| 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 can stably and high-effectively block the expression of target gene to 'silencing' this gene. DsRNA and siRNA have already been widely and successfully utilized on study of gene function and disease therapy research.Survivin is a unique member of the inhibitorof apoptosis protein family (IAP), which was found by Ambrosini et al in 1997. Its molecular weight is small, but survivin is powerful. The survivin is so far the strongest apoptosis inhibitor. Survivin is expressed in embryo and immature-differentiated tissues. In adult, it is trace expression in thymus and placenta and does not exist in differentiated tissues, including peripheral blood leukocytes, lymph nodes, spleen, pancreas, kidney, skeletal muscle, heart, liver, lung and brain. However, it becomes re-expressed in a variety of cancers. Survivin expression in tumours has been associated with increased aggressiveness and decreased patient survival.In the present study, our programs are as follows: Firstly, we detect the protein expression of survivin in pituitary adenomas and investigate the biological relationships between survivin and the invasiveness of pituitary adenomas. Secondly, we design and synthesize plasmid vectors mediated siRNA recombinant targeting survivin that can stably and high-effectively express in rat pituitary tumor cell (GH3). After transfecting GH3 cells with those vectors, we investigate targeting gene silence effect of this recombinant. In order to investigate whether survivin will be regarded as an effective gene target for pituitary tumor gene therapy in the future, the variation of cells proliferation and cell apoptosis level because of survivin knocking down are also dectected.Part 1. Relationship of survivin expression and biological Behavior in Pituitary TumorsObjectiveSurvivin is a structurally unique member of the inhibitor of apoptosis protein (IAP) family. Survivin expression in tumours has been associated with increasing invasiveness and decreasing patient survival. The aim of the present study is to detect the protein expression of survivin in pituitary adenomas, investigate the biological relationship between survivin and the invasiveness of pituitary adenomas, and evaluate the clinical magnificance.Methods1. TissuesPituitary adenomas tissues are obtained by transsphenoidal surgery from 66 patients at the Department of Neurosurgery in Shandong Provincial Hospital from July 2006 to March 2008. The subjects include 29 women (mean age 65.3±4.3 years, range 29-77 years) and 37 men (mean age 68.1±2.5 years, range 34-82years). Parasellar invasiveness of pituitary adenomas is evaluated by preoperative magnetic resonance imaging (MRI) and verified by intraoperative inspection of the medial wall of the cavernous sinus using micrological technique: 39 cases are found to be invasive, and 27 tumours are noninvasive. All pituitary adenomas are evaluated histologically according to the WHO-classification.2. Immunohistochemical staining procedure The protein expression of survivin is detected by the S-P method with Ultrasensitive S-P kit.3. Analysis of immunohistochemical resultsSurvivin immunoreactivity is evaluated semiquantitatively based on the intensity of staining. 5 fields at a magnification of 400×at every slice are observed at random. The percent of positive staining cells in 200 tumor cells is calculated. The slides are analysed independently by four observers blinded for expression of survivin. For the discrepancies a second evaluation course is running to reach agreement.The survivin expression is scored as: strong (+++): if more than 50% of cells are positive or a strong diffuse reaction is seen; moderate (++): if less than 50% of cells are positive, or a moderate diffuse reaction is observed; slightly positive (+): if immunoreactions are found in less than 10% of tumour cells or the diffuse reaction is weak; and negative (-): if no survivin is stained.4. Statistical analysisStatistical analysis of the sample groups is accomplished using the chisquare test. SPSS Version 13.0 is used for statistical analyses. P <0.05 is considered significant value.ResultsIn the pituitary adenomas, a cytoplasmic survivin reaction predominates and only scattered cells exhibited nuclear reaction. Survivin is expressed in 69. 7% (46/66) of the investigated pituitary adenomas. Among invasive pituitary adenomas survivin staining is positive 89. 7% (35/39), while only 40. 7% (11/27) specimens are positive in noninvasive tumours. The chisquare test demonstrates a statistically difference in survivin expression between invasive and noninvasive pituitary adenomas (x~2=14.309, P =0.0002<0. 