南方医科大学学报

南方医科大学学报 ›› 2018, Vol. 38 ›› Issue (07): 807-.

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siRNA介导的LPXN沉默可抑制SHI-1 细胞增殖及增强SHI-1 细胞药物的敏感性

朱国华，戴海萍，段元勋，余泽霖   

  • 出版日期:2018-07-20 发布日期:2018-07-20

Small interfering RNA-mediated LPXN silencing suppresses proliferation and enhances drug sensitivity of human acute monocytic leukemia SHI-1 cells in vitro

  • Online:2018-07-20 Published:2018-07-20

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摘要： 目的探讨LPXN表达下调对人急性单核白血病SHI-1 细胞增殖及药物敏感性的影响。方法将荧光标记的不同浓度 FAM-siRNA序列转染SHI-1细胞后FCM检测转染效率，并优化转染条件。合成LPXN基因特异的siRNA序列（LPXN-siRNA） 并转染SHI-1 细胞，Western blot 筛选能有效干扰LPXN蛋白表达的siRNA 序列以及细胞内p-MAPK的表达。CCK-8 检测 LPXN-siRNA转染后SHI-1细胞的增殖以及细胞对姜黄素（Cur）或阿糖胞苷（Ara-C）药物敏感性的改变。结果当细胞接种密 度为4×105/mL 且siRNA/Lipofectamine 2000 混合比例为200 pmol/1 μL 时，FAM-siRNA 转染入SHI-1 细胞的最高效率达到 74.5%，且筛选出L2-siRNA为有效下调LPXN表达的siRNA序列。在N-siRNA转染的阴性对照组SHI-1 细胞增殖抑制率为 8.247±1.003，而在下调LPXN表达的L2-siRNA转染组细胞增殖抑制率增加至（27.043±2.051）%（P<0.05）；并且各组进一步添加 （0~25 μmol）Cur或（0~2.0 μmol）Ara-C药物后，L2-siRNA转染组的细胞增殖抑制率、p-JNK和p-P38 MAPK的表达均明显高于 N-siRNA对照转染组。而p-ERK的表达水平则各组基本一致。结论siRNA特异性干扰下调LPXN蛋白表达后，可能通过激活 MAPK家族的JNK及P38 MAPK蛋白酶，从而抑制人急性单核白血病SHI-1细胞的增殖，增强SHI-1细胞对Cur或Ara-C药物 的敏感性。

Abstract: Objective To investigate the effect of silencing LPXN expression by RNA interference on the proliferation and drug sensitivity of human acute monocytic leukemia SHI-1 cells in vitro. Methods Small interfering RNA (siRNA) sequences targeting LPXN were designed and transiently transfected in SHI-1 cells via Lipofectamine 2000, and the most efficient siRNA sequence for LPXN silencing was identified using Western blotting. The protein expression levels of LPXN, p-JNK, p-P38 MAPK and p-ERK were in the cells transfected with the selected siRNA were detected using Western blotting, and the cell proliferation changes were assessed using CCK-8 reagent. Results LPXN silencing by siRNA transfection resulted in significant proliferation suppression in SHI-1 cells with an inhibition rate of（27.04±2.05）% (P<0.05). Western blotting showed that treatment of the siRNA-transfected SHI-1 cells with 0-25 μmol/L curcumin or with 0-2.0 μmol/L Ara-C further increased the cell inhibition rate and obviously enhanced the expressions of p-P38 MAPK and p-JNK without significantly affecting p-ERK expression. Conclusions Down-regulation of LPXN expression by siRNA transfection can suppress the proliferation and increase the drug sensitivity of SHI-1 cells probably by activating JNK and P38 MAPK.