趋化因子 CX3CL1/CX3CR1生物轴促进铂类药物诱导卵巢癌细胞凋亡的研究∗

摘要

Objective To study the relationship between the chemokine CX3CL1/ CX3CR1 and platinum drug resistance, as well as prognosis in ovarian cancer patients. To dynamically detect the resistance nude mice model of CX3CL1 and its receptor CX3CR1 in the course of platinum resistance. To analyze the expression of CX3CL1, CX3CR1 and the correlation of Fas signaling pathway. Methods The cancer gene atlas(TCGA) database of patients with ovarian cancer genome-wide expression was employed to analyze the relation-ship of CX3CL1/ CX3CR1 in platinum sensitivity or resistant patients with clinical features. Ovarian cancer SKOV3 sensitive cells(SK-OV3-GFP) with green fluorescent marker and cisplatin resistant cells(SKOV3-GFP / DDPⅢ) were subcutaneously injected in nude mice. After xenograft was constracted, cisplatin was intraperitoneally injected. Real time fluorescence quantitative-PCR(qRT-PCR) was used to detect the expression of CX3CL1, CX3CR1 and genes on Fas signal pathway after 0, 2, 5, 8 times of cisplatin injection. Results CX3CL1 expression was negatively correlated with FIGO and platinum resistance(r=-0. 112,P= 0. 030;r=-0. 106,P= 0. 044), CX3CR1 expression was negatively correlated with progression-free survival time(r = -0. 130,P = 0. 029) and positively correlated with 1-year re-lapse rate(r= 0. 119,P = 0. 045). The average expression of CX3CL1 in platinum sensitive group was 3. 96±0. 039, higher than 3. 64± 0. 55 of platinum resistant group(P= 0. 000). After the intervention of cisplatin, xenograft grew gradually. The weight of xenograft in SK-OV3-GFP / DDPⅢ group was higher than SKOV3-GFP group, and xenograft in the both two groups shrank at the fifth time of infection. After cisplatin stimulation, the expression of CX3CL1/ CX3CR1 in SKOV3-GFP group increased significantly(P= 0. 001, P= 0. 002), but it remained low in SKOV3-GFP / DDPⅢ group. The expression of Fas and FADD of Fas signal pathway in SKOV3 GFP / DDPⅢ group was also significantly lower than them in SKOV3-GFP group(P<0. 001). However, PARP1 gene significantly rose in SKOV3GFP / DDPⅢgroup than that in SKOV3-GFP group(P<0. 001). The expression of CX3CL1 and CX3CR1 was positively correlated with Fas and FADD, while it was negatively correlated with PARP1 of Fas signaling pathways. Conclusion The down-regulation of CX3CL1 and CX3CR1 is associated with platinum resistance. They may play a role in maintain the sensitivity of tumor cells to platinum. The low expression of CX3CL1 and CX3CR1 may suppress Fas signal pathway through some mechanism, resulting in transduction disorder, inhibiting cell apop-tosis and inducing platinum resistance of ovarian cancer. The lower expression of drug-resistant cells may be a mechanism inhibiting Fas signaling pathways, which make its conduction misbalance, finally inhibiting cell apoptosis, inducing the resistance of ovarian cancer. It may be a platinum promote ovarian cancer therapy targeting molecules.%目的：探讨趋化因子 CX3CL1及其受体 CX3CR1与卵巢癌患者耐药及预后的关系，以及裸鼠耐药模型中CX3CL1、CX3CR1在铂类耐药过程中的变化，分析其表达水平与 Fas 信号通路的相关性。方法利用癌症基因图集（TCGA）数据库中卵巢癌患者的全基因组表达谱，分析 CX3CL1和 CX3CR1在铂类敏感和耐药患者中的表达与临床病理特征的关系。利用已构建的带有绿色荧光标记的卵巢癌 SKOV3敏感细胞（SKOV3-GFP）和顺铂耐药细胞（SKOV3-GFP ／ DDPⅢ）进行裸鼠皮下种植，成瘤后分次给予顺铂体内注射；qRT-PCR 检测第0、2、5、8次顺铂注射后移植瘤组织中 CX3CL1、CX3CR1与 Fas 信号通路上基因的表达水平。结果 CX3CL1表达与卵巢癌 FIGO 分期和铂类耐药产生呈负相关（ r ＝－0．112，P ＝0．030；r ＝－0．106，P＝0．044）；CX3CR1表达与无进展生存时间呈负相关（r ＝－0．130，P ＝0．029）和1年复发率呈正相关（r ＝0．119，P ＝0．045）。铂类敏感组卵巢癌患者的卵巢组织中 CX3CL1的平均表达量为3．96±0．039，显著高于耐药组的3．64±0．55（P ＝0．000）。顺铂干预后，裸鼠皮下移植瘤体质量随时间推移而增加，SKOV3-GFP ／ DDP Ⅲ组形成的移植瘤体质量始终高于SKOV3-GFP 组，在第5次给药后移植瘤开始逐渐变小。顺铂干预后，SKOV3-GFP 组 CX3CL1、CX3CR1表达水平明显升高（P ＝0．001，P＝0．002），SKOV3-GFP ／ DDPⅢ组 CX3CL1、CX3CR1基因表达始终处于较低水平。 SKOV3-GFP ／ DDPⅢ组中 Fas 信号通路上的 Fas、FADD 的表达较 SKOV3-GFP 组明显降低（P＜0．001）；而 PARP1基因的相对表达较 SKOV3-GFP 组明显上调（P＜0．001）。 CX3CL1和 CX3CR1的表达与 Fas 信传导通路上节点基因 Fas、FADD 表达呈正相关，与下游 PARP1基因的表达呈负相关。结论 CX3CL1、CX3CR1表达下调与铂类耐药形成相关，两者可能具有维持肿瘤细胞对铂类药物敏感性的作用。CX3CL1、CX3CR1在耐药细胞中的低表达可能通过某种机制抑制 Fas 信号传导通路，使其传导失调，抑制细胞凋亡及诱发卵巢癌对铂类耐药。