Effects And Mechanisms Of Silencing Peroxiredoxin Ⅰ Gene By Rna Interference On The Radiosensitivity Of Breast Carcinoma Cell Line MCF-7

Breast cancer is the most common malignancy of women. About 120 million new cases are diagnosed and 50 million women die of breast cancer each year around the world. Despite the comprehensive treatment of breast cancer has made great progress, there are still 20% to 30% of breast cancer patients occur local recurrence and distant metastasis after treatment Therefore, it is the breakthrough to explore new therapeutic tools for further improving the curative effect of breast cancer.Radiotherapy is one of the major means of treating malignant tumors, however, the radiosensitivity of tumor cells is a key factor to determine radiotherapy effect. In recent years, with thorough study in the radiation biology of the cancer, researchers have found that the expression of some genes at the molecular level affected the radiosensitivity of tumor cells. Gene therapy strategy enable us to knockout these genes that are specific or differential expression between tumor cells and normal cells, and achieve controlled expression of special gene in vivo so as to reverse resistance to the treatment of tumor cell and increase their therapeutic responsiveness.Peroxiredoxins (Prxs) are a newly discovered peroxidase that play an important role in scavenging reactive oxygen species of the body and protect cells from damaging by overoxidized. PeroxiredoxinⅠ(PrxⅠ),a member of the Prxs family, which is overexpressed in many cancers including breast cancer cells. PrxⅠhave been implicated in regulating many cellular processes such as cell proliferation, differentiation and apoptosis, moreover, the levels of its expression is closely related to the metastasis, recurrence, chemosensitivity and radiosensitivity of tumor cells,and can serve as a molecular marker for tumor diagnosis. Thus, PrxⅠmay be an ideal target for gene therapy.DNA strand break is an important mechanism which ionizing radiation treat tumor. DNA damage can activate the repair system of body and can be repaired in time to ensure the stability of organism genome. Researches have found thatγ-H2AX and Rad51 proteins may play an important role in DNA damage repair.We combine gene therapy with radiotherapy in this study: First, we construct specific shRNA eukaryotic expression vector targeting PrxⅠgene in order to down-regulate the expression of PrxⅠ, then we observe the radiosensitivity changes of breast cancer cells MCF-7 cells and investigate its related mechanisms. The purpose of this study is to clarity the role of PrxⅠgene in radiotherapy and provide a new target for gene therapy of breast cancer.Methods and results:Part one Construction and identification of shRNA eukaryotic expression vector targeting PrxⅠgeneMethods:According to the principle of RNA interference construction, we designed and synthesized four shRNA sequences targeting PrxⅠand one sequence for negative control. First the shRNA connected to linear pGPU6/GFP/Neo plasmid vector which was digested by BbsⅠand BamHⅠrestriction enzyme. then the recombination plasmids were identified by BamHⅠor PstⅠdigestion and DNA sequencing after transformation, screening,amplifing and extracting,and transfected into breast carcinoma MCF-7 cells by lipofectamine respectively. Transfection efficiency was detected by Fluorescence microscopy and Flow Cytometry, the mRNA and protein expression levels of PrxⅠwere evaluated by RT-PCR and western blot.Results : we successfully constructed four eukaryotic expression vectors targeting PrxⅠgene:pGPU6-Prx1,pGPU6-Prx2,pGPU6-Prx3,pGPU6-Prx4 and negative expression vectors pGPU6-HK which were successfully transfected into MCF-7 cells by Lipofectamine. Transfection efficiencies were about 80%. The mRNA and protein expressions of PrxⅠin pGPU6-Prx1,pGPU6-Prx2,pGPU6-Prx3 and pGPU6-Prx4 were significantly inhibited compared with control group and pGPU6-HK group(P<0.05). The inhibition rate of pGPU6-Prx3 at mRNA and protein levels were 82.6% and 80.5% respectively. So we chose it for experimental study.Part two Effects and mechanisms of silencing PrxⅠgene by RNA interference on the radiosensitivity of breast carcinoma cell line MCF-7Methods:The pGPU6-HK and pGPU6-Prx3 recombinant plasmids were transfected into breast carcinoma cell line MCF-7 by Lipofectamine respectively. Two stable cell lines were establish by G418 screening which were named as pGPU6-HK and pGPU6-PrxⅠrespectively. The mRNA and protein expression of PrxⅠwere detected by RT-PCR and Western blot. Four groups were divided in the experiment: pGPU6-HK group, pGPU6-PrxⅠgroup, pGPU6-HK+IR group and pGPU6-PrxⅠ+IR group. IR groups were irradiated with a single dose 6Gy X-ray. Intracellular reactive oxygen species, cell cycle and cell apoptosis of each group at 48 hour after irradiation were detected by Flow Cytometry. Cell proliferation was disclosed MTT assay. Morphological changes of cell nucleus were observed by hoechst assay to indirectly measure cell apoptosis. Colony efficiency was discovered by colony formation assay after 0,2,4,6,8,10Gy irradiation,cell survival curves