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Effect of STAT1-shRNA-mediated Gene Silencing on Biological Characteristics in the Cervical Cancer Cell Line Hela

Author: RuanShaSha
Tutor: WangZeHua；ZhaoHong
School: Huazhong University of Science and Technology
Course: Obstetrics and Gynaecology
Keywords: STAT1 Cervical cancer RNAi Cervical cancer cell line Hela proliferation migration invasion
CLC: R737.33
Type: Master's thesis
Year: 2011
Downloads: 8
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Abstract


PartⅠEffects on STAT1 gene in the tissue of human normal cervix, cervical precancerous lesions and cervical cancer Objective: To detect the effects of STAT1-shRNA on biologic of STAT1 gene on the tissue of human normal cervix, cervical precancerous lesions and cervical cancer.Methods: The expression of STAT1 gene in 1 normal cervical tissue, 11 cervical precancerous lesions and 28 cervical cancer cases were detected by SABC immunohistochemistry using monoclonal antibody of STAT1.Results: STAT1 protein in normal cervical tissue was absent,partly expressed in cervical precancerous lesions, while it was detected both in atypia cells and stromal cells of cervical cancer. The positive rate was significantly higher in cervical cancer than in the tissue of human normal cervix, cervical precancerous lesions.Conclusion: The expression of STAT1 associated with the development of cervical cancer.PartⅡShort hairpin RNA (shRNA) targeting STAT1 genes silence its expression in cervical cancer cell line Hela.Objective: To silence genes STAT1 in cervical cancer cell line Hela using short hairpin RNA (shRNA) interference technology, and to establish stable transfected cell lines.Methods: STAT1-shRNA expression vector mediated by Liposomes was transfected into Hela cells. Transfected cells were selected by G418 to become stably transfected cells by using the biotechnologies of cloning. The expression levels of STAT1 mRNA and protein were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting.Results: STAT1-shRNA expression vector was transfected into Hela cells successfully. The expression level of STAT1 mRNA and protein is significantly decreased(P<0.05). Conclusion: STAT1-shRNA expression vector effective in inhibiting the expression of STAT1 in cancer cell line Hela. Successfully established stable transfected STAT1-shRNA eukaryotic expression vector cell line (STAT1-shRNA-Hela) and stable cell line transfected with empty vector (Mock-Hela) laying the foundation for subsequent experiments.PartⅢEffects on biological behaviors of Hela cells by RNA interference targeting STAT1 genes silencingObjective: To explore the effects of STAT1-shRNA on biological behaviors of Hela cells, such as proliferation migration and invasion, by RNA interference targeting STAT1 gene silencing.Methods: cellular proliferation was examined by MTT assay. Transwell chamber model was used to assess the effect of the STAT1-shRNA on the migration and invasion of Hela cells.Results: (1) Compared with groups including transfection reagent control (Mock-Hela) and blank control(Hela cells), the proliferation of experimental group (STAT1-shRNA-Hela) was increased(P<0.05). (2) In vitro migration assay, the number of transmembrane cells of experimental group was (25.66±1.15), while the number of transfection reagent control and control group was (18.33±1.52), (16±1) respectively. The number of penetrating cells in experimental group is more than control groups (P <0.05). There was no significant difference between control groups (P> 0.05). (3)In vitro invasion assay, the number of transmembrane cells of experimental group was(10±1), while the number of transfection reagent control and control group was (5.33±0.58), (4±1) respectively. The number of penetrating cells in experimental group was more than control groups (P <0.05). There was no significant difference between control groups (P> 0.05).Conclusion: ShRNA targeting STAT1 gene can promote the proliferation of Hela cells, and enhance the ability of migration and invasion of Hela cells. STAT1 may be a potential tumor suppressor gene in cervical cancer.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Uterine tumors
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