Expression Of PIAS3 In Gliomas And Its Effects On Apoptosis And Cell Cycle Of Glioma Cells.

Background and ObjectiveThe dysregulation of signaling pathway is associated with tumorigenesis. As an important signaling regulator, protein inhibitor of activated STAT 3 (PIAS3) regulates the activity of many transcription factors, including phosphorylated signal transducer and activator of transcription 3 (pSTAT3). It has been demonstrated that increased PIAS3 expression was observed in a variety of human malignancy. The pSTAT3 contributes to both cell proliferation and the secretion of immunosuppressive factors by tumor cells in tumorigenesis. Glioma is the most common type of primary tumor in human brain. The expression of PIAS3 in gliomas has not been absolutely definited, as well as its effects on apoptosis and cell cycle of glioma cells. In this study, it is examined whether the endogenous PIAS3 expression in gliomas is correlated with the malignant intensity by immunohistochemistry. To investigate the effects of PIAS3 on human glioma cells,the changes of pSTAT3, apoptosis and cell cycle were observed after the transfection of glioma cells with the constructed PIAS3 eukaryotic expression plasmid and RNAi vector.Methods1. The glioma samples and injuried brain tissue were collected and divided into groups according to the WHO standard. The expression of PIAS3 was examined in glioma samples and human glioma cell lines, U251 and CHG-5, by immunohistochemistry. It was analyzed whether its expression was correlated with the malignant intensity of gliomas by cell counting and statistics methods.2. The PIAS3/EGFP eukaryotic expression plasmid was designed and constructed. Two human glioma cell lines, U251 and CHG-5, were transfected with the constructed plasmid by liposome. The expression of fusion protein labeled with green fluorescence was observed by fluorescence microscope 48h later. Expressions of PIAS3 mRNA and protein were examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot in glioma cells divided into three groups, including non-transfected, mock-transfected and transfected cells. Apoptosis in three groups was measured by flow cytometry with a double-labeling means of FITC-conjugated annexin V and PI. Flow cytometry was also applied to assay the cell cycle of non-transfected and transfected cells.3. Three RNAi vectors were constructed and transfected into CHG-5 cells by liposome. The most efficient RNAi vector was selected through examining the expressions of PIAS3 mRNA in the transfected cells by semiquantitative RT-PCR. U251 and CHG-5 cells were transfected with the selected RNAi vector by liposome. After transfection 48h, the expressions of PIAS3 mRNA and protein were examined by semiquantitative RT-PCR and Western blot in glioma cells divided into three groups, including non-transfected, mock-transfected and transfected cells. The pSTAT3 level was detected by Western blot in the transfected cells with PIAS3 eukaryotic expression plasmid and selected RNAi vector, compared with control group. Apoptosis in three groups was measured by flow cytometry with a double-labeling means of FITC-conjugated annexin V and PI. Flow cytometry was also applied to assay the cell cycle of non-transfected and transfected cells.Results1. No expression of PIAS3 is observed in normal brain tissue. The positive rate of PIAS3 expression in total samples is 56% (28/50). The positive staining of PIAS3 is primarily in the cell nucleus, while a little of PIAS3 is in the cytoplasm. The expression of PIAS3 is implicated with glioma pathological grade. The positive rate of PIAS3 in gradeⅣis significantly higher than that in gradeⅠ～Ⅲ(P<0.01). Labeling indices in high grades (Ⅲ,Ⅳ), 22.6±14.4, is higher than that in low grades (Ⅰ,Ⅱ) , 5.7±6.5. (P<0.01). PIAS3 is also detected in the human glioma cell lines, U251 and CHG-5.2. The constructed PIAS3/EGFP eukaryotic expression plasmid is verified to be correct by restriction enzymes and sequence determination. The expression of fusion protein labeled with green fluorescence is observed in the U251 and CHG-5 cells transfected with constructed plasmid. The expressions of PIAS3 mRNA and protein in the transfected cells are significantly higher than that in the non-transfected and mock-transfected cells (P<0.01). The results suggest that PIAS3 gene transfection could increase the expression of PIAS3 in glioma cells. The rounding and floating cells are observed by microscope after transient transfection. Compared with non-transfected and mock-transfected cells, overexpression of PIAS3 can increase the apoptosis of transfected U251 (P<0.01) and CHG-5 cells (P<0.05). The percentage of S phase of transfected cells is higher than that of non-transfected cells, while the percentage of G2 phase is lower (P<0.05).3. The RNAi vector is constructed and selected, which could inhibit the expression of PIAS3 mRNA specially and efficiently. Compared with non-transfected and mock-transfected cells, RNAi down-regulates PIAS3 mRNA expression (P<0.01) in two lines of transfected cells and PIAS3 protein expression in the transfected U251 (P<0.01) and CHG-5 cells (P<0.05), which shows that RNAi could inhibit the PIAS3 expression in glioma cells efficiently. The pSTAT3 is detected in U251 and CHG-5 cells by immunocytochemistry. Compared with non-transfected cells, the pSTAT3 is decreased in U251 and CHG-5 cells (P<0.05) after overexpression of PIAS3, and it is increased (P<0.05) after RNAi. No marked morphological changes are observed by microscope except for more cells after RNAi. RNAi induces U251 and CHG-5 cells to be more resistant to apoptosis (P<0.01), and the changes of cell cycle are induced. Bigger percentage of G2 phase and smaller percentage of S phase is detected in U251 cells (P<0.05), meanwhile in the CHG-5 cells is bigger percentage of G1 phase and smaller percentage of G2 phase (P<0.05) compared with non-transfected cells.Conclusions1. No expression of PIAS3 is detected in human normal tissue. PIAS3 is expressed in glioma samples and glioma cell lines. The PIAS3 expression (positive rate and labeling indices) is correlated with glioma pathological grade (malignant intensity).2. The transfection with constructed PIAS3 eukaryotic expression plasmid could increase the expression of PIAS3 in glioma cells. PIAS3 gene transfection increases the apoptosis of transfected U251and CHG-5 cells and leads to bigger percentage of S phase and smaller percentage of G2 phase.3. RNAi could inhibit the expression of PIAS3 in glioma cells. The PIAS3 gene transfection and RNAi respectively decreases and increases pSTAT3 in U251 and CHG-5 cells. RNAi induces U251 and CHG-5 cells to be more resistant to apoptosis, followed by the changes of cell cycle. They are bigger percentage of G2 phase and smaller percentage of S phase in U251 cells, and bigger percentage of G1 phase and smaller percentage of G2 phase in the CHG-5 cells.