An Experimental Study On Survivin And VEGF Expression And Intervention Therapy In Human Thyroid Carcinoma

Objectives1. investigate the expression of survivin (an apoptosis inhibitor) in thyroid carcinoma and its relationship with vascular endothelial growth factor ( VEGF) expression.2. To investigate the inductive effect of survivin ( an inhibitor of apoptosis gene) antisense oligonucleotides (ASODN) on thyroid carcinoma cell apoptosis.3. To investigate the inhibitive effect of antisense oligonucleotide (ASODN) on vascular endothelial growth factor (VEGF) expression and endothelial cell growth in thyroid carcinoma.4. To evaluate effects of survivin and VEGF antisense oligodeoxyribonucle-otide ( ASODN ) on human medullary thyroid cancer ( MTC) inoculated into nude mouse.Methods1. 68 cases of thyroid carcinoma, 12 cases of thyroid adenoma and 10 cases of normal thyroid tissue were involved. Immunohistochemistry ( SABC method) was used to detect the expression of survivin, caspase -3 and VEGF, and then their relationship with the major clinical pathological parameters of thyroid carcinoma was analysed.2. Targeted ASODN of survivin was designed and synthesized and thentransfected to human medullary thyroid carcinoma cell lines at different concentration and time;and at the same time normal control and positive control (sense oligonucleotides, SODN group) were set for comparison. Reverse tan-scriptionase - Polymerase chain reaction ( RT — PCR) , Western blot and immu-nocytochemical technique were used to detect the expression of survivin mRNA and protein in each group;DNA ladder, acridine orange / ethidium bromide ( AO/EB) staining and flow cytometer (FCM) were used to detect the level and apoptotic morphology in each group;MIT assay was used to detect cell growth suppression.3. Targeted ASODN of VEGF was designed and synthesized, then transfected to TT ( medullary thyroid carcinoma) cell line and the culture supernatant was collected in which ECV304 (endothelial cell line) was seeded. At the same time positive control [ sense oligonucleotides, ( SODN) group ] and normal control were set for comparison. Cell growth condition was observed under microscope;RT - PCR and immuocytochemistry were used for the detection of VEGF mRNA and protein expression in TT cells;MTT assay for cell growth inhibition ratio (IR) of IT and ECV304 cells;flow cytometry (FCM) for apoptotic index (AI) of ECV304 cells;acridine orange/ ethidium bromide ( AO/EB) staining for apoptotic morphology of ECV304 cells.4. Twenty eight nude mouse models bearing human MTC were constructed and randomly assigned to control groups and therapeutic groups. Control groups include untreated group, SODN ( sense oligodeoxyribonucleotide) groups (VEGF SODN group and survivin SODN group) and combined SOND group;therapeutic groups include ASODN groups (VEGF ASODN group and survivin ASODN group) and combined ASODN group. Each group was given an intratu-moral injection with serum — free F12 or corresponding transfection medium ever-y day for seven days. All nude mice were sacrificed one week after treatment termination;tumors were fully resected from each mouse and measured for weight and inhibitory rate (IR) of tumor growth;paraffin sections of each tumor were respectively stained with HE, immunohistochemical or Tdt - mediated dUTP nick end labeling ( TUNEL) method to evaluate the variation of his-topathological construction, VEGF and survivin protein expression, and apopto-SIS.Results1. No survivin was expressed in normal thyroid tissue, but survivin was weakly expressed in thyroid adenoma (16. 7% ) and strongly expressed in thy-roid carcinoma (57.4% );caspase —3 was obviously presented in three kinds of tissue (70% , 75% , 69. 1% ) , and VEGF was expressed in 75% of thyroid carcinoma and 41. 7% of thyroid adenoma. In thyroid carcinoma, survivin expression had no correlation with caspase — 3 expression. but significantly positive correlation existed between survivin and VEGF( r = 0. 302, p < 0.05 ) according to histological classification, clinical staging and lymph node metastasis.2. Compared with normal control and positive control, the expression of survivin mRNA and protein in cells of each transfection ASODN group was obviously decreased, apoptosis rate significantly increased, and cell growth inhibited correspondingly. The effects mentioned above were in concentration — dependent manner, and the most obvious effect was shown in ASODN group transfected after 24 hours;there was no obvious difference between normal control and positive control at different time.3. As compared with positive and normal control groups. VEGF mRNA and protein expressions in IT cells of ASODN transfection groups were obviously decreased ( P <0.01);cell growth was not influenced apparently in ECV3O4 cells with direct ASODN administration;but ECV304 cell growth in ASODN conditioned medium was significantly inhibited and IR (0.21 ±0.03^0.31 ±0.01^0.42±0.22) was significantly higher than that of SODN group (0.05 ±0.03, P <0.01) , with the presence of apparent apoptosis. The effect mentioned above was in a dose — dependent manner.4. Therapeutic groups had a trait of obviously depressed angiogenesis. Compared with control groups, each therapeutic group possessed significant traits of lower expression of VEGF and survivin protein, higher apoptosis, lower tumor weight, and higher IR (each P <0.01) ? furthermore, combined ASODN group held more significant than either of ASODN groups for those traits (each P <0.05) , but no significance was found between control groups or ASODN groups for those traits (each P>0.05).Conclusion1. Survivin is specifically expressed in thyroid carcinoma and is probably involved in the pathogenesis of thyroid carcinoma via inhibiting apoptosis and promoting angiogenesis with the synergetic effect of VEGF.2. Antisurvivin oligodeoxyribonucleotide can down - regulate the expression of survivin gene in thyroid carcinoma specifically, inducing tumor cell apoptosis and suppressing cell proliferation.3. Antisense oligodeoxyribonucleotide of VEGF can suppress endothelial cell growth and inhibit tumor angiogenesis possibly by specifically blocking VEGF expression in thyroid carcinoma.4. Our study demonstrated that survivin and VEGF targeting single or double genetic silence therapy could achieve significantly inhibitor}' effect on tumor growth in human MTC, moreover, the combined one showed more significant effect, thus providing experimental proof for developing gene therapy of MTC.