心钠素对多发性骨髓瘤细胞增殖的影响及作用机制探讨

The effects of atrial natriuretic peptide in proliferation of myeloma cells and its potenial mechanisms

目的:探讨心钠素(atrial natriuretic peptide,ANP)对多发性骨髓瘤细胞株(MM1.S)的增殖抑制及其作用机制。方法:MM1.S置于含10%胎牛血清的RPMI1640培养基培养,ANP浓度分别为1、10、50及100μmol/L。收集各组不同培养时间(24、48、72 h)细胞,CKK-8方法检测细胞的增殖抑制作用,流式细胞术检测细胞周期,Western blot法检测MM1.S细胞NPR-A、NPR-C蛋白的表达,qRT-PCR检测不同浓度ANP作用MM1.S细胞24 h后miR-21的表达。结果 :ANP对MM1.S细胞具有增殖抑制作用,且呈浓度依赖性(P<0.01)。细胞周期检测为G1期细胞比例较对照组明显增加(P<0.01),S期、G2期细胞比例下降(P<0.05),4个浓度组间两两比较有统计学差异(P<0.05)。MM1.S细胞存在NPR-A、NPR-C蛋白的表达。ANP不同浓度(1.0,10,50,100μmol/L)作用MM1.S细胞24 h后miR-21表达下调,相对表达量较阴性对照组分别下降了8.6%、24.2%、37.5%和49%(P<0.05)。结论:ANP能抑制MM1.S增殖,其作用机制可能是通过阻滞细胞周期于G1期和下调miR-21表达发挥其抗肿瘤效应。

Objective To study the effects of atrial natriuretic peptide （ANP） in proliferatiin inhibit and mechanism of myeloma cells （MM1.S）. Methods The human MM1.S cells were placed in RPMI1640 containing 10% FBS and incubated in cell incubator. The ANP concentration were 1.0, 10, 50 and 100 μmol/L, and each group had different incubation time （0,24,48,72 h） respetively. CCK-8 assay was used to detect the inhibition of ANP on MM1.S cell proliferation. The flow cytometry was used to analyze the cell cycle of MM1.S. The Western blotting was needed to detect whether the natriuretic peptide receptor-A （NPR-A, NPR-C） were expressed in MM1. S cells. The qRT-PCR was employed to analyze expression levels of miR-21 in MM1.S with various concentrations of ANP for 24 h. Results ANP could inhibit proliferation of MM1.S in significantly dose-dependent manner （P 〈 0.01） ,and the inhibition was the most obvious after treated with ANP by 24 hours. Percentage of cells in G1 phase increased significantly accompanying cell cycle arrest in G1 phase comparing with the control group （P 〈 0.01）, while the percentage of S and G2 phase cells decreased（P 〈 0.05）. Significant differences were observed among the four ANP-treated MM1.S cells groups （P 〈 0.05）. Natriuretic peptide receptor （NPR-A/C） were expressed in MM1. S cells. After the treatment of 1.0, 10, 50 and 100 μmol/L ANP in MM1.S cells for 24 h, qRT-PCR examination indicated that miR-21 was down-regulated comparing with the control group （P 〈 0.05）. ANP concentration in the four groups reduced the expression of miR-21 by 8.6%, 24.2%, 37.5%and 49% respectively. Conclusion ANP has anti-proliferative effect in myeloma cells by blocking cell cycle in G1 phase and inhibiting the miR-21 expression.