The Significance And Pathomechanism Of S100A13 Gene In Thyroid Carcinomas

Thyroid cancer is one of the most common cancers in the endocrine system,and the mechanism of which is not yet fully understood .S100A13 is a new member of S100 calcium-binding protein family. S100A13 shares the commonness of S100 protein family, and it also has its own features on the structure and function. FGF-1 is an important member of fibroblast growth factor protein family. Increased expression of FGF-1 is related to the tumor growth and metastasis. S100A13 plays important roles in tumorgenesis, inflammation, and arteriosclerosis through mediating transmembrane export of FGF-1 and IL-1a.We found and verified that the expression of S100A13 gene is higher in thyroid cancer tissues than that in normal thyroid tissues in the process of sieving genes of differential expression in thyroid cancer tissues and normal thyroid tissues by the suppression subtractive hybridization （SSH）.To explore the role of S100A13 gene in the progress of thyroid cancer, we observed the expression of S100A13 in thyroid cancer tissues and determined the effect of S100A13 overexpression on the cell growth of thyroid cancer TT cells and on the growth of xenografts of human thyroid cancer cell line TT in nude mice by the use of immunohistochemistry and gene transfection.The results show that the expression of S100A13 and FGF-1 in carcinoma was significantly higher than that in adenomas and normal tissues, and the exogenous S100A13 gene expression in TT cells could accelerate cell proliferation, and promote cell cycle progression from G0/G1 phase to S and G2/M phase.Subsequently, the inhibition effect of small hairpin RNA-mediated S100A13 and Ca²⁺-chelating agents on the release of FGF-1 in human thyroid cancer cells was also observed .we found that the inhibition of S100A13 could reduce the release of FGF-1 in TT cells and Ca²⁺-Chelating agent could inhibit the rise of [Ca²⁺] and reduce the and metastasis by promoting the expression and the release of FGF-1. release of S100A13 and FGF1 induced by serum-deprivation stress in TT cells. It is believed that increased expression of S100A13 in thyroid is crucial in tumorigenesis Part1 Significances of S100A13 and FGF-1 Expression in Thyroid CarcinomaObjective: To investigate the expression and clinical significance of S100A13 and FGF-1 in the thyroid carcinoma. Methods: The expression of S100A13 and FGF-1 were detected by immunohistochemistry in 71 cases of thyroid tumor （56 carcinomas and 15 adenomas） and 14 of normal thyroid. Results: The positive rate of S100A13 is 91.1%,66.7% and 64.3% and the positive rate of FGF-1 is 89.3%、60.0%、57.1% respectively in thyroid carcinoma,thyroid adenoma and normal thyroid tissues. The expression of S100A13 and FGF-1 in carcinoma was significantly higher than that in adenomas and normal tissues（P<0.05）. S100A13 and FGF-1 showed a higher positive immunostaining percentage in thyroid carcinoma with lymph node metastasis compared with that in thyroid carcinoma without lymph node metastasis. Conclusion: The expression of S100A13 and FGF-1 in thyroid carcinoma was significantly higher than that in thyroid adenoma and normal thyroid tissues. The percentage of S100A13 and FGF-1 expression may be correlated with the carcinoma metastasis.Part2 The Effect of S100A13 on Proliferation of Human Thyroid Cancer Cell Line TTObjective: To obtain eukaryotic express vector of human S100A13 gene and to investigate the effect of exogenous S100A13 expression on the proliferation of human thyroid cancer cell line. Methods: S100A13 cDNA was amplified by RT-PCR from papillary thyroid carcinoma tissues, and PCR products was cloned into pGEM-T vector and sequenced. The recombinant ORF tagged with six histidine at 5’min terminal was subcloned into pcDNA3.1/NT-GFP-topo vector and sequenced. The eukaryotic expression plasmid pcDNA3.1/NT-GFP-S100A13 and empty vector pcDNA3.1/NT-GFP were transfected into TT cells and the cells were selected by G418. The expression of green fluorescent protein （GFP） in TT cells was observed by laser scanning microscope and the expression of S100A13 mRNA and protein was detected by real-time PCR and Western blot. The effects of S100A13 on TT cell proliferation and cell cycle progression were measured by cell growth curve and flow cytometry. Results: S100A13 gene tagged with six histidine at 5’min terminal was indentified to be inserted into pcDNA3.1/NT-GFP- vector correctly.TT-S100A13-GFP and TT-GFP cells with the stable exogenous expression of GFP-S100A13 and GFP were established successfully. TT-S100A13-GFP cells grew faster than TT-GFP and TT cells [（2.30±0.24）×105 vs. （1.40±0.25）×105 and （1.50±0.22）×105 at the 7th of cell culture, P<0.05]; both S phase proportion and G2/M phase proportion were significantly higher in TT-S100A13-GFP cells than that in TT-GFP and TT cells [（6.47±0.14）% vs. （5.86±0.23）% and （5.99±0.28）% at S phase, P<0.05; （50.27±0.66）% vs. （39.39±0.23）% and （39.64±0.64）% at G2/M phase, P<0.05]. Conclusion: The eukaryotic expression vector of human S100A13 gene was successfully constructed. Exogenous S100A13 gene expression in TT cells could accelerate cell proliferation, and promote cell cycle progression from G0/G1 phase to S and G2/M phase.Part3 