| ObjectiveResently studies showed that thyroid hormone could promote angiogenesis. Plasma membrane receptor integrinαvβ3played an important role in it,but the exact signaling pathway was not clear.In our study,human umbilical endothelial cells (HUVEC) were used to investigate that binding to membrane receptor integrin αvβ3, thyoid hormone exerts pro-angiogenic effects through epigenetic regulation and increasing angiogenic factor expression. And protein kinase D(PKD)/histone deacetylases5(HDAC5) signaling pathway plays an important role in it. Meanwhile, human glioblastoma cells (SNB19) were used to study the proliferation and apoptosis of tumor cell induced by thyroid hormone and its analogue (Tetrac).Methods(1) Study of pro-angiogenesis induced by thyroid hormone. HUVEC were cultured,and divided into normal control group,T4group (100nmol/L),Tetrac+T4group(Tetrac100nmol/L+T4100nmol/L),protein kinase C (PKC) inhibitor (2.5μmol/L) group,PKC inhibitor (2.5μmol/L)+T4(100nmol/L) group.Total cell proteins were extracted, and Western blot was used to measure phophorylation of PKD and HDAC5. RNA interfere was used,and HUVEC were transfected with PKD siRNA. Total cell protein were extracted after treated with T4for15min,30min, and Western blot was used to measure the phosphorylation of PKD and HDAC5. Nuclear and Cytoplasmic Extraction Kit was used to extract protein in cytoplasm.Phosphorylated HDAC5in cytoplasm of HUVEC under different treatment was measured by Western blot. Cultured HUVEC were divided into normal control group, T4group, Tetrac group, Tetrac+T4group,total RNA were extracted at2h,4h,6h, and bFGF mRNA was analyzed by RT-PCR. Meanwhile,cultured HUVEC were divided into normal control group, T4group, Tetrac group, Tetrac+T4group.Wound healing assay was used to record the migration distance of HUVEC at0h,8h,12h,24h.Cultured HUVEC were divided into normal control group, T4group, and tubulogenesis Assay in Matrigel was used to measure the number of tube-like structures in HUVEC.(2) SNB19were cultured and divided into normal control group, group (100nmol/L),Tetrac group(100nmol/L),Tetrac+T4group(different concentration Tetrac+100nmol/L T4).MTT was applied to assay the survival rate of SNB19treated for24h,48h,72h.Flow cytometry was used to detect the apoptostic rate of SNB19treated for48h.Results(1) Compared with normal control group, phosphrylation of PKD in T4group increased (P<0.05). Compared with T4group, phosphrylation of PKD in Tetrac group,Tetrac+T4group decreaed (all P<0.05), and effect of Tetrac was in a dose-dependent manner (P<0.05).Compared with T4group, phosphorylation of PKD in PKC inhitor group and PKC inhibitor group decreaed (all P<0.05). Compared with normal control group, phosphorylation of HDAC5in Tetrac group and Tetrac+T4group decreased (all P<0.05), but the effect of Tetrac was not in a dose-dependent manner. PKD siRNA could significantly inhibit PKD mRNA and protein,meanwhile phosphorylation of HDAC5in HUVEC transfected with PKD siRNA decreased (P<0.05). Compared with normal control group, phosphorylated HDAC5in T4group increased in cytoplasm, while decreased in PKC inhibitor+T4group and Tetrac+T4group (P<0.05).Compared with normal control group,bFGF mRNA increased in T4group (P<0.05), while decreased in Tetrac group and Tetrac+T4group (P<0.05).Compared with normal control group,migration distance of HUVEC in T4group was longer (P<0.05), compared with T4group, migration distance of HUVEC in Tetrac group and Tetrac+T4group were shorter (P<0.05). Compared with normal control group,number of tubular like structure in T4group was more (P<0.05).(2) The survival rate of SNB19in T4group was similar to that in normal control group.Compared with normal control group, the survival rate of SNB19in Tetrac group decreased in a dose-dependent manner (P<0.05).Compared with normal control group,apoptosis rate of SNB19in T4group was decreased a bit,but apoptosis rate of SNB19in Tetrac group increased in a dose-dependent manner (P<0.05).Conclusions(1) Thyroid hormone increased the expression of bFGF mRNA via integrin αvβ3/PKC/PKD/HDAC5singaling transduction pathway, played pro-angiogenesis in epigenetics. As to cultured HUVEC in vitro, thyroid hormone could promote the migration and tubulogenesis.(2) Thyroid hormone may promote the proliferation of tumor cell in some extent,inhibit the apoptosis of tumor cell.Tetrac,the deaminated thyroid hormone analogue, could inhibit the proliferation of tumor cell,increase the apoptosis of tumor cell by blocking the binding of thyroid hormone to integrin αvβ3. |
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