| 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 isalways on a high level in tumor tissues and can be detected at the early stage oftumorigenesis. The overexpression of survivin results in the inhibition ofapoptosis, the progression of tumor angiogenesis and the invasion of tumortowards other tissues. And, survivin appears to be involved in the resistance oftumor tissues towards radiotherapy and chemical drugs. Accordingly, survivin isbecoming a helpful potential target for tumor treatment. In this project, RNAi isused to interfer the expression of survivin, in order to investigate its biologicaleffect and to explore the biological mechanism of survivin on apoptosis andchromosome instability in HeLa cells induced by X-rays. Results of the studywill be helpful to elucidate the influence of RNAi on survivin radiobiologicaleffect.1. Survivin expression in cells from different tissuesThe expression of survivin was detected by FCM in six sorts of humancarcinoma 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 levelin 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 inHL-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 survivin2.1.1 Dose-effect of X-rays on survivin expression in HeLa cellsFCM was applied to detect the change of survivin expression in HeLa cellsat 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that theexpression of survivin in 6.0 Gy groups increased significantly, compared withsham-irradiated groups (P>0.05).2.1.2 Time course changes of survivin expression in HeLa cells exposed to 4.0 GyX-raysFCM was applied to detect the change of survivin expression in HeLa cellsduring 2~72 h exposed to 4.0 Gy X-rays. Results showed that the expression ofsurvivin was on a higher level at 4 h, 8 h, 48 h and 72 h, and they weresignificant difference with the control groups (P<0.01~ P<0.001).2.2 Effects of ionizing radiation on mRNA level of survivin2.2.1 Dose-effect of X-rays on survivin mRNA level in HeLa cellsRT-PCR assay was employed for the measurement of mRNA in HeLa cellsat 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed thatsurvivin mRNA tended to increase in 1.0~6.0 Gy groups compared with thecontrol groups.2.2.2 Time course changes of survivin mRNA level in HeLa cells exposed to 4.0 GyX-raysRT-PCR assay was employed for the measurement of mRNA in HeLa cellsduring 2~72 h exposed to 4.0 Gy X-rays. Results showed that survivin mRNAincreased at 8 h, 12 h and 24 h after 4.0 Gy irradiated.2.3 Effects of ionizing radiation on apoptosis of HeLa cells2.3.1 Dose-effect of X-rays on apoptosis in HeLa cellsFCM was applied to detect the change of apoptosis in HeLa cells at 24 hexposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the numberof apoptosis in 2.0 Gy groups declined significantly, compared with thesham-irradiated groups (P<0.05). However, the number of apoptosis had nosignificant difference between the irradiation groups with other doses and thesham-irradiated groups (P>0.05). It indicated that 0.5~6.0 Gy X-rays could notinduced apparently the increase of apoptosis in HeLa cells.2.3.2 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 HeLacells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that it was nosignificant difference between the irradiation groups and the control groups atdifferent observed time (P>0.05).2.4 Effects of ionizing radiation on the distribution of cell cycle in HeLa cells2.4.1 Dose-effect of X-rays on the distribution of cell cycle in HeLa cellsFCM was applied to analyze the distribution of cell cycle in HeLa cells at24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that thepercentage in G2+M phase was increased significantly in HeLa cells exposed to2.0 Gy, 4.0 Gy and 6.0 Gy, which was significant different with thesham-irradiated groups (P<0.001, respectively).2.4.2 Time course changes of the distribution of cell cycle in HeLa cells exposed to 4.0Gy X-raysFCM was applied to analyze the distribution of cell cycle in HeLa cellsduring 2~72 h exposed to 4.0 Gy X-rays. Results showed that the percentage inG2+M phase rose from12 h to 72 h. The increase was sharper than that of thecontrol groups (P<0.05~ P<0.001).2.5 Effects of ionizing radiation on the number of chromosome of HeLa cells2.5.1 Dose-effect of X-rays on the number of chromosome in HeLa cellsFCM was applied to detected the changes of the number of chromosome inHeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Resultsshowed that the percentage of tetraploid cells was increased significantly inHeLa cells exposed to 2.0 Gy, 4.0 Gy and 6.0 Gy, compared with thesham-irradiated groups (P<0.01~P<0.001), and the percentage of octoploid cellsin these groups was also more than those of the sham-irradiated groups(P<0.05~P<0.01).2.5.2 Time course changes of the number of chromosome in HeLa cells exposed to 4.0Gy X-raysFCM was applied to detected the number of chromosome in HeLa cellsduring 2~72 h exposed to 4.0 Gy X-rays. Results showed that the percentage oftetraploid cells remained the increase in HeLa cells during 2 h~48 h. They weresignificant difference with the control groups (P<0.05~ P<0.001). And, thepercentage of octoploid cells was also significantly increased at 4 h, 24 h, 48 hand 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 vector3.1.1 Construction of the recombinant plasmidSucceed to construct the RNAi vector, pSUPER-anti-SVV, which was therecombinant plasmid including an insert fragment of shRNA.3.1.2 Identification of the recombinant plasmidTo identify the sequence veracity of shRNA, the plasmid was treated bycleavage of endonucleases, PCR and sequencing process. Results indicated thatthe 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 theintensity of green fluorescence in HeLa cells transfected with pcDNA3.1-GFPwhich acted as the control vector, in order to elevate the efficiency oftransfection of pSUPER-anti-SVV indirectly. It was showed that the efficiencywas up to 64.64% at 48 h.3.3 Effects of shRNA on mRNA level of survivin in HeLa cellsFCM was applied to measure the change of survivin on mRNA level inHeLa cells at 24 h after 4.0 Gy X-irradiation or/and transfected withpSUPER-anti-SVV for 48 h. Results showed that mRNA was depressedapparently in the groups of transfected with pSUPER-anti-SVV, while there wasspecial product in the other control groups, such as, the untreated groups,irradiated groups, irradiated groups transfected with pSUPER.basic.3.4 Effects of shRNA on the expression of survivin in HeLa cellsFCM was applied to detect the time course of changes of survivinexpression in HeLa cells transfected with pSUPER-anti-SVV for 48 h andexposed to 4.0 Gy X-irradiation. Results showed that the treated groupstransfected with pSUPER-anti-SVV had the lower level on the expression ofsurvivin at 12 h, 24 h and 48 h, compared with the control groups transfectedwith pSUPER.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 cellstransfected with pSUPER-anti-SVV for 48 h and exposed to 4.0 GyX-irradiation. Results showed that the treated groups transfected withpSUPER-anti-SVV showed an apparently increase in apoptosis spanning 12 h to24 h (P<0.05 and P<0.001, respectively). It suggested that the interference ofsurvivin 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 cellstransfected with pSUPER-anti-SVV for 48 h and exposed to 4.0 GyX-irradiation. Results showed that the percentage in G2+M phase of the treatedgroups transfected with pSUPER-anti-SVV were increased at 12 h and 48 h,which was significant different with the the control groups transfected withpSUPER.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 cellstransfected with pSUPER-anti-SVV for 48 h and exposed to 4.0 Gy X-irradiation. Resultsshowed that the percentage of tetraploid cells was increased in the treated groupstransfected with pSUPER-anti-SVV at 24 h and 24 h. They were significant difference withthe control groups transfected with pSUPER.basic (P<0.01 and P<0.05, respectively). And,the percentage of octoploid cells in the treated groups was also significantly increased at 12h, 24 h and 48 h (P<0.01~P<0.001).Results of the project will provide the possible evidence for the further research onsurvivin biological effects.
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