中国癌症杂志 ›› 2017, Vol. 27 ›› Issue (12): 921-927.doi: 10.19401/j.cnki.1007-3639.2017.12.001

• 论著 • 上一篇    下一篇

Piwil2调控宫颈癌细胞恶性生物学行为的机制探讨

冯定庆,闫克芹,张 筱,邓 琳,凌 斌   

  1. 中日友好医院妇产科,北京 100029
  • 出版日期:2017-12-30 发布日期:2018-01-11
  • 通信作者: 凌 斌 E-mail:lingbin.ling@vip.sina.com
  • 基金资助:
    国家自然科学基金面上项目(81372779;81372777)。

The role of Piwil2 in regulating the malignant process of cervical cancer

FENG Dingqing, YAN Keqin, ZHANG Xiao, DENG Lin, LING Bin   

  1. Department of Obstetrics and Gynecology, China-Japan Friendship Hospital, Beijing 100029, China
  • Published:2017-12-30 Online:2018-01-11
  • Contact: LING Bin E-mail: lingbin.ling@vip.sina.com

摘要: 背景与目的:Piwil2在肿瘤干细胞及癌前干细胞中高表达,通过转录和转录后调控机制调控多种基因表达,与肿瘤发生、发展密切相关。该研究探讨Piwil2与宫颈癌细胞恶性生物学行为之间的关系及可能的调控机制。方法:构建慢病毒载体pLenti-CMV-Piwil2-SV40-EGFP和shPiwil2质粒,分别转染HeLa和SiHa细胞,建立过表达/沉默Piwil2表达稳转细胞株,反转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)及蛋白[质]印迹法(Western blot)鉴定转染效果;采用CCK-8法检测细胞增殖能力;PI染色后,流式细胞术(fluorescence-activated cell sorting,FACS)检测细胞周期;Western blot检测细胞增殖及周期相关调控蛋白表达;采用维拉帕米(verapamil)处理细胞,Hoechst 33342染色,FACS检测侧群(side polulation,SP)细胞比例;CCK-8法检测顺铂对细胞的杀伤活性。结果:过表达Piwil2上调HeLa和SiHa细胞S期及G2/M期细胞比例,进而促进细胞增殖,与对照组比较差异均有统计学意义(P均<0.05);沉默Piwil2表达则细胞发生G0/G1期阻滞,抑制细胞增殖(P<0.05);Piwil2上调cyclin D1表达,提高Stat3磷酸化水平,下调p53表达;Piwil2显著上调HeLa和SiHa细胞中SP细胞比例(P<0.01),提高宫颈癌细胞对顺铂的耐药性(P<0.01);沉默Piwil2则相反,显著提高对顺铂的敏感性。结论:Piwil2上调SP细胞比例,促进宫颈癌进展;Piwil2可作为宫颈癌的潜在治疗靶点。

关键词: Piwil2, 子宫颈癌, 侧群细胞, 耐药

Abstract: Background and purpose: Piwil2 is highly expressed in precancerous and cancer stem cells, which plays a key role in the process of tumorigenesis and progression through the transcriptional and post-transcriptional regulation of gene expression. This study aimed to elucidate the role of Piwil2 in regulating the malignant process of cervical cancer. Methods: In order to generate cell line with overexpression or silence of Piwil2, HeLa and SiHa cells were transfected with lentiviral pLenti-CMV-Piwil2-SV40-EGFP or plasmid shPiwil2, respectively. Cell proliferation assays using CCK-8 were performed in 96-well format in duplicate. Cell cycle and side population (SP) cells were analyzed by fluorescence-activated cell sorting (FACS). The proteins related to cell growth and cell cycle were measured by Western blot. CCK-8 assay was also used to assess the killing effects of cisplatin. Results: Overexpression of Piwil2 promoted cervical cancer cell proliferation and the entry of cells from G0/G1 phase into S phase, as compared to the control cells (P<0.05). On the contrary, Piwil2 knockdown suppressed proliferation of both cells and increased the number of cells during G0/G1 phase markedly (P<0.05). Western blot analyses confirmed that Piwil2 overexpression led to an upregulation of cyclin D1 and p-Stat3 but a significantly decreased level of p53. Furthermore, overexpression of Piwil2 significantly increased the SP cell populations in both HeLa and SiHa cells (P<0.01), sequentially enhanced resistance in cancer cells to cisplatin (P<0.01). Instead, Piwil2 gene knockdown induced an apparent downregulation of cyclin D1 and p-Stat3, significantly increased p53 expression, and decreased proportion of SP cells, which, to some extent, contributed to the improved sensitivity to cisplatin. Conclusion: Piwil2 plays an essential role in the progression of cervical cancer via increasing the proportion of SP cells. Therefore, targeting Piwil2 may be an effective therapeutic option for patients with cervical cancer.

Key words: Piwil2, Cervical cancer, Side population cells, Drug resistance