Oncogenic high‐risk human papillomavirus (HR‐HPV) infection causes a majority of cases of cervical cancer and pre‐cancerous cervical lesions. However, the mechanisms underlying the direct evolution from HPV‐16/18‐infected epithelium to cervical intraepithelial neoplasia (CIN) III, which can progress to cervical cancer, remain poorly identified. Here, we performed RNA‐seq after laser capture microdissection, and found that APOBEC3B was highly expressed in cervical cancer specimens compared with CIN III with HPV‐16/18 infection. Furthermore, immunohistochemical analysis confirmed that high levels of APOBEC3B were correlated with lymph node metastasis in cervical cancer. Subsequent experiments revealed that HPV‐16 E6 could upregulate APOBEC3B through direct binding to the promoter of APOBEC3B in cervical cancer cells. Silencing of APOBEC3B by stable short hairpin RNA‐mediated knockdown reduced the proliferative capacity of Caski and HeLa cells in vitro and in vivo, but had only a small effect on the migration and invasion of two cervical cancer cell lines. Finally, we identified the changes in gene expression following APOBEC3B silencing in Caski cells by microarray, demonstrating a biological link between APOBEC3B and CCND1 in cervical cancer cells. Importantly, through methyl‐capture sequencing and pyrosequencing, APOBEC3B was found to affect the levels of the downstream protein Cyclin D1 (which is encoded by the CCND1 gene) through hypomethylation of the CCND1 promoter. In conclusion, our study supports HPV‐16 E6‐induced APOBEC3B expression associates with proliferation of cervical cancer cells and hypomethylation of Cyclin D1. Thus, APOBEC3B may be a potential therapeutic target in human cervical cancer.

中文翻译：

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HPV-16 / 18 E6诱导的APOBEC3B表达与宫颈癌细胞的增殖和细胞周期蛋白D1的低甲基化有关

致癌性高风险人乳头瘤病毒（HR‐HPV）感染导致大多数宫颈癌和癌前宫颈病变病例。然而，从感染HPV-16 / 18的上皮直接发展为宫颈上皮内瘤样变（CIN）III的直接机制可能会发展为宫颈癌，目前仍知之甚少。在这里，我们在激光捕获显微切割后进行了RNA序列分析，发现APOBEC3B在宫颈癌标本中的表达水平高于感染HPV-16 / 18的CIN III。此外，免疫组织化学分析证实，高水平的APOBEC3B与宫颈癌的淋巴结转移有关。随后的实验表明，HPV-16 E6可通过直接结合到APOBEC3B的启动子上调APOBEC3B。在子宫颈癌细胞中 稳定的短发夹RNA介导的敲低沉默APOBEC3B会降低Caski和HeLa细胞在体外和体内的增殖能力，但对两种宫颈癌细胞系的迁移和侵袭影响很小。最后，我们通过微阵列鉴定了Caski细胞中APOBEC3B沉默后基因表达的变化，证明了宫颈癌细胞中APOBEC3B和CCND1之间的生物学联系。重要的是，通过甲基捕获测序和焦磷酸测序，APOBEC3B被发现影响下游蛋白细胞周期素D1（其由编码的水平CCND1通过的低甲基化基因）CCND1启动子。总之，我们的研究支持HPV-16 E6诱导的APOBEC3B表达与宫颈癌细胞的增殖和细胞周期蛋白D1的低甲基化有关。因此，APOBEC3B可能是人类宫颈癌的潜在治疗靶标。