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  • Title: [S100A7 promotes the metastasis and epithelial-mesenchymal transition on HeLa and CaSki cells].
    Author: Tian T, Hua Z, Wang LZ, Wang XY, Chen HY, Liu ZH, Cui ZM.
    Journal: Zhonghua Fu Chan Ke Za Zhi; 2018 Feb 25; 53(2):99-105. PubMed ID: 29534378.
    Abstract:
    Objective: To elucidate the impact of over-expression of S100A7 on migration, invasion, proliferation, cell cycle, and epithelial-mesenchymal transition (EMT) in human cervical cancer HeLa and CaSki cells. Methods: (1) Immunohistochemistry of SP was used to examine the expression of S100A7 in 40 cases of squamous cervical cancer tissues and 20 cases of normal cervical tissues. (2) The vectors of pLVX-IRES-Neo-S100A7 and pLVX-IRES-Neo were used to transfect human cervical cancer HeLa and CaSki cells, and the positive clones were screened and identified. Next, transwell migration assay, cell counting kit-8 (CCK-8) assay and fluorescence activating cell sorter (FACS) were used to detect the effect of S100A7-overexpression on the migration, invasion, proliferation and cell cycle of cervical cancer cells. Furthermore, western blot was performed to observe the expression of epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin, vimentin, and fibronectin) of EMT. Results: (1) S100A7 expression was significantly higher in cervical squamous cancer tissues (median 91.6) than that in normal cervical tissues (median 52.1; Z=-2.948, P=0.003) . (2) Stable S100A7-overexpressed cells were established using lentiviral-mediated gene delivery in HeLa and CaSki cells. S100A7 was detected by real-time quantitative reverse transcription PCR, S100A7 mRNA of S100A7-overexpressed cells were 119±3 and 177±16, increased significantly compared with control groups of median (P<0.01) . Compared with the control cells, the number of S100A7-overexpressed HeLa and CaSki cells that passed the transwell membrane assay were increased significanatly (572±51 vs 337±25, P<0.01; 100±8 vs 41±4, P<0.01) .Matrigel invasion assay showed that the number of S100A7-overexpressed HeLa and CaSki cells that passed the transwell membrane were respectively 441±15 and 110±14, elevated significantly compared with control cells (156±21 and 59±7; P<0.05) . However, S100A7 overexpression didn't influence the proliferation and cell cycle progression of HeLa and CaSki cells (P>0.05) . Expression of E-cadherin was dramatically decreased, while N-cadherin, vimentin, and fibronectin increased in S100A7-overexpressed cells. Conclusion: S100A7 enhances the migration, invasion and EMT of HeLa cells and CaSki cells, and may be plays an important role in the development of cervical cancer. 目的：探讨S100钙结合蛋白A7（S100A7）在子宫颈癌组织中的表达及其对子宫颈癌HeLa和CaSki细胞迁移、侵袭、增殖及上皮间质转化（EMT）的影响。 方法： （1）选择2007年4月至2008年3月青岛大学附属医院妇科收治的因子宫颈鳞癌进行手术治疗的癌组织标本共40份作为子宫颈癌组，以同期因妇科良性肿瘤行子宫全切除术的正常子宫颈组织20份作为对照组，应用免疫组化SP法检测两组子宫颈组织中S100A7蛋白的表达。（2）通过慢病毒包装系统将重组质粒pLVX-IRES-Neo-S100A7和空质粒pLVX-IRES-Neo分别转染子宫颈癌HeLa和CaSki细胞（分别为转染组和对照组），筛选稳定表达S100A7基因的HeLa和CaSki细胞系并进行鉴定。穿膜（transwell）小室体外实验检测两组子宫颈癌细胞的迁移和侵袭能力，活细胞计数（CCK-8）法检测两组子宫颈癌细胞的增殖能力，流式细胞仪检测两组子宫颈癌细胞的细胞周期比例，蛋白印迹（western blot）法检测两组子宫颈癌细胞中EMT标志分子——上皮型钙黏蛋白（E-cad）、神经型钙黏蛋白（N-cad）、波形蛋白（Vim）和纤连蛋白（FN）的表达。 结果： （1）免疫组化SP法检测显示，子宫颈癌组、对照组子宫颈组织中S100A7蛋白表达水平的中位数分别为91.6和52.1，两组比较，差异有统计学意义（Z=-2.948，P=0.003）。（2）本研究成功构建稳定的过度表达S100A7基因的HeLa和CaSki细胞系，转染组HeLa和CaSki细胞中S100A7 mRNA的表达水平分别为119±3和177±16，均明显高于各自的对照组（均设为1，P均<0.01）；转染组HeLa和CaSki细胞中S100A7蛋白的表达强度均明显高于各自对照组HeLa和CaSki细胞。transwell小室体外迁移实验显示，转染组HeLa和CaSki细胞的穿膜细胞数分别为（572±51）和（100±8）个，均明显高于各自的对照组[分别为（337±25）和（41±4）个；P均<0.01]；应用matrigel基质的transwell小室进行细胞侵袭能力检测，结果显示，转染组HeLa和CaSki细胞的穿膜细胞数分别为（441±15）和（110±14）个，均明显高于各自的对照组[分别为（156±21）和（59±7）个；P均<0.05]。CCK-8法和流式细胞仪检测显示，转染组HeLa和CaSki细胞的增殖率和细胞周期比例分别与各自的对照组比较，差异均无统计学意义（P均>0.05）。western blot法检测显示，与各自的对照组比较，转染组HeLa和CaSki细胞中E-cad蛋白的表达强度均明显减弱，而N-cad、Vim和FN蛋白的表达强度均明显增强。 结论： S100A7基因促进子宫颈癌HeLa和CaSki细胞的侵袭转移和EMT，可能与子宫颈癌的发生、发展有关。.
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