核因子NF-κB p65下调对蛋白激酶抑制宫颈癌HeLa细胞增殖及侵袭的影响

Effect of down-regulation of nuclear factor NF-κB p65 on protein kinase R to induce cellular proliferation and invasion of cervical cancer HeLa cells

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 of down-regulation of nuclear factor-κB （NF-κB）p65 on protein kinase R （PKR） to induce cellular proliferation and invasion of cervical cancer HeLa cells, and to explore their possible mechanism. Methods： Recombinant plasmid vector which contained short hairpin RNA （shRNA） interfering sequence aiming at the target of NF-κB p65 gene was constructed and transfected into HeLa cells. The expression levels of NF-κB p65 and PKR mRNAs were detected by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） assay. The expression levels of NF-κB p65, PKR, phospho-PKR （p-PKR） and phospho-eukaryotic initiation factor 2α （p-eIF2α） were detected by Western blotting. The proliferative ability of HeLa cells after NF-κB p65 gene silencing was detected by MTT assay. The invasive ability of HeLa cells after NF-κB p65 gene silencing was detected by Transwell invasion assay. Results： The recombinant plasmid vector pSilencer-NF-κB p65-shRNA was successfully constructed. Based on interfering the expressions of NF-κB p65 mRNA and protein, the relative expressions of PKR mRNA and protein （including p-PKR） of pSilencer-NF-κB p65-shRNA transfection group were significantly higher as compared with those of the pSilencer-negative control （NC）-shRNA transfection group and the blank control group （only transfection reagent was added） （all P 〈 0.05）, and the relative expression of p-eIF2α protein of pSilencer-NF-κB p65-shRNA transfection group was also significantly higher （P 〈 0.05）; but the proliferative and invasive abilities of HeLa cells of pSilencer-NF-κB p65-shRNA transfection group were significantly decreased （both P 〈 0.05 ）. Conclusion：NF-κB p65 can down-regulate the expression and activity of PKR to restrain the activation of PKR/eIF2α signal transduction pathway, and promote the proliferation and invasion of cervical cancer HeLa cells. Therefore, NF-κB p65 may play an important role in the generation and development of cervical cancer.