| Pei etc. discovered a new proto-oncogene PTTG which is 13kb long, when they detected pituitary tumor cells by differential display PCR methods. They found this gene highly expressing in pituitary tumor cells, on the other hand, no expression could be identified in normal pituitary cells. Vitro experiment showed that nude mouse would develop a tumor 3 weeks after the PTTG transfected fibroblasts were planted subcutaneously. Many researchers found that PTTG not only expressed in pituitary tumor cell, cells of normal tissues with high proliferation activity would also see PTTG high expression, like testis, thymus and embryo liver, etc. At the same time, PTTG could hardly be positively detected in most of other tissues such as colon, liver, ovary, pituitary, pancreatic gland, brain, kidney and leucocyte of peripheral blood. The researches focusing at the relationship of PTTG expression and tumor showed that most tumors had PTTG high presentation, including pituitary tumor, colon carcinoma, hepatoma, ovary tumor, womb tumor, lymphoma, breast cancer, renal carcinoma, leukemia and all the tumor cell series (lymphoid cell series, myeloid cell series, mesenchymal cell series, epithelium cell series, etc.). Zhang etc. found that PTTG mRNA levels of 46 pituitary tumor samples were 50% higher than that of control group when they investigated 54 pituitary tumor samples and normal pituitary tissues by RT-PCR method. They also found that, among 23 hormone-secreting pituitary tumors of these samples, PTTG mRNA level of tumors who infiltrating sphenoid was significantly higher than that of who limited in sellar fossa. According to these findings, we could see that PTTG generally participated in the whole process of pituitary tumor genesis and development, playing an important role in its production. RNA interference is a newly rising and increasing full-grown gene silencing technique, which is considered to be a very effective research tool in gene engineering fields. Operating this technique, we could high performance and stable block the expression of target gene, so that 'silencing' this gene. DsRNA and siRNA have already been widely and successfully utilized for study of gene function and disease therapy research. RNAi could precisely suppress the proto-oncogene when it was applied for some gene therapy strategy. Just microcrystalline siRNA would depress the level of virulent gene product more than 90%, even could knocking down this gene. Then, RNAi possessed a strictly series specificity, could directly silence target gene. Different gene could be depressed simultaneously without any mutual interference. At the same time, RNAi could be cascaded amplified and had high penetrability. All of these characters suggested it was fitful for cancer gene therapy research. On the other hand, RNAi mechanism was proved existing in tumor cells. RNAi mediated by chemosynthesis, plasmid/virus or other mode could effectively depress endogenous RNA expression.In our study, we designed and produced lenvirus mediated shRNA recombinant targeting rPTTG that could stably and high-effectively express in rat pituitary tumor cell (GH3), after infecting GH3 cells with this vshRNA, we investigated targeting gene silence effect of this recombinant. The variation of cells proliferation cycle and apoptosis level because of PTTG knocking down were also dectected, in order to investigate whether PTTG could be regarded as an effective gene target for pituitary tumor gene therapy in the future.Part 1. Preparation of vshRNA recombinants targeting PTTG andeffective vshRNA screeningOperating RNAi sequence designing software downloaded from public website, based on RNAi structure principle, we designed four different shRNA arrays aiming directly at rPTTG, which were labeled as 1#, 2#, 3# and 4# shRNA. Then these shRNAs were inserted into lenvirus vectors, building up four kinds of recombinants. These four products were transferred into E Coli competent cell. Clones of these different infected cells were collected for RT-PCR and sequence analysis. Results showed that these four recombinants were successfully constructed.Four recombinants and two lenvirus-packing helpers were extracted. All of these things infected 293T cells separated by four groups, supernate that rich of virus particles were then collected and concentrated. The virus MOI levels were demarcated in 293T cells.Four recombinants were then separately operated to infect tool cell (NRK cell series), transfection effect was then detected in order to find the most effective vshRNA among these four.Objective cells were cultured in good condition and planted in 12-well plate one day before virus infecting. According to group designing, vshRNA recombinants of different MOI levels were separately added into the plate for infection. Fluorescence microscope were employed to observe GFP expression three days after the transfection. 4-5 days later, we detected the PTTG RNA level of these cells with real time PCR method to inspect the depressing results of different vshRNA. Outcome showed that all these four vshRNA could decrese the gene presentation. 2#,3# and 4# could decrease it more than 70% in high MOI level. 