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Effect on Proliferation and Invasion of Laryngeal Cancer Hep-2 Cells by Stable Transfection of Recombinant STAT3 Gene

Author: SunHongCun
Tutor: WangJunGe
School: Hebei Medical University
Course: Otorhinolaryngology
Keywords: Laryngeal RNA interference STAT3 Stably transfected Cell Proliferation Cell invasion
CLC: R739.6
Type: Master's thesis
Year: 2010
Downloads: 48
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Abstract


Objective: RNAi (RNA interference) technology to silence STAT3 (signal transducer and transcription activator), screened cell lines stably expressing siRNA-STAT3 gene was observed Hep-2 cell proliferation and invasion force, to further explore the possible mechanism of STAT3 role in the proliferation and invasion of Hep-2 cells at the protein level. To find inhibition of laryngeal cancer proliferation and invasion of a new method for the clinical treatment of laryngeal cancer by gene silencing technology to provide experimental support and theoretical basis. Methods: To construct the eukaryotic expression vector of recombinant plasmid PGPU6/GFP/Neo-siRNA-STAT3 recombinant expression plasmid liposome LipofactaminTM2000 human laryngeal carcinoma Hep-2 cells transfected by G418 last monoclonal screening, Western-blot blot method identification, thereby screening out the stable cell lines expressing siRNA-STAT3. The experiment was divided into three groups: siRNA-STAT3 group, siRNA-shNC group and the control group. Using flow cytometry detection of Cyclin D1, P21, ICAM-1, the expression of VEGF protein levels and cell cycle; MTT assay inoculated plates 1d, 2d, 3d, 4d, 5d, after 6 days of laryngeal cancer Hep- 2 cell proliferation; MTT was used to observe the changes of adhesion; cells scratches were observed migration; the Transwell method to detect the invasive ability of. Results: (1) Construction the recombinant pGPU6/GFP/Neo-siRNA-STAT3 of plasmid vectors by template annealing and Vectors linearization step, to construct a recombinant plasmid expression vector. The digestion results show that all are positive recombinant plasmid vectors. (2) cell transfection and G418 to Filter 6 hours after transient transfection Multiple green fluorescent protein (GFP) expression in Hep-2 cells was observed under a fluorescence microscope, GFP expression was significantly increased after 24 hours. G418 selection results showed that all cells of the control group died, siRNA-STAT3 transfection group and the siRNA-shNC turn transfection group of cells appear obvious and a large number of cell clones colony formation. (3) Western-blot identification of p-STAT3 expression results showed that siRNA-STAT3 group of p-STAT3 expression was significantly reduced, the siRNA-shNC group and blank control group cells p-STAT3 expression there was no decline. (4) MTT cell proliferation status Hep-2 cells stably transfected with the siRNA-STAT3, independent plasmid group compared with the control group and transfection of siRNA-shNC, two days later began to exhibit more pronounced inhibitory effect (P lt ; 0.05), for 6 days in the control group cell growth slows, siRNA-STAT3 group Hep-2 cell growth inhibition was still evident (P lt; 0.05), its inhibitory effect with time is more obvious, and showed some time inhibiting effect relationship. (5) colony formation assay after 2 weeks was observed that the three groups have certain monoclonal colony formation. The results showed that siRNA-STAT3 group Hep-2 cell colony formation rate (15.17 ± 2.32)%, significantly lower than the the siRNA-shNC group (24.33 ± 1.97)% and the control group (26.33 ± 2.16)% (P lt; 0.05). (6) by flow cytometry (FCM) Cell cycle applications by FCM cell cycle distribution of transfected recombinant siRNA-STAT3 plasmid Hep-2 cells in G1 phase ratio increased from (60.7 ± 0.9)% to (66.0 ± 2.0)% (P lt; 0.05), S of the percentage of (34.8 ± 1.1)% to (24.6 ± 1.9)% (P lt; 0.05) (7) FCM detection of Cyclin D1, P21, ICAM-1, VEGF protein expression observed siRNA-STAT3 group cyclin DL, ICAM-1, VEGF protein expression fluorescence index were 0.71 ± 0.08 (P lt; 0.05), 0.83 ± 0.06 (P lt; 0.05), 0.74 ± 0.02 (P lt; 0.05) were lower than siRNA-shNC group, siRNA-STAT3 group P21 protein fluorescence index (1.39 ± 0.10) is higher than the siRNA-shNC group (P lt; 0.05). (8) The cell adhesion test by MTT assay cells on Matrigel matrix adhesion inoculated 96-well culture plates observed 20 minutes after the adhesion rate of cells in each group (P gt; 0.05) or less, but there are certain trends resistance, adhesion rate of siRNA-STAT3 group after 40 minutes less than siRNA-shNC group and the control group (P lt; 0.05), this inhibitory effect is more pronounced after 60 minutes (P lt; 0.05) (9) cell migration test grown in culture plates after 48 hours, see the blank control group and of siRNA-shNC group Hep-2 cells under a fluorescence microscope is significantly faster than the growth rate of the scratch area siRNA-STAT3 group, while the control group and the the siRNA-shNC group Hep-2 cells to the scratch area growth rate was not significantly different. 400 × optical microscope (10) each group of cells were inoculated on Transwell chambers indoor 24 hours in vitro invasion assay siRNA-STAT3 group through the Matrigel matrix and membrane of cells (85.20 ± 4.92) was significantly less than observed in five fields of view of the random the control group (122.8 ± 7.50) and siRNA-shNC group (126.4 ± 8.73) (P lt; 0.05). Conclusion: 1, was successfully constructed recombinant pGPU6/GFP/Neo-siRNA-STAT3 plasmid vector. 2, the application G418 selection stably expressing siRNA-STAT3 gene in laryngeal carcinoma Hep-2 cell lines. Hep-2 cell cycle, inhibition of STAT3 activity formed G1 arrest, the proliferation ability and monoclonal forming ability decreased significantly. Description of laryngeal cancer cells with unlimited proliferative capacity and high expression of STAT3 have some relevance. 4, the experimental application of RNA interference to silence STAT3 activity found to inhibit laryngeal cancer Hep-2 cell adhesion, migration and invasion. Speculated that the expression of STAT3 gene in laryngeal carcinoma cell adhesion, migration and matrix degradation and increased invasion and reach the surrounding tissue and distant metastasis and colonization opportunities for growth. STAT3 plays an important role in the invasion and metastasis of laryngeal cancer. Blocking STAT3 signaling pathway downstream of STAT3 regulation protein expression of CyclinD1 of VEGF, ICAM-1 expression reduced p21 protein expression was increased. The prompt expression of CyclinD1, P21, VEGF, ICAM-1 protein may participate in the induction of STAT3 STAT3 signaling pathway in laryngeal squamous cell carcinoma cell proliferation, invasion, invasion and metastasis.

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