Effect of HPV E6 on Proliferation，Invasion and Migration of Cervical Cancer Cells Through Rap1 Signaling Pathway
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摘要:
目的 探讨人乳头瘤病毒E6蛋白（human papillomavirus E6 protein，HPV E6）对宫颈癌Hela细胞增殖、侵袭和迁移的影响及潜在分子机制。 方法 基于Illumina Hiseq 4000转录组测序技术检测12例不同程度的宫颈病变组织之间的基因表达特征，筛选与HPV相关的差异基因；利用GO、KEGG Pathway 方法对差异基因进行生物学功能富集分析；Western blotting检测正常宫颈上皮细胞HCerEpic、宫颈癌细胞Hela中Rap、Rap1GAP蛋白表达水平；通过平板克隆实验、Transwell实验和划痕实验检测其增殖、侵袭及迁移能力；通过sh-E6慢病毒转染下调细胞内HPV E6表达，分为对照组（Hela细胞）、空载组（sh-NON）和sh-E6组（低表达HPV E6）；下调HPV E6表达，采用平板克隆和CCK-8实验分别检测每组细胞增殖能力和细胞活力；Transwell和划痕实验检测细胞侵袭、迁移能力；Western blotting检测各组细胞中E6、Rap1、Rap1GAP、CyclinD1、E-cadherin、N-cadherin蛋白的表达水平。在sh-E6组基础上过表达Rap1，Western blot检测细胞中Rap1通路及相关蛋白的表达水平；平板克隆实验、Transwell实验和划痕实验分别检测细胞增殖、侵袭和迁移能力。 结果 测序结果显示，与正常宫颈HPV阴性比较，HPV阳性的正常宫颈、CIN 、宫颈癌组织中差异上调表达的基因分别有340、864和 1036 个；3个数据集寻找共有差异基因共24个。GO|KEGG 富集分析24个差异表达基因主要富集在Rap1相关信号通路。与HCerEpic细胞相比，Hela细胞内Rap1表达升高，Rap1GAP表达降低（P < 0.01）；其细胞的增殖、侵袭和迁移能力增强（P < 0.01）；下调HPV E6表达后，与Hela组比较，sh-E6组细胞的增殖、细胞集落形成、侵袭和迁移能力降低（P < 0.001）；Western blotting表明，与Hela组比较，sh-E6组细胞内Rap1、CyclinD1、N-cadherin蛋白表达降低，Rap1GAP和E-cadherin蛋白表达升高（P < 0.001）。与sh-E6组相比，过表达Rap1组（sh-E6/OE Rap1）细胞内Rap1/Rap1GAP、CyclinD1、E-cadherin和N-cadherin蛋白表达被恢复（P < 0.001），细胞增殖、侵袭迁移能力被恢复（P < 0.01）。结论 在宫颈癌发展过程中，HPV E6通过激活Rap1信号通路促进宫颈癌细胞增殖、侵袭及迁移。 Abstract:Objective To investigate the effect of human papillomavirus E6 protein （HPV E6） on the proliferation, invasion and migration of Hela cells of cervical cancer and its potential molecular mechanism. Methods Illumina Hiseq 4000 transcriptome sequencing technology was employed to identify the gene expression profiles of 12 cases featuring various degrees of cervical lesions and to screen for HPV-related differential genes. The biological function enrichment of the differential genes was analyzed using GO and KEGG Pathway. Western blotting was utilized to detect the expression levels of Rap and Rap1GAP proteins in normal cervical epithelial cells HCerEpic and cervical cancer cells Hela. The capabilities of proliferation, invasion, and migration were examined by plate cloning, Transwell, and scratch tests. Intracellular HPV E6 expression was down-regulated through sh-E6 lentivirus transfection and classified into the control group （Hela cells）, the no-load group （sh-NON）, and the sh-E6 group （with low expression of HPV E6）. The expression of HPV E6 was diminished, and the proliferation ability and cell viability of each group were determined by plate cloning and CCK-8 assay. Transwell and scratch assays were adopted to detect cell invasion and migration. Western blotting was conducted to detect the expression levels of E6, Rap1, Rap1GAP, CyclinD1, E-cadherin, and N-cadherin in cells of each group. Rap1 was overexpressed based on the sh-E6 group, and the expression levels of the Rap1 pathway and related proteins were measured by Western blot. Cell proliferation, invasion, and migration were evaluated respectively by plate cloning, Transwell assay, and scratch assay. Results The sequencing results indicated that, in contrast to normal cervical HPV-negative tissues, 340, 864 and 1036 differentially upregulated genes were respectively present in HPV-positive