA interference on the distribution of cell cycle in EL-4 and CHO cellsEL-4 and CHO cells were transfected with pSilencer3.1-H1 neo-p21. At 48 h after transfection, the cells were exposed to 4.0 Gy X-rays. FCM was applied to analyze the distribution of cell cycle at 24 h and 48 h after X-irradiation. It was showed that, the distribution of cell cycle in EL-4 cells transfected with pSilencer 3.1-H1 neo-p21 had no apparent difference with the control. In CHO cells transfected with pSilencer 3.1-H1 neo-p21, the percentage in S phase increased and that in G2/M phase decreased significantly (P<0.05).10. Effects of siRNA interference on the number of the chromosome in EL-4 and CHO cellsEL-4 and CHO cells were transfected with pSilencer3.1-H1 neo-p21. At 48 h after transfection, the cells were exposed to 4.0 Gy X-rays. FCM was applied to detecte the changes of the number of chromosome at 24 h and 48 h after X-irradiation. It was showed that, compared with the controls, at 24 h after X-irradiation, both in EL-4 and CHO cells of the transfection groups, the percentage of diploid cells decreased significantly and the percentage of octoploid cells increased significantly (P<0.05-P<0.01). At 48 h after X-irradiation, in EL-4 cells of transfection groups, the percentage of octoploid cells increased significantly. In CHO cells of transfection groups, the percentage of diploid cells decreased significantly and that of octoploid cells increased significantly (P<0.05-P<0.01).11. Effects of siRNA interference on apoptosis in EL-4 and CHO cellsEL-4 and CHO cells were transfected with pSilencer3.1-H1 neo-p21. At 48 h after transfection, the cells were exposed to 4.0 Gy X-rays. FCM was applied to detect the changes of the number of apoptosis at 24 h and 48 h after X-irradiation. It was showed that, compared with the controls, the percentage of apoptosis in EL-4 and CHO cells transfected with pSilencer3.1-H1 neo-p21 increased significantly (P<0.05-P<0.01).
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