Wang Huan, Zhu Liancheng, Gao Jian, Hu Zhenhua, Lin Bei
Department of Obstetrics and Gynecology, Shengjing Hospital Affiliated to China Medical University, Heping, Shenyang, Liaoning 110004, P.R. China.
Oncol Rep. 2015 Jan;33(1):403-12. doi: 10.3892/or.2014.3549. Epub 2014 Oct 17.
Available evidence on the proliferation-promoting effect of HE4 remains controversial, and few studies have been carried out on the molecular mechanism of chemoresistance mediated by HE4. The aim of the present study was to investigate the influence of exogenous recombinant HE4 protein on proliferation and resistance to carboplatin in ovarian cancer cells. The human ovarian cancer cell line (SKOV-3) was exposed to recombinant HE4 protein (0-1 µg/ml) for different durations based on the schemes. Cell viability was evaluated by Cell Counting Kit-8 and colony formation assays. Cell cycle distribution and apoptosis were analyzed by flow cytometry. Markers of apoptosis (Bax and Bcl-2) were assessed by western blotting. Furthermore, Affymetrix microarray analysis was performed to investigate transcriptome profiling. The differential expression of four genes was verified by quantitative real-time PCR. The HE4 protein enhanced cell viability, promoted accumulation of cells in the G2/M phase and attenuated carboplatin-induced apoptosis. HE4 markedly decreased the Bax/Bcl-2 ratio. Candidate genes (387) (236 upregulated and 151 downregulated) were obtained by microarray analysis. Among those upregulated, several Gene Ontology (GO) terms related to cell cycle regulation and proliferation were significantly overrepresented and those within the downregulated dataset included genes involved in several aspects of the DNA damage response such as positive regulation of apoptosis. No information concerning the EGFR-MAPK pathways in a recent report on HE4 was acquired. The mRNA expression of the candidate genes determined by quantitative real-time PCR was significantly correlated with the microarray data. The present study indicates that the HE4 protein plays a promotive role in the proliferation and resistance to carboplatin in ovarian cancer cells, implicating the value of HE4 to predict tumor growth potential and resistance to platinum-based chemotherapy in epithelial ovarian cancer (EOC).
关于HE4促增殖作用的现有证据仍存在争议，且针对HE4介导的化疗耐药分子机制的研究较少。本研究旨在探讨外源性重组HE4蛋白对卵巢癌细胞增殖及顺铂耐药性的影响。根据实验方案，将人卵巢癌细胞系（SKOV-3）暴露于不同浓度（0 - 1µg/ml）的重组HE4蛋白中不同时间。通过细胞计数试剂盒-8和集落形成实验评估细胞活力。采用流式细胞术分析细胞周期分布和凋亡情况。通过蛋白质免疫印迹法检测凋亡标志物（Bax和Bcl-2）。此外，进行了Affymetrix基因芯片分析以研究转录组图谱。通过定量实时PCR验证了四个基因的差异表达。HE4蛋白增强了细胞活力，促进细胞在G2/M期的积累，并减弱了顺铂诱导的凋亡。HE4显著降低了Bax/Bcl-2比值。通过基因芯片分析获得了候选基因（387个）（236个上调和151个下调）。在那些上调的基因中，几个与细胞周期调控和增殖相关的基因本体（GO）术语显著富集，而在下调的数据集中包括参与DNA损伤反应多个方面的基因，如凋亡的正调控。在最近一篇关于HE4的报告中未获得有关EGFR-MAPK途径的信息。通过定量实时PCR测定的候选基因的mRNA表达与基因芯片数据显著相关。本研究表明，HE4蛋白在卵巢癌细胞的增殖和顺铂耐药中起促进作用，提示HE4在预测上皮性卵巢癌（EOC）的肿瘤生长潜力和铂类化疗耐药性方面具有价值。