normal cervix, CIN and cervical cancer tissues. A total of 24 differential genes were identified across the 3 datasets. GO|KEGG enrichment analysis of the 24 differentially expressed genes primarily focused on the RAP1-related signaling pathway. Compared with HCerEpic cells, Rap1 expression was elevated and Rap1GAP expression was reduced in Hela cells （P < 0.01）. The proliferation, invasion and migration of the cells were enhanced （P < 0.01）. After down-regulating the expression of HPV E6, the proliferation, colony formation, invasion and migration of sh-E6 cells were decreased compared with the Hela group （P < 0.001）. Western blotting showed that the protein expressions of Rap1, CyclinD1 and N-cadherin in sh-E6 group were lower than those in Hela group, and the protein expressions of Rap1GAP and E-cadherin were higher than those in hela group （sh-E6/OE Rap1） were restored （P < 0.001）, and the cell proliferation, invasion and migration were restored （P < 0.01）.Conclusion During the development of cervical cancer, HPV E6 promotes the proliferation, invasion and migration of cervical cancer cells by activating Rap1 signaling pathway. -
Key words:
- HPV E6 /
- Cervical cancer /
- Rap1 pathway /
- Proliferation /
- Invasion /
- Migration
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图 1 HPV 16感染宫颈病变组织中差异基因的筛选和功能富集分析
A：可视化差异排序图 B:3组数据间差异基因的交集 C：差异表达基因KEGG通路分析注：N_HPV_P_vs_NC_HPV_N：正常宫颈（HPV 16阳性）vs 正常宫颈（HPV阴性）；N_HPV_N_vs_CIN_HPV_P：正常宫颈（HPV阴性）vs CIN（HPV 16阳性）；N_HPV_N_vs_CA_HPV_P：正常宫颈（HPV阴性）vs 宫颈癌（HPV 16阳性）。
Figure 1. Screening and functional enrichment analysis of differential genes in HPV 16-infected cervical lesions
图 2 HPV 16感染激活宫颈癌Hela细胞内Rap1/Rap1GAP通路（$ \bar x \pm s$，n = 3）
A：Western blot检测结果；B：HeLa、HCerEpiC细胞中Rap1/Rap1GAP通路关键蛋白相对表达水平；与HCerEpiC组相比，*P < 0.05，**P < 0.01。
Figure 2. HPV 16 infection activates the Rap1/Rap1GAP pathway in Hela cells of cervical cancer （$ \bar x \pm s$，n = 3）
图 3 HPV 16感染促进宫颈癌Hela细胞增殖、侵袭和迁移能力（$\bar x \pm s$，n = 3）
A：细胞平板克隆实验结果；B：Transwell实验结果（标尺为100 μm）；C：细胞划痕实验结果（标尺为100 μm）；与HCerEpiC组相比，**P < 0.01。
Figure 3. HPV 16 infection promotes the proliferation，invasion and migration of Hela cells （$\bar x \pm s$，n = 3）
图 4 宫颈癌Hela细胞慢病毒转染效率（$\bar x \pm s $，n = 3）
A：荧光倒置显微镜下观察各组细胞的荧光表达情况（标尺为100 μm）；B：Western blot检测慢病毒转染Hela细胞后HPV E6蛋白表达；与Hela组相比，***P < 0.001。
Figure 4. Lentivirus transfection efficiency of Hela cells of cervical cancer （$\bar x \pm s $，n = 3）
图 5 下调HPV E6表达后各组宫颈癌细胞增殖、侵袭和迁移能力（$\bar x \pm s $，n=3）
A：细胞平板克隆实验结果；B：CCK-8实验结果；C：Transwell实验结果（标尺为100 μm）；D：细胞划痕实验结果（标尺为100 μm）；与Hela组相比，***P < 0.001。
Figure 5. Proliferation，invasion and migration of cervical cancer cells in each group after down-regulating HPV E6 expression （$\bar x \pm s $，n=3）
图 6 HPV E6通过Rap1信号通路调控宫颈癌细胞增殖、侵袭和迁移（$\bar x \pm s $，n = 3）
A：Western blot检测Hela细胞下调HPV E6后Rap1信号通路中相关蛋白的表达；B：Western blot检测下调HPV E6后再次过表达Rap1细胞内Rap1信号通路中相关蛋白的表达；C：细胞平板克隆实验结果；D：Transwell实验结果（标尺为100 μm）；E：细胞划痕实验结果（标尺为100 μm）；与sh-E6组相比，***P < 0.001；与sh-NON组相比，###P < 0.001，##P < 0.01。
Figure 6. HPV E6 by Rap1 signaling pathways regulating the cervical cancer cell proliferation，invasion and migration （$\bar x \pm s $，n = 3）
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