| Survivin is a new member of the inhibitior of apoptosis protein (IAP) family. It possesses specific biological effects, such as, depressing apoptosis, regulating cell cycle and progressing angiogenesis. The expression of survivin is always on a high level in tumor tissues and can be detected at the early stage of tumorigenesis. The overexpression of survivin results in the inhibition of apoptosis, the progression of tumor angiogenesis and the invasion of tumor towards other tissues. And, survivin appears to be involved in the resistance of tumor tissues towards radiotherapy and chemical drugs. Accordingly, survivin is becoming a helpful potential target for tumor treatment. In this project, RNAi is used to interfer the expression of survivin, in order to investigate its biological effect and to explore the biological mechanism of survivin on apoptosis and chromosome instability in HeLa cells induced by X-rays. Results of the study will be helpful to elucidate the influence of RNAi on survivin radiobiological effect.1. Survivin expression in cells from different tissuesThe expression of survivin was detected by FCM in six sorts of human carcinoma from different tissues, such as, HeLa, HCT-8, SKOV-3, K562, SGC-7901, B16 and in one sort of cell from human normal hepatogenic tissue, HL-7702. Results showed that the expression of survivin was on the high level in six sorts of tumor cells, respectively, under the standard culture conditions (P<0.05~P<0.001). However, the expression of survivin could not be detected in HL-7702 cells (P>0.05).2. Effects of ionizing radiation on the expression of survivin and its biological effect2.1 Effects of ionizing radiation on the expression of survivinFCM was applied to detect the change of survivin expression in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the expression of survivin in 6.0 Gy groups increased significantly, compared with sham-irradiated groups (P>0.05).FCM was applied to detect the change of survivin expression in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that the expression of survivin was on a higher level at 4 h, 8 h, 48 h and 72 h, and they were significant difference with the control groups (P<0.01~ P<0.001).2.2 Effects of ionizing radiation on mRNA level of survivinRT-PCR assay was employed for the measurement of mRNA in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that survivin mRNA tended to increase in 1.0~6.0 Gy groups compared with the control groups.RT-PCR assay was employed for the measurement of mRNA in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that survivin mRNA increased at 8 h, 12 h and 24 h after 4.0 Gy irradiated.2.3 Effects of ionizing radiation on apoptosis of HeLa cellsFCM was applied to detect the change of apoptosis in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the number of apoptosis in 2.0 Gy groups declined significantly, compared with the sham-irradiated groups (P<0.05). However, the number of apoptosis had no significant difference between the irradiation groups with other doses and the sham-irradiated groups (P>0.05). It indicated that 0.5~6.0 Gy X-rays could not induced apparently the increase of apoptosis in HeLa cells.FCM was applied to detect the change of the number of apoptosis in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that it was no significant difference between the irradiation groups and the control groups at different observed time (P>0.05).2.4 Effects of ionizing radiation on the distribution of cell cycle in HeLa cellsFCM was applied to analyze the distribution of cell cycle in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the percentage in G2+M phase was increased significantly in HeLa cells exposed to 2.0 Gy, 4.0 Gy and 6.0 Gy, which was significant different with the sham-irradiated groups (P<0.001, respectively).FCM was applied to analyze the distribution of cell cycle in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that the percentage in G2+M phase rose from12 h to 72 h. The increase was sharper than that of the control groups (P<0.05~ P<0.001).2.5 Effects of ionizing radiation on the number of chromosome of HeLa cellsFCM was applied to detected the changes of the number of chromosome in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the percentage of tetraploid cells was increased significantly in HeLa cells exposed to 2.0 Gy, 4.0 Gy and 6.0 Gy, compared with the sham-irradiated groups (P<0.01~P<0.001), and the percentage of octoploid cells in these groups was also more than those of the sham-irradiated groups (P<0.05~P<0.01).FCM was applied to detected the number of chromosome in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that the percentage of tetraploid cells remained the increase in HeLa cells during 2 h~48 h. They were significant difference with the control groups (P<0.05~ P<0.001). And, the percentage of octoploid cells was also significantly increased at 4 h, 24 h, 48 h and 72 h (P<0.01~P<0.001).3. Effects of shRNA on the expression of survivin and the biological effects3.1 Construction of the RNAi vectorSucceed to construct the RNAi vector, pENTR-anti-SVV, which was the recombinant plasmid including an insert fragment of shRNA. To identify the sequence veracity of shRNA, the plasmid was treated by sequencing process. Results indicated that the sequence of shRNA was consistent with what was designed.3.2 The efficiency of transfection in HeLa cells transfected with shRNAUnder the same transfected conditions, FCM was used to examine the intensity of green fluorescence in HeLa cells transfected with pcDNA3.1-GFP which acted as the control vector, in order to elevate the efficiency of transfection of pENTR-anti-SVV indirectly. It was showed that the efficiency was up to 64.64% at 48 h.3.3 Effects of shRNA on mRNA level of survivin in HeLa cells FCM was applied to measure the change of survivin on mRNA level in HeLa cells at 24 h after 4.0 Gy X-irradiation or/and transfected with pENTR-anti-SVV for 48 h. Results showed that mRNA was depressed apparently in the groups of transfected with pENTR-anti-SVV, while there was special product in the other control groups, such as, the untreated groups, irradiated groups, irradiated groups transfected with pENTR-basic.3.4 Effects of shRNA on the expression of survivin in HeLa cellsFCM was applied to detect the time course of changes of survivin expression in HeLa cells transfected with pENTR-anti-SVV for 48 h and exposed to 4.0 Gy X-irradiation. Results showed that the treated groups transfected with pENTR-anti-SVV had the lower level on the expression of survivin at 12 h, 24 h and 48 h, compared with the control groups transfected with pENTR-basic (P<0.001, respectively).3.5 Effects of shRNA on apoptosis in HeLa cellsFCM was applied to detect the percentage of apoptosis in HeLa cells transfected with pENTR-anti-SVV for 48 h and exposed to 4.0 Gy X-irradiation. Results showed that the treated groups transfected with pENTR-anti-SVV showed an apparently increase in apoptosis spanning 12 h to 24 h (P<0.05 and P<0.001, respectively). It suggested that the interference of survivin expression could induce the incident of apoptosis in HeLa cells.3.6 Effects of shRNA on the distribution of cell cycle in HeLa cellsFCM was applied to analyze the distribution of cell cycle in HeLa cells transfected with pENTR-anti-SVV for 48 h and exposed to 4.0 Gy X-irradiation. Results showed that the percentage in G2+M phase of the treated groups transfected with pENTR-anti-SVV were increased at 12 h and 48 h, which was significant different with the the control groups transfected with pENTR-basic (P<0.05 and P<0.01, respectively).3.7 Effects of shRNA on the number of the chromosome in HeLa cellsFCM was applied to detect the change of the number of chromosome in HeLa cells transfected with pENTR-anti-SVV for 48 h and exposed to 4.0 Gy X-irradiation. Results showed that the percentage of tetraploid cells was increased in the treated groups transfected with pENTR-anti-SVV at 24 h and 24 h. They were significant difference with the control groups transfected with pENTR-basic (P<0.01 and P<0.05, respectively). And, the percentage of octoploid cells in the treated groups was also significantly increased at 12 h, 24 h and 48 h (P<0.01~P<0.001).Results of the project will provide the possible evidence for the further research on survivin biological effects.
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