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目的 :探讨下调核因子-κB(nuclear factor-kappa B,NF-κB)p65对宫颈癌HeLa细胞体外增殖和侵袭能力的影响及可能的分子机制。方法 :构建靶向NF-κB p65基因的短发夹RNA(short hairpin RNA,shRNA)重组载体pSilencer-NF-κB p65-shRNA,并用脂质体法转入HeLa细胞;采用半定量RT-PCR法检测转染pSilencer-NF-κB p65-shRNA后对NF-κB p65及蛋白激酶R(protein kinase R,PKR)mRNA表达的影响;蛋白质印迹法检测对NF-κB p65和PKR蛋白,以及PKR和其下游底物真核细胞翻译启始因子2α(eukaryotic initiation factor 2α,eIF2α)蛋白磷酸化的影响;采用MTT法检测NF-κB p65基因沉默后对HeLa细胞增殖活性的影响;Transwell小室法检测对HeLa细胞侵袭能力的影响。结果 :成功构建了重组载体pSilencer-NF-κB p65-shRNA;在沉默NF-κB p65 mRNA及蛋白表达的基础上,PKR mRNA及蛋白(包括磷酸化-PKR)的表达水平均明显上调(P均<0.05),磷酸化-eIF2α蛋白的表达水平亦明显上调(P<0.05),而HeLa细胞的增殖及侵袭能力显著降低(P<0.05和P<0.01)。结论 :NF-κB p65可通过下调PKR的表达及活性,抑制PKR/eIF2α转导通路的激活,促进HeLa细胞的增殖及侵袭能力,在宫颈癌的发生和发展过程中可能起重要作用。
Objective: To investigate the effect and possible molecular mechanism of downregulation of nuclear factor-kappa B (p65) on the proliferation and invasion of cervical cancer HeLa cells in vitro. Methods: The recombinant plasmid pSilencer-NF-κB p65-shRNA targeting short hairpin RNA (shRNA) of NF-κB p65 was constructed and transfected into HeLa cells by lipofectamine. The expression of NF-κB p65 and protein kinase R (PKR) mRNA in transfected pSilencer-NF-κB p65-shRNA was detected by Western blotting. The expression of NF-κB p65 and PKR proteins, The effect of NF-κB p65 gene silencing on HeLa cell proliferation was detected by MTT assay. Transwell chamber assay was used to detect the effect of HeLa Impact of cell invasiveness. Results: The recombinant vector pSilencer-NF-κB p65-shRNA was successfully constructed. The expression of PKR mRNA and protein (including phospho-PKR) was significantly up-regulated on the basis of silencing NF-κB p65 mRNA and protein <0.05). The expression of phosphorylated-eIF2α protein was also significantly increased (P <0.05), while the proliferation and invasion ability of HeLa cells was significantly decreased (P <0.05 and P <0.01). CONCLUSION: NF-κB p65 may play an important role in the development and progression of cervical cancer by down-regulating the expression and activity of PKR, inhibiting the activation of PKR / eIF2α transduction pathway and promoting the proliferation and invasion of HeLa cells.