4# was the best sequence of these shRNA. Knocking-down effect of high virus MOI would be better than low MOI, but non-specific virus toxicity of high MOI was significantly higher than that of low MOI in nonsense control group comparing with blank control group.In this part, we successfully built up and packed four specific targeting rPTTG vshRNA recombinants, virus MOI were also demarcated in 293T cells. Besides of these, we managed to pick out the most effective vshRNA of these four, and clarified the non specific virus toxicity of different MOI. These results provided us reliable evidence to carry on RNAi experiment targeting PTTG in GH3 cells.Part 2. Investigation of RNAi effect targeting PTTG in GH3 cellswe had successfully built up four specific targeting rPTTG vshRNA recombinants, evaluated and screened 4# vshRNA as the best sequence with high infecting efficiency and silencing effect. In this part of experiment, we employed 4# vshRNA to infect GH3 cells to identify the knock-down ability of this recombinant.Objective cells (GH3) were cultured in good condition and planted in 6-well plate one day before virus infecting. According to group designing, vshRNA recombinants were separately added into the plate for infection. Fluorescence microscope were employed to observe GFP expression three days after the transfection. 5 days later, harvesting cells, we detected the PTTG mRNA and protein level of these cells with real time PCR and western-blotting methods to inspect the depressing results.Results showed that 4# vshRNA could swimmingly infect GH3 cells. Real time PCR data suggested that this recombinant could depress rPTTG mRNA level of GH3 cells obviously to more than 70%, similar results could be seen with western blotting method. At the same time, 5 days after stable transfection, comparing with blank control group and nonsense control group, we could see cells infected by 4# vshRNA emerging foliating stacking and shrinkage by fluorescence microscope. Large-area apoptosis could be observed 8 days later.Part 3. Biological behavior variation of GH3 cell after RNAiIn order to identify the effect of RNAi targeting PTTG in GH3 cell, we apply MTT and flow cytometry (FCM) to detect the changes of cell proliferation cycle and apoptosis level.(1). Investigation of cell proliferation-ability variation by MTT: Objective cells (GH3) were cultured in good condition and planted in 96-well plate one day before virus infecting. According to group designing, vshRNA recombinants and controls were separately added into the plate for infection. Fluorescence microscope were employed to observe GFP expression three days after the transfection. 4-5 days later, MTT method was employed to contrast the changes of cell proliferation cycle.(2). Investigation of cell apoptosis level variation by PI dyeing FCM: Objective cells (GH3) were cultured in good condition and planted in 96-well plate one day before virus infecting. According to group designing, vshRNA recombinants and controls were separately added into the plate for infection. Fluorescence microscope were employed to observe GFP expression three days after the transfection. 5 days later, PI dyeing FCM method was employed to contrast the changes of cell apoptosis levels.Results:(1). Comparing with other two groups, cell proliferation cycle of silencing group appeared obvious delay, which suggested that pituitary tumor cells' multiplication ability turned to be weak, growth ability of tumor cell was depressed remarkably.(2). Analyzing the proportion of all period cells in cell colony by FCM, we found that, after PTTG silenced, the proportion of apoptosis cell increased obviously, comparing with those two groups.Numerous research revealed that the occurrence and development of the majority of disease owed to the gene mutation and modification, so that how to repair or cut out the virulence gene specifically has become an interesting topic in the research for the gene therapy. Due to the high specificity and high performance of RNA interference that the previous tools can not provide, it becomes the most frequently used means in the gene therapy research increasingly. At the same time, similar to the previous researching tools, it's difficult to maintain the stable and long-term effect of intracellular RNA interference.In our study, we employed lenvirus vector to construct shRNA recombinants targeting rPTTG gene, and successfully achieved high-effectively, specifically and stably PTTG silencing effect in rat pituitary adenoma cells (GH3). Because of the silencing of PTTG, we could see that many biological behavior variation appeared such as cell proliferation cycle delay, apoptosis proportion level of tumor cells increasing, etc. Based on these results, we thought that PTTG played an important role in the process of pituitary tumor cells' survival and proliferation indeed, this gene could probably be considered as an effective gene target for pituitary tumor gene therapy in the future. At the same time, the system of lentivirus vector can assist the intracellular stable expression of shRNA fragment. It's an ideal vector for the gene therapy research.
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