The Influence Of Survivin Specific SiRNA On Heavy-ion Radiation Induced Biological Effects In Human Hepatoma SMMC-7721 Cells

Compared with low-LET X- or 3,-rays, high-LET heavy ion beam could induce more intensive biological effects. The main physical characteristic of heavy ion beam is its inverted depth-dose distribution, i.e. a high-dose peak (so called Bragg peak) presented at the end of its range. Moreover, high relative biological effectiveness (RBE), which is the most important biological characteristic of heavy ion beam, would be presented within the Bragg peak. Heavy ion beam could kill cancerous cells in the target volume effectively while the healthy tissues surrronding the target voume are spared as much as possible. Therefore, high-LET heavy ion beam is superior to conventional low-LET radiations in cancer treatment. Survivin is a member of inhibitor of apoptosis family (IAPs) and is expressed in embryonic tissues as well as in the majority of human cancers, but not in most normal adult tissues. It is proved that over-expression of survivin in some kinds of malignant cancerous cell lines is related to the radioresistance induced by radiotherapy. In order to investigate the biological effects and relavent mechanisms such as the relationship between survivin expression after (12)^C6+ irradiation and radiosensitivity, the present study used human hepatoma SMMC-7721 cells as experimental material and transfected with survivin-specific siRNA. Finally, the ransfected cells were irradiated with a carbon ion beam of 68 MeV/u (corresponding to a LET value of 35 keV/μm), which was supplied by the Heavy Ion Research Facility in Lanzhou (HIRFL). Based on the methods above, the influence of survivin-specific siRNA on heavy-ion radiation induced biological effects in SMMC-7721 cells was studied. Several important experimental results were acquired as summarized as below:1. Heavy ion irradiation improved the radiosensitivity of SMMC-7721 cells, changed the cell cycle distributions, and induced cell apoptosis effectivelyWe used high-LET (12)^C6+ ion beam and low-LET X-rays to irradiate the SMMC-7721 cells and applied the standard colony-forming assay to analyze and draw survival curve; apoptotic cells and cell cycle distribution after irradiation were analyzed by running into flow cytometry (FCM). Results indicated that SMMC-7721 cells are more sensitive to high-LET heavy ion beam radiation than low-LET X-rays irradiation; cells restrained at G₂/M phase obviously and the apoptosis enhanced after heavy ion beam radiation, both changements presented dose-dependent manner.2. The radiosensivity of SMMC-7721 cells to the (12)^C6+ ion irradiation was enhanced obviously after trasfected with survivin-specific siRNASurvivin-specific siRNA oligonucleotides were synthesized based on the survivin sequence in Genebank. SMMC-7721 cells were transfeced with survivin-specific siRNA to make survivin expressed transiently. Transfected cells showed more obviously enhanced radiosensitivy to (12)^C6+ ion irradiation than that to the (12)^C6+ ion irradiation alone. This suggested that survivin is one of reasons why SMMC-7721 cells possess the radioresistance.3. survivin expression was inhibited after treatment with survivin-specific siRNA combinded with (12)^C6+ ion beamReal-time PCR assay method and western bolting were used to determinded the survivin expression at transcriptional and post-transcriptional levels respectively. Survivin expression was restrained both transcriptional and post-transcriptional levels after treatment with survivin-specific siRNA, this indicated that survivin-specific siRNA could make survivin gene silent effectively so as to decrease survivin expression.4. The cell proliferation activity of SMMC-7721 cells decreased after treatment with survivin-specific siRNA combined with (12)^C6+ ion beamMTT assay was used to analyze the proliferation activity of SMMC-7721 cells after the combinational treatment with siRNA transfection and (12)^C6+ ion irradiation. The combinational treatment could inhibit proliferation activity of SMMC-7721 ceils. The expression of survivin gene could facilitate cancerous cells go into the cell division process through mal-mitosis induced by the apoptosis inhibition effect of survivin and the ceil proliferation capability is enhanced. When survivin-specific siRNA was used to knockdown the survivin gene in SMMC-7721 cells, the proliferation acvtivity was weakened and the cells showed decreased proliferation activity.5. Both Gz/M cells and the apoptosis cells increased after treatment with survivin-specific siRNA combinded with (12)^C6+ ion irradiationThe cell cycle distributions of SMMC-7721 cells after combined treatment were detected by FCM and Annexin V assay was used to determine apoptotic rate. Compared with the simple irradiation, combinational treatment cells appeared more obvious G₂/M block and increased celt apoptotic rate. The anti-apoptosis effect possessed by survivin also manifests in aspect of the cell cycle distribution changement. Survivin over-expresses in G₂/M phase and helps the cancerous cells to overcome G₂/M checkpoint and go into cell differentiation through mitosis. The goal of survivin-specific siRNA is to counteract the anti-apoptosis effect and make the radiated cells restrain at G₂/M phase, which is the most sensitive to radiations, and more number of cells apoptosis.After survivin-specific siRNA was applied, the radiosensitivity of SMMC-7721 cells to heavy ion beam was improved obviously, cell proliferation activity was depressed, radiation-induced G2/M block and apoptosis were both increased markedly. All of the experimental results herein provide basic data for heavy-ion cancer therapy. At the same time a new cancer treatment modality, i.e. heavy ion radiotherapy in combination with gene therapy, could be figured out.