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SLC1A5-TM4SF1复合物对食管鳞状细胞癌细胞顺铂耐药的调控作用及机制

Regulating effect and mechanism of SLC1A5-TM4SF1 complex on cisplatin resistance in esophageal squamous cell carcinoma cells

    摘要
  • 摘要:
    目的 探讨氨基酸转运载体溶质载体家族1成员5(SLC1A5)-四跨膜蛋白超家族成员1(TM4SF1)复合物对食管鳞状细胞癌(ESCC)细胞顺铂耐药的调控作用及分子机制。
    方法 通过慢病毒载体构建SLC1A5稳定过表达的Eca109细胞, 通过细胞活力试验检测SLC1A5对细胞顺铂敏感性的影响。采用蛋白质免疫印迹法(WB)检测SLC1A5在Eca109细胞及顺铂耐药Eca109细胞(Eca109-R)中的表达。在Eca109-R细胞中通过慢病毒载体敲低SLC1A5表达，检测敲低SLC1A5表达后细胞对顺铂的敏感性。采用实时荧光定量聚合酶链反应(qRT-PCR)检测SLC1A5敲低对Eca109-R细胞中DNA损伤修复关键基因表达的影响。在SLC1A5敲低的Eca109-R细胞中，通过细胞活力试验检测过表达RAD50后细胞对顺铂的敏感性。在Eca109细胞中，利用慢病毒载体分别或共同过表达SLC1A5、TM4SF1, 通过细胞活力试验检测SLC1A5-TM4SF1复合物对RAD50表达及细胞顺铂耐药的影响。
    结果 与对照细胞相比，过表达SLC1A5的Eca109细胞对顺铂的耐药性增强，差异有统计学意义(P < 0.05)。Eca109-R细胞的SLC1A5蛋白表达升高，且与对照细胞相比，敲低SLC1A5表达的细胞对顺铂更敏感，差异有统计学意义(P < 0.05)。顺铂处理条件下, RAD50基因表达水平在敲低SLC1A5后显著下调(P < 0.05)。敲低SLC1A5会抑制RAD50蛋白表达，且与对照细胞相比，在SLC1A5敲低细胞中过表达RAD50可以大幅度恢复细胞对顺铂的耐药，差异有统计学意义(P < 0.05)。SLC1A5与TM4SF1同时过表达可进一步上调RAD50表达，且与对照细胞相比，细胞对顺铂的耐药性增强，差异有统计学意义(P < 0.05)。
    结论 SLC1A5-TM4SF1复合物通过上调RAD50表达，促进ESCC细胞对顺铂的耐药。

     

    Abstract:
    Objective To investigate the regulatory effect and molecular mechanism of solute carrier family 1 member 5 (SLC1A5) -tetraspanin superfamily member 1 (TM4SF1) complex on cisplatin resistance in esophageal squamous cell carcinoma (ESCC) cells.
    Methods SLC1A5-overexpressing Eca109 cells were constructed using lentiviral vectors, and the effect of SLC1A5 on cisplatin sensitivity was assessed through cell viability assays. Western blotting (WB) was employed to detect SLC1A5 expression in Eca109 cells and cisplatin-resistant Eca109 cells (Eca109-R). SLC1A5 expression was knocked down in Eca109-R cells using lentiviral vectors, and cisplatin sensitivity was examined thereafter. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to analyze the influence of SLC1A5 knockdown on the expression of key genes involved in DNA damage repair in Eca109-R cells. In SLC1A5-knockdown Eca109-R cells, cell viability assays were performed to evaluate the sensitivity to cisplatin after RAD50 overexpression. Additionally, Eca109 cells were separately or co-overexpressed with SLC1A5 and TM4SF1 using lentiviral vectors, and the effect of the SLC1A5-TM4SF1complex on RAD50 expression and cisplatin resistance was examined through cell viability assays.
    Results Compared with control cells, Eca109 cells overexpressing SLC1A5 exhibited enhanced cisplatin resistance (P < 0.05). Eca109-R cells showed increased SLC1A5 protein expression, and knockdown of SLC1A5 cells were more sensitivity to cisplatin (P < 0.05). RAD50 gene expression was significantly downregulated upon SLC1A5 knockdown under cisplatin treatment (P < 0.05). Knockdown of SLC1A5 inhibited RAD50 protein expression, and overexpression of RAD50 in SLC1A5-knockdown cells significantly restored cisplatin resistance (P < 0.05). Co-overexpression of SLC1A5 and TM4SF1 can further up-regulate the expression of RAD50, and the drug resistance of the cells to cisplatin was enhanced compared with the control cells(P < 0.05).
    Conclusion The SLC1A5-TM4SF1 complex promotes cisplatin resistance in ESCC cells by upregulating RAD50 expression.

     

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