| Objective: To investigate the effect of exogenous S100A13 overexpression on the proliferation of human thyroid cancer cell line TT. Then further studied whether the release of FGF-1 changed after inhibition of S100A13 gene and serum-deprivation.METHODS: The eukaryotic expression plasmid pcDNA3.1/NT-GFP-S100A13 was transfected into TT cells. The cells were selected by G418. The expression of green fluorescent protein (GFP) was observed under laser scanning microscope, and the expression of S100A13 mRNA and protein was detected by Real Time polymerase chain reaction (PCR) and Western blot. The effects of S100A13 on cell proliferation and cell cycle progression were measured by cell growth curve and flow cytometry. The S100A13-shRNA pENTRTM/U6 entry vector was transfected into TT cells. The expression of S100A13 mRNA and protein was detected by immunoflurescence,Real Time PCR and Western blot. Then TT cells were treated with S100A13 gene inhibition and serum-deprivation. The changes of release of FGF-1 were detected by indirect immunoflurescence,RT-PCR and ELISA.RESULTS: TT-S100A13-GFP and TT-GFP cells, which separately expressed S100A13 and pcDNA3.1/NT-GFP vector, were constructed succes??ly. 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, P<0.05)]; both S phase proportion and G2/M phase proportion were significantly higher in TT-S100A13-GFP cells than 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)].To assess whether knockdown of S100A13 gene expression affects on thyroid cancer cells, the specific S100A13 short-hairpin silence RNA (shRNA) was utilized to inhibit endogenous S100A13 expression in TT cells. Real Time PCR, Western blot and immunofluorescence analysis showed that S100A13 shRNA transfected TT cells (S100A13 RNAi cells) had a reduction of S100A13 gene and protein expression by 50%. A control shRNA, SR-PSOX shRNA had no effect on the S100A13 expression. Furthermore, immunofluorescence analysis revealed there were no differences in FGF-1 localization between S100A13 RNAi cells and TT cells in normal culture condition, in which most FGF-1 evenly distributed in the cytosol and nuclei. In contrast, S100A13 RNAi cells had a slight higher cytosol FGF-1 expression than TT cells did after 6 h serum-deprivation cultures. FGF-1 in culture media measured by ELISA also showed that the secretion of FGF-1 significantly reduced in S100A13 RNAi cells compared to that in TT cells (P<0.05) under serum-deprivation condition, indicating that knockdown S100A13 gene expression might prevent FGF-1 secretion in thyroid cancer TT cells. CONLUSION: Overexpression exogenous S100A13 gene could accelerate cell proliferation, and promote cell cycle progression of TT cells from G0/G1 phase to S and G2/M phase. The S100A13-shRNA pENTRTM/U6 entry vector transfected into TT cells could inhibit the expression of S100A13. FGF-1 release from TT cells a?? serum-deprivation. The inhibition of S100A13 could reduce the release of FGF-1. |
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