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.
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。
Keywords: SHI-1 cells; acute monocytic leukemia; drug sensitivity; leupaxin; small interfere RNA.