Zhang Chu-Yue, Bao Wei, Wang Li-Hua
Department of Obstetrics and Gynecology, International Peace Maternity and Child Health Hospital of China Welfare Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, P.R. China.
Int J Mol Med. 2014 Jun;33(6):1577-85. doi: 10.3892/ijmm.2014.1731. Epub 2014 Apr 7.
Studies have suggested that p16(ink4a) may be a surrogate biomarker for the diagnosis of cervical cancer; however, the function of p16(ink4a) in human cervical cancer cells remains largely unknown. Therefore, in this study, we aimed to investigate the role of p16(ink4a) in human cervical cancer cells. Immunocytochemistry was used to examine invasive squamous cell carcinoma and its precancerous lesions. p16(ink4a)-siRNA was transfected into SiHa and HeLa cells to deplete its expression. The cellular levels of p16(ink4a) mRNA and protein were detected by qRT-PCR and western blot analysis. Proliferation rates were assessed by methyl thiazolyl tetrazolium (MTT) and plate colony formation assays. Cellular migration and invasion ability were assessed by a wound healing assay and Transwell assay. Cellular apoptosis and the cell cycle were measured by flow cytometry. The protein levels of retinoblastoma (Rb), phosphorylated Rb (phospho-Rb), cyclin D1 and caspase-3 were determined by western blot analysis. The results revealed that p16(ink4a) was overexpressed in the cervical cancer and precancerous lesions (P<0.05). The downregulation of p16(ink4a) in the SiHa and HeLa cells inhibited their proliferation, migration and invasion. In the SiHa cells, p16(ink4a)-siRNA also induced G1 cell cycle arrest and apoptosis. Western blot analysis revealed that the downregulation of p16(ink4a) in the SiHa cells markedly induced caspase-3 activation and decreased cyclin D1 expression. These data suggest that the overexpression of p16(ink4a) appears to be useful in monitoring cervical precancerous lesions, which supports that the hypothesis that p16(ink4a) is a surrogate biomarker for the diagnosis of cervical cancer. The therapeutic targeting of overexpressed p16(ink4a) in the p16(ink4a)-cyclin-Rb pathway may be a useful strategy in the treatment of cervical cancer.
研究表明，p16(ink4a) 可能是宫颈癌诊断的替代生物标志物；然而，p16(ink4a) 在人宫颈癌细胞中的功能仍 largely 未知。因此，在本研究中，我们旨在研究 p16(ink4a) 在人宫颈癌细胞中的作用。免疫细胞化学用于检查浸润性鳞状细胞癌及其癌前病变。将 p16(ink4a)-siRNA 转染到 SiHa 和 HeLa 细胞中以耗尽其表达。通过 qRT-PCR 和蛋白质印迹分析检测 p16(ink4a) mRNA 和蛋白质的细胞水平。通过甲基噻唑基四唑 (MTT) 和平板集落形成试验评估增殖率。通过伤口愈合试验和 Transwell 试验评估细胞迁移和侵袭能力。通过流式细胞术测量细胞凋亡和细胞周期。通过蛋白质印迹分析确定视网膜母细胞瘤 (Rb)、磷酸化 Rb（磷酸 -Rb）、细胞周期蛋白 D1 和半胱天冬酶 -3 的蛋白质水平。结果显示，p16(ink4a) 在宫颈癌和癌前病变中过表达（P<0.05）。SiHa 和 HeLa 细胞中 p16(ink4a) 的下调抑制了它们的增殖、迁移和侵袭。在 SiHa 细胞中，p16(ink4a)-siRNA 还诱导 G1 期细胞周期停滞和凋亡。蛋白质印迹分析显示，SiHa 细胞中 p16(ink4a) 的下调明显诱导了半胱天冬酶 -3 的激活并降低了细胞周期蛋白 D1 的表达。这些数据表明，p16(ink4a) 的过表达似乎有助于监测宫颈上皮内瘤变，这支持了 p16(ink4a) 是宫颈癌诊断的替代生物标志物这一假设。在 p16(ink4a)-细胞周期蛋白 -Rb 途径中对过表达的 p16(ink4a) 进行治疗靶向可能是治疗宫颈癌的一种有用策略。