Experimetal Study on the Role of S100A13 in the Growth of Xenografts of Human Thyroid Cancer Cell Line TT in Nude MiceObjective: To investigate the effect of S100A13 expression on the growth of nude mice xenograft tumors, and to clarify the preliminary mechanism of the effect of S100A13 on the growth of thyroid cancer. Methods: The stable TT cell line with over-expression of S100A13 （TT-S100A13-GFP）,the TT cell transfected with empty vector （TT-GFP） and the TT cell were cultured respectively. The TT-S100A13-GFP/ TT-GFP/TT cell line was inoculated subcutaneously in nude mice. To estimate the the effect of S100A13 expression on the growth of nude mice xenograft tumors, the volume and weight of xenograft tumor was measured and the expression of Cyclin E、FGF-1 and CD31 protein was detected in nude mice transplantation tumor by using the immuno- histochemical method. Results: The nude mice xenograft tumors models of TT-S100A13-GFP,TT-GFP and the TT cell were established successfully. Compared with the TT-GFP and the TT cell group, the nude mice xenograft tumor growth ability was greatly increased in TT-S100A13-GFP group （P<0.05）. Among the three groups, the weight of TT-S100A13-GFP group xenograft tumor was the heaviest （P<0.05）, and the expression of Cyclin E、FGF-1 and CD31 protein of the TT-S100A13-GFP group was up-regulated in TT-S100A13- GFP group xenograft tumor （P<0.05）. Conclusion: Over-expression of S100A13 could promote the growth of nude mice xenograft tumor of thyroid cancer TT cell line, and enhance the expression of Cyclin E、FGF-1、CD31 protein in nude mice xenograft tumor.Part4 Effects of Small Hairpin RNA-mediated S100A13 Inhibition on the Release of FGF-1 in Human Thyroid Cancer Cell Line TTObjective: To investigate whether the release of FGF-1 could be changed by the inhibition of S100A13 gene and serum-deprivation. Methods: The S100A13-shRNA pENTRTM /U6 entry vector was transfected into TT cells. The expression of S100A13 mRNA and protein were detected by immunoflurescence、real-time RT-PCR and Western blot. The changes of release of FGF-1 were detected by indirect immunoflurescence and ELISA. Results: S100A13 was shown to be down regulated by 80% in TT cells transfected with S100A13 shRNA （S100A13 RNAi cells） .Indirect immunofluorescence indicated that FGF-1 was mostly localized in the cytosol and neucleus of TT cells in primary culture. When serum-deprivation strees was given to TT cells,FGF-1 in cytoplasm almost disappeared in the cells at 6h. ELISA also showed that the release of FGF-1 was inhibited by S100A13 down regulation （P<0.05）. Conclusion: These results indicated that the S100A13-shRNA pENTRTM/U6 entry vector could inhibit the expression of S100A13 in TT cells. The inhibition of S100A13 could reduce the release of FGF-1 in TT cells.Part5 Effects of Ca²⁺-chelating Agents on the Release of S100A13 and FGF-1 in Human Thyroid Cancer Cell Line TT under Serum-deprivationObjective: To investigate the effect of Ca²⁺-chelating agents on the release of S100A13 and FGF1 in human thyroid cancer cell line TT（TT cell） under serum-deprivation.To understand the role of Ca²⁺ in the release of S100A13 and FGF1 in TT cells. Methods: The effect of BAPTA-AM（2.5μmol/L） and EGTA （2.5mmol/L） on the changes of [Ca²⁺]in TT cells under serum-deprivation were deter分钟ed by confocal laser scanning microscopy. The release of FGF-1 at the time points of 0, 1, 2, 4 and 6h after serum-deprivation were detected by Western Blot in TT cells. The effect of BAPTA-AM and EGTA on distributions of S100A13 and FGF1 were detected by indirect immunofluorescence under serum-deprivation for 6h in TT cell, and the release of both proteins were detected by Western Blot and ELISA. Results: Confocal laser scanning of Ca²⁺ imaging indicated that [Ca²⁺]i of normal group cells were bout 0.1～0.3μmol/L at all the scanning times, and [Ca²⁺]i of serum-deprivated cells were also stable within 23 min, but accelerated to 1.6μmol/L at time of 23 min and then decreased to 0.3～0.6μmol/L at time from 40min to 60 min, and the average [Ca²⁺]i was higher than that in normal group （P<0.05）. Both of BAPTA-AM and EGTA could inhibit serum-deprivation induced the rise of [Ca²⁺], and the average [Ca²⁺]i was similar with normal group （P>0.05）.Western Blot showed that both expressions of S100A13 and FGF1 were downregualted at the time points of 4h and 6h after serum-deprivation. Both expression of the two proteins at the time points of 4h and 6h after serum-deprivation showed obvious difference compared to the time points of 0,1,2h （P<0.05, P<0.01）.Indirect immunofluorescence indicated that the florescence intensitys of both S100A13 and FGF-1 at 6h after serum-deprivation were weakened in TT cells and BAPTA-AM and EGTA could reverse the changes. Both expressions of S100A13 and FGF-1 were down regulated by the treatment of serum-deprivation for 6h, and the treatment of serum-deprivation together with BAPTA-AM or EGTA showed on effect on the expressions of S100A13 and FGF-1 in TT cells. ELISA showed that the level of FGF-1 in cell medium at 6h after serum-deprivation was higher than other 3 groups（P<0.05）. Conclusion: Ca²⁺-Chelating agent could inhibit the rise of [Ca²⁺] and reduce the release of S100A13 and FGF1 induced by serum-deprivation stress in TT cells.