Influence Of RNAi On Livin Radiobiological Effects

Livin is a new member of the inhibitior of apoptosis protein (IAPs) family. It possesses specific biological effects, such as, depressing apoptosis and regulating cell cycle. The expression of livin is always on a high level in tumor tissues and can be detected at the early stage of tumorigenesis. The overexpression of livin results in the inhibition of apoptosis. And, livin appears to be involved in the resistance of tumor tissues towards radiotherapy and chemical drugs. Accordingly, livin is becoming a helpful potential target for tumor treatment. In the present study, RNAi is used to interfere the expression of livin, in order to investigate its biological effect and to explore the biological mechanism of livin on apoptosis in HeLa cells induced by X-rays. Results of the study will be helpful to elucidate the influence of RNAi on livin radiobiological effect.1. Effects of X-rays on the protein expression of livin1.1 Dose-effect of X-rays on livin protein expression in HeLa cellsWestern Blot was applied to detect the change of livin expression in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the protein expression of livin in 1.0 Gy,2.0 Gy,4.0 Gy groups increased, compared with sham-irradiated groups.1.2 Time course changes of livin protein expression in HeLa cells exposed to 4.0 Gy X-raysWestern Blot was applied to detect the change of livin expression in HeLa cells during 0~48 h exposed to 4.0 Gy X-rays. Results showed that the protein expression of livin was on a higher level at 12 h, 24 h, and they were difference with the control groups .2. Effects of X-rays on mRNA level of livin 2.1 Dose-effect of X-rays on livin mRNA level in HeLa cellsRT-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 livin mRNA tended to increase in 0.5~4.0 Gy groups compared with the control groups.2.2 Time course changes of livin mRNA level in HeLa cells exposed to 4.0 Gy X-raysRT-PCR assay was employed for the measurement of mRNA in HeLa cells during 12~24 h exposed to 4.0 Gy X-rays. Results showed that livin mRNA increased at 12 h after 4.0 Gy irradiated.3. Effects of X-rays on the distribution of cell cycle in HeLa cells3.1 Dose-effect of X-rays on the distribution of cell cycle in HeLa cells FCM 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 1.0 Gy 2.0 Gy, 4.0 Gy and 6.0 Gy, which was significant different with the sham-irradiated groups (P<0.001, respectively).3.2 Time course changes of the distribution of cell cycle in HeLa cells exposed to 4.0 Gy X-raysFCM was applied to analyze the distribution of cell cycle in HeLa cells during 0~48 h exposed to 4.0 Gy X-rays. Results showed that the percentage in G2/M phase rose from 4 h, 8 h, 12 h, 24 h, 48 h. The increase was sharper than that of the control groups (P<0.001, respectively).4. Effects of X-rays on apoptosis of HeLa cells4.1 Dose-effect of X-rays on apoptosis in 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 had no significant difference between the irradiation groups with 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.4.2 Time course changes of apoptosis in HeLa cells exposed to 4.0 Gy X-raysFCM was applied to detect the change of the number of apoptosis in HeLa cells during 0~48 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).5. Construction and identification of the recombinant plasmid5.1 Construction of the RNAi vectorSucceed to construct the RNAi vector, pSilencer3.1-H1 neo-livin, which was the recombinant plasmid including an insert fragment of shRNA.5.2 Identification of the recombinant plasmidTo identify the sequence veracity of shRNA, the plasmid was treated by cleavage of endonucleases, PCR and sequencing process. Results indicated that the sequence of shRNA was consistent with what was designed.6. Effects of shRNA on mRNA level of livin in HeLa cellsFCM was applied to measure the change of livin on mRNA level in HeLa cells stably transfected with pSilencer3.1-H1 neo-livin and exposed to 4.0 Gy X-irradiation. Results showed that mRNA was depressed apparently in the groups of transfected with pSilencer3.1-H1 neo-livin at 12h, 24 h, 48h after 4.0 Gy X-irradiation, while there was special product in the other control groups, such as, the untreated groups, irradiated groups, irradiated groups transfected with pSilencer3.1-H1 neo.7. Effects of shRNA on the protein expression of livin in HeLa cellsWestern blot was applied to detect the time course of changes of livin protein expression in HeLa cells stably transfected with pSilencer3.1-H1 neo-livin and exposed to 4.0 Gy X-irradiation. Results showed that the treated groups transfected with pSilencer3.1-H1 neo-livin had the lower level on the protein expression of livin at 12 h, 24 h and 48 h, compared with the control groups transfected with pSilencer3.1-H1 neo. 8. Effects of shRNA on the distribution of cell cycle in HeLa cellsFCM was applied to analyze the distribution of cell cycle in HeLa cells stably transfected with pSilencer3.1-H1 neo-livin and exposed to 4.0 Gy X-irradiation. Results showed that the percentage in G2/M phase of the treated groups stably transfected with pSilencer3.1-H1 neo-livin were increased at 12 h, which was significant different with the the control groups transfected with pSilencer3.1-H1 neo (P<0.05, respectively).9. Effects of shRNA on apoptosis in HeLa cellsFCM was applied to detect the percentage of apoptosis in HeLa cells stably transfected with pSilencer3.1-H1 neo-livin and exposed to 4.0 Gy X-irradiation. Results showed that the treated groups stably transfected with pSilencer3.1-H1 neo-livin showed an apparently increase in apoptosis spanning 12h, 24h, 48h (P<0.001, respectively). It suggested that the interference of livin expression could induce the incident of apoptosis in HeLa cells.Results of the project will provide the possible evidence for the further research on livin biological effects.