Silencing of augmenter of liver regeneration inhibited cell proliferation and triggered apoptosis in U266 human multiple myeloma cells

Augmenter of liver regeneration (ALR) is a thermostable cytokine that was originally identified to promote the growth of hepatocytes. This study was conducted to explore the expression and function of ALR in multiple myeloma (MM), a common hematologic malignancy. Real-time PCR and western blot analysis were performed to detect the expression of ALR in U266 human MM cells and healthy peripheral blood mononuclear cells (PBMCs). U266 MM cells were exposed to 20 or 40 μg/mL of recombinant ALR and tested for cell proliferation. Small interfering RNA-mediated silencing of ALR was done to investigate the role of ALR in cell proliferation, apoptosis, and cytokine production. Compared to PBMCs, U266 MM cells exhibited significantly higher levels of ALR at both the mRNA and protein levels. The addition of recombinant ALR protein significantly promoted the proliferation of U266 cells. In contrast, knockdown of ALR led to a significant decline in the viability and proliferation of U266 cells. Annexin-V/PI staining analysis demonstrated that ALR downregulation increased apoptosis in U266 MM cells, compared to control cells (20.1±1.1 vs 9.1±0.3%, P<0.05). Moreover, ALR depletion reduced the Bcl-2 mRNA level by 40% and raised the Bax mRNA level by 2-fold. Additionally, conditioned medium from ALR-depleted U266 cells had significantly lower concentrations of interleukin-6 than control cells (P<0.05). Taken together, ALR contributed to the proliferation and survival of U266 MM cells, and targeting ALR may have therapeutic potential in the treatment of MM.

肝再生增强因子（Augmenter of liver regeneration，ALR）是一种热稳定细胞因子，最初被发现可促进肝细胞生长。本研究旨在探究肝再生增强因子（ALR）在常见血液系统恶性肿瘤多发性骨髓瘤（multiple myeloma，MM）中的表达与功能。实验采用实时荧光定量PCR（Real-time PCR）与蛋白免疫印迹（Western blot）分析，检测人多发性骨髓瘤U266细胞与健康人外周血单个核细胞（peripheral blood mononuclear cells，PBMCs）中ALR的表达水平。将U266骨髓瘤细胞分别暴露于20 μg/mL与40 μg/mL的重组ALR（recombinant ALR）中，检测其细胞增殖情况。通过小干扰RNA（small interfering RNA，siRNA）介导的ALR基因沉默，探究ALR在细胞增殖、凋亡及细胞因子产生中的作用。与健康人外周血单个核细胞相比，U266骨髓瘤细胞中ALR的mRNA与蛋白表达水平均显著升高。添加重组ALR蛋白可显著促进U266细胞的增殖；与之相反，ALR基因敲低会显著降低U266细胞的存活率与增殖能力。膜联蛋白V-碘化丙啶（Annexin-V/PI）染色分析显示，与对照组细胞相比，ALR表达下调可促进U266骨髓瘤细胞的凋亡（20.1±1.1% vs 9.1±0.3%，P<0.05）。此外，ALR敲低可使Bcl-2的mRNA水平降低40%，并使Bax的mRNA水平升高2倍。另外，ALR敲低的U266细胞的条件培养基中，白细胞介素-6（interleukin-6，IL-6）的浓度显著低于对照组（P<0.05）。综上，ALR可促进U266骨髓瘤细胞的增殖与存活，靶向ALR或许有望成为多发性骨髓瘤的潜在治疗策略。