Objective

This study aims to explore the role of MARK2 in chemotherapeutic resistance and potential mechanism within cisplatin resistance models of CD133⁺ MG-63 and MNNG/HOS cells.

Methods

CD133⁻ and CD133⁺ MG-63 and MNNG/HOS cells were differentiated and obtained by MACS(Magnetic bead sorting). Cell activity was determined by CCK-8 assay. siRNA was employed to down regulate the Microtubule Affinity Regulated Kinase 2 (MARK2) expression. Immunofluorescence detection and RT-qPCR were used to measure the expressions of MARK2 and DNA-PKcs at both protein and mRNA levels. Western blot was applied to test the levels of MARK2, γH2AX (S139), DNA-PKcs, Phospho-PI3 Kinase p85 (Tyr458), Akt, phospho-Akt (T308) antibodies, mTOR, phospho-mTOR (Ser2448).

Results

Compared with CD133⁻ MG-63 cells, CD133⁺ MG-63 cells showed significantly strong cisplatin resistance, with high levels of MARK2, DNA-PKcs and potent DNA damage repair ability (p<0.05). Down regulation of MARK2 reduced the cisplatin resistance of CD133⁺ MG-63 cells, with deceasing expression of DNA-PKcs (p<0.05). PI3K/Akt/mTOR pathway was potentially activated in CD133⁺ MG-63 cells, and involved in the cisplatin resistance of MG-63 cells. The similar results were observed in CD133⁺ MNNG/HOS cells. The reduction of MARK2 retarded the activity of PI3K/Akt/mTOR pathway and further impeded the cisplatin resistance in CD133⁺ MG-63 and MNNG/HOS cell.

Conclusion

Our data suggested that MARK2 was related to cisplatin resistance in CD133⁺ MG-63 and MNNG/HOS cells. The decrease of MARK2 restricted the cisplatin resistance of CD133⁺ MG-63 and MNNG/HOS cells by down regulating the expression of DNA dependent protein kinase catalytic subunit (DNA-PKcs) and inhibiting activity of PI3K/Akt/mTOR signaling pathway, which provides new clues for the osteosarcoma chemotherapy strategy.

中文翻译：

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MARK2的抑制通过调节DNA损伤和修复来抑制骨肉瘤干细胞的顺铂耐药。

客观的

本研究旨在探讨 MARK2 在 CD133 ⁺ MG-63 和 MNNG/HOS 细胞顺铂耐药模型中化疗耐药的作用和潜在机制。

方法

CD133 ^-和CD133 ⁺ MG-63和MNNG/HOS细胞通过MACS（磁珠分选）进行分化和获得。通过CCK-8测定确定细胞活性。使用 siRNA 来下调微管亲和调节激酶 2 (MARK2) 的表达。免疫荧光检测和 RT-qPCR 用于测量 MARK2 和 DNA-PKcs 在蛋白质和 mRNA 水平上的表达。应用蛋白质印迹检测 MARK2、γH2AX (S139)、DNA-PKcs、磷酸化 PI3 激酶 p85 (Tyr458)、Akt、磷酸化-Akt (T308) 抗体、mTOR、磷酸化-mTOR (Ser2448) 的水平。

结果

与CD133 ^- MG-63细胞相比，CD133 ⁺ MG-63细胞表现出显着强的顺铂耐药性，具有高水平的MARK2、DNA-PKcs和强大的DNA损伤修复能力（p <0.05）。MARK2的下调降低了CD133 ⁺ MG-63细胞的顺铂耐药性，DNA-PKcs的表达降低（p <0.05）。PI3K/Akt/mTOR通路可能在CD133 ⁺ MG-63细胞中被激活，并参与MG-63细胞的顺铂耐药。^{在 CD133 +} MNNG/HOS 细胞中观察到类似的结果。MARK2 的减少延缓了 PI3K/Akt/mTOR 通路的活性，进一步阻碍了 CD133 ^{+顺铂耐药}MG-63 和 MNNG/HOS 电池。

结论

我们的数据表明 MARK2 与 CD133 ⁺ MG-63 和 MNNG/HOS 细胞中的顺铂耐药有关。MARK2 的降低通过下调 DNA 依赖性蛋白激酶催化亚基 (DNA-PKcs) 的表达和抑制 PI3K/Akt/mTOR 信号通路的活性来限制 CD133 ⁺ MG-63 和 MNNG/HOS 细胞的顺铂耐药，从而提供骨肉瘤化疗策略的新线索。