| Objectives: We selected out cell lines stably expressing siRNA-STAT3 to observe the proliferative and invasive ability of Hep-2 cells by RNAi(RNA interference) technology further explore the possible mechanisms at the protein level that STAT3(Signal transducer and activator of transcription 3) gene was involved in proliferation and invasion of laryngeal cancer Hep-2 cells to find a new method that proliferative and invasive ability of laryngeal cancer cells could be inhibited to provide experimental support and theoretical basis for the clinical treatment of laryngeal cancer.Methods: Constructed the recombinant plasmid vector(PGPU6/GFP/Neo- siRNA-STAT3). Transfected the recombinant plasmid into human laryngeal cancer Hep-2 cells by LipofactaminTM2000. Identificated the expression of STAT3 protein after G418 continued monoclonal selection by western-blot analysis. And then we selected out the cell lines stably expressing siRNA-STAT3 gene. This study was divided into three groups: siRNA-STAT3 group, siRNA-shNC group and blank control group. Examined CyclinDl, P21, ICAM-1, VEGF protein expression and cell cycle analysis by Flow CytoMeter and proliferative ability after Hep-2 cells were inoculated in culture plate for 1d, 2d, 3d, 4d, 5d, 6d by MTT assay. Examined their adhesive ability by MTT assay, migratory ability by scratch assay and invasive ability by transwell invasion assay respectively.Results:(1) Construction of the recombinant plasmid vector (pGPU6/GFP /Neo-siRNA-STAT3). By template annealing and vector linearization, we constructed the recombinant plasmid vector. Enzyme digestion results explained all were positive recombinant plasmid vector.(2) Cell transfection and G418 selection. We observed many Hep-2 cells with green fluorescent protein (GFP) under fluorescence microscopy after transient transfection for 6 hours. 24 hours later, Hep-2 cells with GFP significantly increased. G418 selection results showed that cells in the blank control died while the celld in two other groups had apparent cell colonies with a large number of cells.(3) Identification of p-STAT3 expression. The western-blot analysis results showed that p-STAT3 expression in siRNA-STAT3 group significantly decreased while p-STAT3 expression in siRNA-shNC group and blank control group still maintained unchanged.(4) Detection of cell proliferation by MTT assay. Hep-2 cells grew much more slowly after two days because they were transfected with recombinant siRNA-STAT3 plasmid(P <0.05), compared with the siRNA-shNC group and blank control group. Even after 6 days the cells of two controls grew a bit slowly, the Hep-2 cells growth inhibition of siRNA-STAT3 group was still more evident (P<0.05).(5) Plate colony forming assay. After 2 weeks, we observed there was a certain monoclonal colony-forming in each group. The results showed that colony-forming rate in siRNA-STAT3 group (15.17±2.32)% was less than that in siRNA-shNC group (24.33±1.97)% and blank control group (26.33±2.16)% (P<0.05).(6) Cell cycle analysis by FCM. We detected G1 phase ratio of Hep-2 cells in siRNA-STAT3 group increased from (60.7±0.9) to (66.0±2.0)% (P<0.05)while S phase ratio in siRNA-STAT3 group decreased from (34.8±1.1) % to (24.6±1.9)% (P<0.05).(7) CyclinDl, P21, ICAM-1, VEGF protein expression. FCM results shew fluorescence index of CyclinD1 protein (0.71±0.08) (P<0.05), ICAM-1 protein (0.83±0.06) (P<0.05), VEGF protein(0.74±0.02) (P<0.05) in siRNA-STAT3 group was less than that in siRNA-shNC Group while fluorescence index of P21 protein in siRNA-STAT3 group(1.39±0.10) (P<0.05)was more than that in siRNA-shNC Group.(8) Cell adhesion assay. We detected their adhesive ability in Matrigel matrix by MTT assay and found their adhesion rate was not obvious after the cells were inoculated in 96-well culture plates for 20 minutes(P=0.98). However, it was a certain positive trend. After 40 minutes, their adhesion rate in the siRNA-STAT3 group was less than that of siRNA-shNC group and blank control group (P<0.05). This effect was more obvious after 60 minutes (P<0.05).(9) Cell migration assay. Under fluorescence microscopy, we saw the growth of Hep-2 cells to scratch zone in the blank control group and siRNA-shNC group was significantly faster than that in siRNA-STAT3 Group after cells were inoculated in culture plate for 48 hours. But growth of Hep-2 cells to scratch zone between the blank control group and siRNA-shNC group was not significant.(10) Transwell invasion assay in vitro. After Hep-2 cells in each group were inoculated in Transwell chambers for 24 hours, under optical microscope (400×) we observed they in siRNA-STAT3 group (85.20±4.92) (P<0.05) passing through the matrigel were less than that in the blank control group (122.8±7.50) and siRNA-shNC group (126.4±8.73).Conclusions:(1) We successfully constructed recombinant pGPU6/GFP/ Neo-siRNA- STAT3 plasmid vector.(2) We successfully selected out laryngeal cancer Hep-2 cells stably expressing siRNA-STAT3 gene with G418.(3) Hep-2 cells were blocked in the G0/G1 phase for the inhibition of activity of STAT3. The proliferative ability and single-cell colony forming ability of Hep-2 cells was suppressed for the activity of STAT3 was silenced by RNAi technology. It suggest that the strong proliferation of laryngeal cancer have association with high expression of STAT3.(4) In this study we found the adhesive, migratory and invasive ability of Hep-2 cells was even inhibited after the activity of STAT3 was blocked by RNAi technology. We speculated that the expression of STAT3 gene of laryngeal cancer cells by improving its adhesion, migration and degradation of matrix increased the opportunities to invade the surrounding tissue and have a distant metastasis and growth.So it suggest that the STAT3 signaling pathway activation plays an important role in the progression of invasion of laryngeal cancer.(5) We observed ICAM-1, CyclinDl, VEGF protein expression elevated while P21 protein expression reduced after the activity of STAT3 was blocked by RNAi technology. We conclusion that ICAM-1, CyclinDl, P21, VEGF protein induced STAT3may be involved with the regulation of STAT3 signal transduction. So STAT3 could enhance the proliferative ability and the invasive ability of Hep-2 cells. |
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