目的:探讨人乳头瘤病毒(human papillomavirus,HPV)16E6对宫颈癌细胞中高迁移率族蛋白B1(high mobility group box-B1,HMGB1)主动释放及细胞侵袭能力的影响。方法:构建HPV16E6表达质粒,并转染至宫颈癌C33A细胞,FCM法分析细胞存活率;半定量RT-PCR法检测转染24h前后C33A细胞中HPV16E6基因的表达水平;蛋白质印迹法检测转染前后C33A细胞的细胞核、细胞质以及细胞培养液中HMGB1蛋白水平的变化情况;Transwell实验检测细胞侵袭能力的变化。结果:HPV16E6基因转染C33A细胞后,细胞核中HMGB1蛋白水平明显降低(P<0.01),而细胞质及细胞培养液中HMGB1蛋白水平均明显升高(P<0.01)。转染后细胞存活率未见明显差异,但细胞穿膜个数明显多于未转染组(P<0.01)。结论:HPV16E6可诱导宫颈癌细胞内HMGB1的主动释放,提高肿瘤细胞的侵袭能力。
Summary： Cancer stem cells （CSCs） are considered responsible for the high recurrence rate in cervical carcinoma. It has been demonstrated that the signal transducer and activator of transcription 3 （STAT3） is involved in the oncogenesis and takes part in mediating the effects of maintaining stem cell phenotype and pluripotency by regulating the expression of stem cell-related transcription factors. However, the correlation between STAT3 and stem cell-related transcription factors in cervical cancer has not been elucidated. In this study, we established overexpressing plasmid （GV316-STAT3） and siRNA-STAT3 for transfecting Siha cells. Cells negative or positive for Nanog, Oct4, or Sox2 were selected by flow cytometry. Proliferation and differentiation rate of Siha cells was determined by detecting the efficiency of tumor sphere formation. The expression of Nanog, Oct4 and Sox2 （cancer stem cell markers） and STAT3 was detected by quantitative real-time PCR and immunoblotting for Siha cells and by immuno- histochemistry （IHC） for cervical tissues, respectively. The results showed that Nanog＋, Oct4＋, and Sox2＋ Siha-STAT3 over-expressing cells displayed the typical non-adherent spheres. The sphere for- mation efficiency was significantly different between Siha-STAT3 overexpressing cells and siRNA-STAT3 cells （P〈0.05）. Meanwhile, the expression levels of Oct4, Nanog and Sox2 rrtRNA and protein were significantly higher in Siha-STAT3 overexprssing cells than in siRNA-STAT3 cells （P〈0.05）. In addition, the positive rate of STAT3, Nanog, Oct4 and Sox2 in cervical cancer tissues was higher than that in chronic cervicitis group （P〈0.05）. There was a significantly positive relationship between STAT3 and Nanog or Oct4 or Sox2 expression （all P〈0.001）. These results suggested that Oct4＋, Sox2＋, and Nanog＋ cell population possesses stem cell properties in cervical cancer, which may contribute to cervical carcinogenesis and be regulated by STAT3.
