| The incidence of thyroid carcinoma has risen especially Papillary thyroid carcinoma in recent years. Papillary thyroid carcinoma is the most common type of thyroid carcinoma, it accounts for 60-80% of thyroid malignancies and 1-2% of all human malignant tumor. There are biological characters present in papillary thyroid carcinoma such as outcontrol of proliferation and apoptosis arrest, strong capability of invasion, high incidence of lymph nodes metastasis, high recurrence rate after operation, unfavorable result after second surgery, and lack of internal and biological therapy approaches generally.XIAP(X-linked inhibitor of apoptosis protein) is one of the most important members in IAP family, and is well known as hallmark of the family, it also has been recognized as the strongest endogenous caspase suppress factor, key regulate factor of cell death in physiology. The Studies have shown that low or not XIAP expression in normal tissue, but in many malignant neoplasms, such as lung cancer, thymus cancer, pancreatic cancer, cervical cancer, leukemia and oophoroma, have over expression. The experiments in vitro and animal have pointed out XIAP play a tumor gene role in tumor development and progress through suppression of cell apoptosis and promotions of cell proliferation, survival, migration, invasion and tumor vessel formation. Due to the over expression in malignancies and low even not expression in normal human tissue, and the latest research demonstrates mice XIAP-deficient mice do not have any obvious pathology defects that prompt maybe knockout of XIAP has no shortcoming to do with normal cells. On account of all these characteristics above, XIAP gene is considered as a potential oncotherapy target point and has become a popular target gene in tumor therapy and pharmaprojects.RNA interference (RNAi) is a new technique which has been developed in recent years, it can be defined as a phenomenon in which ectogenesis and endogenous double strand RNA (dsRNA) degrade the homologous gene mRNA, then lead to gene silence after transcription. There are many characters in RNA interference, such as sequence specificity, high performance of gene express suppression, stability and transmissibility. There are two RNA interference experiment methods used commonly: direct transfection double strand siRNA and vector (plasmid and virus) express hairpin RNA. Lentivirus can be used in transfecting dividing cells or non-dividing cells, and has the characters such as high transfection efficiency, strong suppression of gene expression, long time duration, it also can cassette hereditary substance into genome of host cells.It becomes possible of prolonged action suppresses target gene expression through lentivirus vector mediated RNA interference.In spite of the proof of the intimate relationship between XIAP and some malignant tumors’development and progression, also XIAP presents potential as a tumor therapy target, but there are few documents about the relationship between XIAP and papillary thyroid carcinoma right now. With regards to all reasons above, we study the expression of XIAP in papillary thyroid carcinoma and the cell biological effect mediated by RNA interference, and expecting to bring up a new biology therapy strategy for papillary thyroid carcinoma.Chapter 1 Overexpression of X-linked inhibitor of apoptosis protein in papillary thyroid carcinoma1. Aim(1) To detect the expression of XIAP in papillary tyroid carcinoma, thyroid adenoma and normal thyroid tissue(2) To analysis the relationship between XIAP and clinical parameters2. Materials and Methods(1) Case data collection: this study was conducted on a total 49 paraffin embeded papillary thyroid carcinoma samples, 33 thyroid adenoma and 25 normal thyroid tissue samples, which were diagnosed by pathology department at our hospital.(2) Immunohistochemistry for XIAP expression using a rabbit monoclonal anti- XIAP antibody.3. Results(1) XIAP expression was strongly positive in 40 of 49 papillary thyroid carcinoma samples(81.6%).