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Objective To discuss the activation and biological effects of STAT3 signaling pathway and its negative regulator PIAS3 in glioma cells. Methods Immunohistochemistry(IHC)was used to test the expression of p?STAT3 and PIAS3 in gliomas. AG490(60μmol/L),the in?hibitor of STAT3 signaling pathway,was used to treat U118 and U87 cells for 24/48 h,and the method of MTT assay was taken to evaluate the pro?liferation after AG490 treatment. The expression of p?STAT3 and PIAS3 was also examined by immunofluorescence(IF). Results There was no obvious significance between p?STAT3 and PIAS3 in nuclei or cytoplasm at different grades of gliomas. Whereas,p?STAT3 and PIAS3 were nega?tively correlated in the nuclei of vary grades malignancy gliomas. After AG490 treatment,U118 cells showed no obvious quantitative changes. How?ever,U87 cells showed obvious growth inhibition. IF results showed that there was no significant change at the levels of p?STAT3 and PIAS3 after AG490 treatment in U118 cells. However,the expression of p?STAT3 in the nuclei was down?regulated,and PIAS3 showed obvious nuclear translo?cation in U87 cells. Conclusion Nuclear translocation of PIAS3 plays the key role in modulating JAK/STAT signaling activation and inhibiting glioma cells proliferation.
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Objective:To observe the effect of the proteininhibitor of activated STAT 3 (PIAS3) on the proliferation and apopto-sis of U251 glioma cells after PIAS3 expression was inhibited by RNAi. Methods: Three RNAi expression vectorstargeting PIAS3 were constructed and transfected into CHG-5 cells by liposomein vitro. The most efficient RNAi vector was subsequently selected by examiningthe mRNA expressions of PIAS3 in the transfected cells by semi-quantitativeRT-PCR. The selected RNAi vector was then transfected into U251 cells. After 48h of transfection, the mRNA and protein expressions of PIAS3 in glioma cellswere examined by semi-quantitative RT-PCR and western blot. Apoptosis wasobserved by flow cytometry using a double-staining method with FITC-con-jugatedannexin V and PI. Flow cytometry was also applied in cell cycle assay. Results:RNAidownregulated the mRNA (P<0.01) and protein (P<0.01) expressionsof PIAS3 in transfected cells.RNAi promoted the resistance of U251 cells to apoptosisand subsequently al-tered the cell cycle. A high percentage of G2 phaseand a low percentage of Sphase were observed in U251 cells. Conclusion:The down-regulation of PIAS3arrested U251 cells in the G2 phase andinduced the resistance of U251 cells to apoptosis.
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Objective To construct the eukaryotic expression recombinant PIAS3 plasmid fused with Myc protein and express the fusion protein Myc-PIAS3.Methods The full length PIAS3 fragment of 1 851bp was amplified by PCR and ligated into pMD18-T vector. The full length PIAS3 fragment was subcloned into eukaryotic pCMV-Myc vector between SalⅠ and NotⅠ sites.The recombinant pCMV-Myc-PIAS3 plasmid was trandfected into PC3 cells.The eukaryotic expression Myc-PIAS3 protein was checked by Western blotting with Myc antibody.Results The recombinant plasmid showed right sequence by the full length sequencing.The recombinant plasmid of pCMV-Myc-PIAS3 was identified by enzyme digestion.As expected,by EcoRⅠ digestion,it showed two bands of 4 357bp and 1 318 bp. By XbaⅠdigestion,it showed two bands of 3 291 bp and 2 384 bp.The sequencing result showed a N-terminal Myc of 13 amino acids followed with PIAS3 gene sequence in right reading frame.The pCMV-Myc-PIAS3 plasmid was transfected into prostate cancer PC3 cells.A specific protein expression band at relative molecular mass 68 000 was obtained by using Myc-antibody with Western blotting method.Conclusion The recombinant plasmid of pCMV-Myc-PIAS3 is sucssesefully constructed,and Myc-PIAS3 fusion protein is sucssesefully expressed.
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0.05),but existed between grades Ⅰ-Ⅲ and grade Ⅳ(P0.05).Conclusion PIAS3 is expressed in human gliomas,closely correlated with the pathological grade and malignant intensity.PIAS3 may be involved in the development and progression of gliomas.