were drawn and the values of radiobiological parameters Do, N, Dq and SF2 were calculated to assess cellular radiosensitivity; A landmark proteinγ-H2AX of DNA double-strand breaks and a key protein Rad51 of DNA damage repair were evaluated by Western blot assay.Results:Two stable cell lines(pGPU6-PrxⅠand pGPU6-HK)were obtained by G418 pressure screening after four weeks. The mRNA and protein expression of pGPU6-PrxⅠwere distinctly decreased compared with pGPU6-HK group(P﹤0.05). The inhibition rates of the mRNA and protein expression were 84.8% and 83.5% respectively. These results of Flow Cytometry, MTT and Hoechst assays demonstrated that ROS levels markedly raised, the G1 phase cell increased,S phase cells decreased,cell apoptosis rate significantly increased,cell proliferation obviously delayed, apoptotic cell which appeared typical apoptotic morphological changes including nuclear condensed and fragmentation also enhanced at 48h after 6Gy irradiation in pGPU6-PrxⅠand combined with IR groups compared with pGPU6-HK group(P﹤0.05).These changes in pGPU6-PrxⅠ+IR group were more obvious compared with pGPU6-PrxⅠand pGPU6-HK+IR(P﹤0.05).Colony formation assay indicated that colony formation rate reduced after irradiation with different doses and the values of radiobiological parameters Do,N,Dq and SF2 of the pGPU-PrxⅠgroup,were 1.354,3.106,1.547 and 0.504 respectively, lower than the pGPU-HK group. The SER which derived from SF2 was 1.353. The protein expression of Rad51 was distinctly inhibited,while the protein expression ofγ-H2AX was markedly enhanced in pGPU6-PrxⅠand combined with IR groups compared with pGPU6-HK(P<0.01).These changes of pGPU-PrxⅠ+ IR group were more obvious in which the protein expression of Rad51 decreased 79.3% and the protein expression ofγ-H2AX increased 3.7 times compared with pGPU6-HK. The significant differences were also observed between pGPU-PrxⅠ+IR group and pGPU6-PrxⅠor pGPU6-HK+IR(P﹤0.05).Part three Effects of silencing PrxⅠgene by RNA interference on the radiosensitivity of nude mice xenografts of breast carcinoma cell line MCF-7Methods:Two stable cells of pGPU6-HK and pGPU6-PrxⅠwere implanted subcutaneously into BALB/c nude mice to establish xenograft model of breast cancer. It was judged as tumorigenesis when the diameter of tumor reached 5mm. Four groups were divided in the experiment: pGPU6-HK group, pGPU6-PrxⅠgroup, pGPU6-HK+IR group and pGPU6-PrxⅠ+IR group. IR groups were irradiated with a total dose 10Gy. Tumor growth was regularly monitored. All BALB/c nude mice were sacrificed at the thirty day after irradiation and and tumor tissues were stripped, weighted. The inhibitory rate of tumor weight was calculated. The protein expressions of PrxⅠand caspase3 were detected by immunohistochemistry. Ultramicrostructure of tumor cell was observed by electron microscopy. The expressions of a landmark proteinγ-H2AX of DNA double-strand breaks and a key protein Rad51 of DNA damage repair were analyzed by western blot.Results:Xenograft model in nude mice were successfully established. The time of tumor formation in pGPU6-HK group and pGPU6-PrxⅠgroup was ten days and fourteen days respectively Compared with pGPU6-HK group, tumor volume and weight were smaller and lighter in pGPU6-PrxⅠgroup and inhibition rate of tumor weight was 37.8%(P﹤0.01). Tumor volume of combined with IR emerged negative growth lessened after 10Gy irradiation while showed positive growth at the third day after irradiation end which were significantly slower than pGPU6-HK and pGPU6-PrxⅠ,especially pGPU6-PrxⅠ+IR group. The inhibition rate reached 79.76% that were significant differences compared with pGPU6-PrxⅠ(34.92%) and pGPU6-HK+IR(56.94%)(P<0.05). Immunohistochemistry and electron microscopy illustrated that the expression of PrxⅠprotein significantly down-regulated,whereas the expression of Caspase3 protein markedly up-regulated,cell apoptosis and necrosis increased,the protein expression of Rad51 distinctly decreased, while the protein expression ofγ-H2AX was markedly increased in pGPU6-PrxⅠand combined with IR groups compared with pGPU6-HK(P<0.01). These changes of pGPU-PrxⅠ+IR group were more obvious compared with pGPU6-PrxⅠgroup and pGPU6-HK+IR group(P < 0.01). The protein expression of Rad51 decreased 84.8% and the protein expression ofγ-H2AX increased 5.6 folds in pGPU-PrxⅠ+ IR group compared with pGPU6-HK.Conclusion:The shRNA eukaryotic expression vector targeting PrxⅠgene were successfully constructed and xenograft model of breast cancer was successfully established. Silencing PrxⅠgene by RNA interference could significantly enhance the radiosensitivity of breast cancer MCF-7 cells in vitro and vivo through inhibiting cell proliferation,promoting cell apoptosis, regulating cell phase redistribution, improving sensitive enhancement ratio(SER) and its mechanisms might be related to reducing the capacity of scavenging ROS,increasing DNA double-strand breaks and decreasing DNA damage repair capacity. PrxⅠgene showed a negative correlation with the radiosensitivity of breast carcinoma cell line MCF-7 and might act as a molecular target for radionsensitization of breast carcinoma.