CLC-3 的过表达受 XRCC5 调控，是胃癌预后不良的生物标志物。
Overexpression of CLC-3 is regulated by XRCC5 and is a poor prognostic biomarker for gastric cancer.
机构信息
Department of Pharmacology, Medical College, Jinan University, Guangzhou, 510632, China.
Department of Pathophysiology, Medical College, Jinan University, Guangzhou, China.
出版信息
J Hematol Oncol. 2018 Sep 14;11(1):115. doi: 10.1186/s13045-018-0660-y.
BACKGROUND
Recently, many potential prognostic biomarkers for gastric cancer (GC) have been identified, but the prognosis of advanced GC patients remains poor. Chloride channels are promising cancer biomarkers, and their family member chloride channel-3 (CLC-3) is involved in multiple biological behaviors. However, whether CLC-3 is a prognostic biomarker for GC patients is rarely reported. The molecular mechanisms by which CLC-3 is regulated in GC are unclear.
METHODS
The expression of CLC-3 and XRCC5 in human specimens was analyzed using immunohistochemistry. The primary biological functions and pathways related to CLC-3 were enriched by RNA sequencing. A 5'-biotin-labeled DNA probe with a promoter region between - 248 and + 226 was synthesized to pull down CLC-3 promoter-binding proteins. Functional studies were detected by MTS, clone formation, wound scratch, transwell, and xenograft mice model. Mechanistic studies were investigated by streptavidin-agarose-mediated DNA pull-down, mass spectrometry, ChIP, dual-luciferase reporter assay system, Co-IP, and immunofluorescence.
RESULTS
The results showed that CLC-3 was overexpressed in human GC tissues and that overexpression of CLC-3 was a poor prognostic biomarker for GC patients (P = 0.012). Furthermore, higher expression of CLC-3 was correlated with deeper tumor invasion (P = 0.006) and increased lymph node metastasis (P = 0.016), and knockdown of CLC-3 inhibited cell proliferation and migration in vitro. In addition, X-ray repair cross-complementing 5 (XRCC5) was identified as a CLC-3 promoter-binding protein, and both CLC-3 (HR 1.671; 95% CI 1.012-2.758; P = 0.045) and XRCC5 (HR 1.795; 95% CI 1.076-2.994; P = 0.025) were prognostic factors of overall survival in GC patients. The in vitro and in vivo results showed that the expression and function of CLC-3 were inhibited after XRCC5 knockdown, and the inhibition effects were rescued by CLC-3 overexpression. Meanwhile, the expression and function of CLC-3 were promoted after XRCC5 overexpression, and the promotion effects were reversed by the CLC-3 knockdown. The mechanistic study revealed that knockdown of XRCC5 suppressed the binding of XRCC5 to the CLC-3 promoter and subsequent promoter activity, thus regulating CLC-3 expression at the transcriptional level by interacting with PARP1.
CONCLUSIONS
Our findings indicate that overexpression of CLC-3 is regulated by XRCC5 and is a poor prognostic biomarker for gastric cancer. Double targeting CLC-3 and XRCC5 may provide the promising therapeutic potential for GC treatment.
背景
最近，许多潜在的胃癌（GC）预后生物标志物已被确定，但晚期 GC 患者的预后仍然较差。氯离子通道是很有前途的癌症生物标志物，其家族成员氯离子通道-3（CLC-3）参与多种生物学行为。然而，CLC-3 是否是 GC 患者的预后生物标志物很少有报道。CLC-3 在 GC 中调节的分子机制尚不清楚。
方法
使用免疫组织化学分析人标本中 CLC-3 和 XRCC5 的表达。通过 RNA 测序富集与 CLC-3 相关的主要生物学功能和途径。合成了一个带有-248 到+226 启动子区域的 5′-生物素标记的 DNA 探针，以拉下拉 CLC-3 启动子结合蛋白。通过 MTS、克隆形成、划痕愈合、Transwell 和异种移植小鼠模型检测功能研究。通过链霉亲和素琼脂糖介导的 DNA 下拉、质谱、ChIP、双荧光素酶报告基因检测系统、Co-IP 和免疫荧光检测机制研究。
结果
结果表明，CLC-3 在人 GC 组织中过度表达，CLC-3 的过度表达是 GC 患者预后不良的生物标志物（P=0.012）。此外，CLC-3 的高表达与肿瘤浸润更深（P=0.006）和淋巴结转移增加（P=0.016）有关，体外敲低 CLC-3 可抑制细胞增殖和迁移。此外，X 射线修复交叉互补基因 5（XRCC5）被鉴定为 CLC-3 启动子结合蛋白，CLC-3（HR 1.671；95%CI 1.012-2.758；P=0.045）和 XRCC5（HR 1.795；95%CI 1.076-2.994；P=0.025）均是 GC 患者总生存期的预后因素。体外和体内结果表明，XRCC5 敲低后 CLC-3 的表达和功能受到抑制，而 CLC-3 过表达可挽救抑制作用。同时，XRCC5 过表达后 CLC-3 的表达和功能得到促进，而 CLC-3 敲低则逆转了这种促进作用。机制研究表明，XRCC5 敲低抑制 XRCC5 与 CLC-3 启动子的结合及其随后的启动子活性，从而通过与 PARP1 相互作用，在转录水平上调节 CLC-3 的表达。
结论
我们的研究结果表明，CLC-3 的过表达受 XRCC5 调控，是胃癌的预后不良生物标志物。双重靶向 CLC-3 和 XRCC5 可能为 GC 治疗提供有前景的治疗潜力。
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