(2) We analysed the clinical data of papillary thyroid carcinoma patients by Chi-Square test and t test. The XIAP expression of male was 76.9%(10/13),and among female patients was 80.6%(29/36), Compared to the group with positive XIAP expression, XIAP expression was not associated with gender(p=0.780). The ages of positive XIAP expression was 46.18±15.2 years, and the ages of negative XIAP expression was 43.22±11.35 years, compared to the group with positive XIAP expression, XIAP expression was not associated with age(p=0.520). XIAP expression of metastatic samples was 86.7%, and the expression of non metastatic samples was 79.4%, there was not significant difference between two groups(p=0.546).The diameters of positive XIAP expression was 1.98±0.59cm, and the diameters of negative XIAP expression was 1.53±0.52cm, compared to the group with positive XIAP expression, XIAP expression was significantly associated with tumor diameter(p=0.041). XIAP expression of extrathyroid invasion samples was 95.4%(21/22), and the expression with non invasion samples was 70.4%(19/27), there is significant difference between two groups(p=0.024). XIAP expression was 69.6%(16/23) among early stage patients, and 92.3%(24/26) among advanced stage patients, there was significant difference between two groups(p=0.040). XIAP expression was 63.2%(12/19) among classical histology, 92.8%(13/14) among follicular variant, and 93.7%(15/16) among others histology classification, there was significant difference among group classic variant and group follicular and others(p=0.045, 0.031).(3) Positive expression of XIAP was associated with tumor diameters(r=0.328, P=0.031), invasion(r=0.317, P=0.037), clinical stage(r=0.289, P=0.046), while not age, gender, metastasis and histology classification by spearman correlation analysis.4. Conclusions(1) XIAP expression was strongly positive in 40 of 49 papillary thyroid carcinoma samples(81.6%).(2) XIAP expression was significantly associated with the tumor diameters invasion, clinical stage and histology classification. In contrast,no correlation was found between gender or age at the expression level of XIAP protein of papillary thyroid cacinoma patients.(3) Positive expression of XIAP was associated with tumor diameters, invasion, clinical stage, while not with gender, age, metastasis and and histology classification by spearman correlation analysis.Chapter 2 Silencing of XIAP in papillary thyroid carcinoma mediated by lentivirus vector1. Aim(1) To construct XIAP RNAi lentivirus vector, and produced high titer lentivirus particals.(2) Select the most effective shRNA through XIAP expression after transfection of papillary thyroid carcinoma cell line.2. Materials and Methods(1) XIAP RNAi lentiviral vectors were constructed by Genechem lentiviral RNAi Expression System(2) XIAP RNAi lentiviral vectors were packaged by co-transfecting 293FT with 3 plasmids. The titer of lentivirus was measured by hole dilution system(3) XIAP mRNA level was analyzed by real time RT-PCR, XIAP protein level was determined by Western Blot.3. Result(1) The lentivirus vector targeting XIAP was successfully constructed,.(2) The concentration of lentivirus were 2×108TU/ml after packaged and produced, the MOI of K1 and CGTH W3 were 10 and 20,respectively.(3) High performance interference sequence was selected, and after transfection, the mRNA level decreased 79%, the protein level decreased 87% in K1 cell line; and the mRNA level decreased 72%, the protein level decreased 76% in CGTH W3 cell line. 4. Conclusion(1) The construction of lentivirus vectorand targeting XIAP was successful.(2) The package and production of high titer lentivirus particle were successful.(3) The selected siRNA sequence demonstrated significant silencing effect of XIAP at both mRNA and protein levels.Chapter 3 Effects of XIAP gene silence on biological behavior of papillary thyroid carcinoma cells1. Aim(1) To measure the function of XIAP in proliferation of papillary thyroid carcinoma cell lines(2) To determine the function of XIAP in cell cycle of papillary thyroid carcinoma cell lines(3) To analyse the function of XIAP in apoptosis of papillary thyroid carcinoma cell lines(4) To elucidate the function of XIAP in invasion of papillary thyroid carcinoma cell lines2. Materials and Methods(1) Proliferation of papillary thyroid carcinoma was measured by MTT(2) Cell cycle analysis is performed by flow cytometry.(3)Apoptosis of cells were determined by flow cytometry with Annexin-V and PI Staining, and we also Western blotting and Real time PCR to test the protein and RNA level which is relevant to apoptosis(4) Invasion of cells were tested by Transwell methods,3. Results(1) The knockdown of XIAP has reduced the proliferation level, 51% and 43%, respectively.(2) After XIAP knockdown, about increased 18.4% and 14.1% of cells were at G2/M phase.(3) XIAP silencing could increase the level of apoptosis, 14.2% and 8.4%, respectively. (4) Knockdown of XIAP decreased the number of invaded cells about 50.7% and 44.9%, respectively.4. Conclusion(1) The knockdown of XIAP could restrain the proliferation of papillary thyroid carcinoma cells, and made it detained at G2/M phase.(2) The silence of XIAP could promote the apoptosis of papillary thyroid carcinoma.(3) The silence of XIAP could suppress the invasion of papillary thyroid carcinoma. |