05). Furthermore, the strong and moderate positives (71.4%, 25/35)are t most common in invasive pituitary adenomas, while in noninvasive pituitary adenomas is slight (72.7%, 8/11).Conclusions 1. There is high expression of survivin in pituitary adenoma.2. There is a statistically difference in survivin expression between invasive and noninvasive pituitary adenomas. Survivin is highly associated with invasive pituitary adenomas3. Survivin can serve as a useful tool for confirmation of invasive pituitary adenoma and survivin gene also may be an effective target for pituitary adenoma gene therapy.Part2. Design and screen effective siRNA vector aiming at survivinObjective3 siRNA vectors aiming at survivin are designed and synthesized, 2 effective ones are screened for the next research.Methods1. Design and synthesize siRNA vectorOperating RNAi sequence designing software is downloaded from public website. Based on RNAi structure principle, we design 3 different siRNA vectors aiming directly at survivin, which are labeled as 1#, 2#, 3# and one negative control vector, which is labeled as 0#. These different vectors are collected for RT-PCR and sequence analysis. Results show that these four vectors are successfully constructed.2. Transfect tool cell (glioma C6)Tool cells (glioma C6) are cultured in good condition and planted in 6-well plate one day before tansfecting. According to group designing, different vectors are separately added into the plate for tansfection. Fluorescence microscope is employed to observe GFP expression 48 hours after the transfection. Transfection efficiency is recorded.3. Detect interference effects and screen effective vectorsThose are picked out, if their transfection efficiency is over 90%. We detect the survivin RNA level of these cells with FQ-PCR method to inspect the depressing results of different siRNA vectors and screen 2 effective ones.Results1. The transfection efficiency of siRNA vector 1 #, 2 #, 3 # is 87.4±6.2%, 92.6±4.5%, 75.3±11.0% respectively. The transfection efficiency of negative control vector 0# is 95.0±4.2%.2. All these 3 siRNA vectors can decrese the gene presentation. Compared to negative control vector 0#, the silencing efficiency of 1 #, 2 #, 3 # is 68.4%, 44.4%, 22.7% respectively. Considering transfection efficiency and silencing efficiency, 1 # and 2 # are better than 3 #.Conclusions1. In this part, we successfully design and synthesize 3 specific targeting survivin siRNA vectors.2. We manage to pick out 2 effective siRNA vectors of these three. These results provide us reliable evidence to carry on RNAi experiment targeting survivin in GH3 cells.Part3. Cell proliferation and apoptosis level variation of pituitary tumor cells by RNA interference survivin geneObjectiveUsing constructed the conditionally replicating vector 1#,2# which can transfect to GH3 effectually and selectively and contain siRNA to rat survivin gene, we investigate the effect of the vector silencing on expression of mRNA and protein of survivin gene in GH3 cells on propagation and apoptosis of pituitary tumor cell lines GH3.Methods1 .Transfect GH3 cells with siRNA vector1#,2# respectively.Objective cells (GH3) are cultured in good condition and planted in 6-well plate one day before transfecting. According to group designing, Vector1#, 2# and 0# is separately added into the plate for transfection.2. Screen stable transfectants with G418Cells are trypsinized and plated into 1000 cells/ml, and concentration ranges (100μg/ml -1mg/ml) G418 is added to the culture medium. We pick out the lowest concentration G418 when all cells die in 10-14 days. G418 is add 24 hours after transfection. With Cells metabolic activity and G418 concentration decreasing, the culture medium containing G418 is changed in 3-5 days. At this time the drug concentration may be reduced to 200μg/ml. Fluorescence microscope is employed to observe GFP expression. 14 days after selection, cell colonies are isolated and expanded for testing.3. Inspect the effects of gene silencingThe survivin mRNA and protein level of these cells are detected with FQ-PCR and western-blotting method is used to inspect the effects of gene silencing.4. Investigation of pituitary tumor cell proliferation-ability variation by MTTStable tansfecion pituitary tumor cells (GH3) are cultured in good condition. According to group designing (siRNA recombinants and controls), cells are planted in 96-well plate. 1 day later, MTT method is employed to contrast the changes of cell proliferation cycle.5. Investigation of pituitary tumor cell apoptosis level variation by PI dyeing FCMStable tansfecion pituitary tumor cells (GH3) are cultured in good condition. According to group designing (siRNA recombinants and controls), cells are collected. PI dyeing FCM method is employed to contrast the changes of cell apoptosis levels.Results1. FQ-PCR data suggests that the 2 siRNA recombinant could depress survivin mRNA level of GH3 cells obviously. Compared to negative control, vector 1#, 2# silencing efficiency is 54.4%,54.0% respectively. Similar results can be seen with western blotting method.2. The proliferation suppressing effects of RNAi targeted to survivin are observed by MTT assay. The proliferation inhibitory rates of 1# and 2# are 42.5% and 38.7% respectively, which suggest that pituitary tumor cells' multiplication ability turns to be weak, and the growth ability of tumor cells is depressed remarkably.3. Analyzing the proportion of all period cells in cell colony by FCM, we found that, after survivin silenced, the proportion of apoptosis cell increased in the two siRNA groups, comparing with control group.Conclusions1. Survivin plays an important role in the process of pituitary tumor cells' survival and proliferation indeed. After survivin silenced, growth ability of tumor cell is depressed remarkably and apoptosis cell increased obviously.2. The RNA interference technology will be important in the application value of pituitary tumor gene therapy in the future. Survivin can probably be considered as an effective gene target for pituitary tumor gene therapy.
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