[Regulation of Ruxolitinib on matrix metalloproteinase in JAK2V617F positive myeloroliferative neoplasms cells]

Abstract

Objective: To investigate the regulation of JAK2 tyrosine kinase inhibitor ruxolitinib on extracellular matrix metalloproteinase (MMP in JAK2V617F positive myeloproliferative neoplasms (MPN) cells. Methods: ①Forty cases of newly diagnosed JAK2V617F positive MPN patients and 15 healthy volunteers as control in Baoding No.1 Hospital between January 2012 and December 2015 were enrolled in this study. JAK2V617F/JAK2 ratio was detected by real-time-PCR; the expression levels of phosphorylation protein tyrosine kinase 2 (p-JAK2) , MMP-2 and MMP-9 in pathological tissues of bone marrow were detected by immunohistochemistry. The bone marrow cells of JAK2V617F positive MPN patients were treated with ruxolitinib, then the migration ability and MMP-2, MMP-9 gene and protein expression levels were detected. ②The human erythroleukemia cell line HEL cells were treated with different concentrations of ruxolitinib (0, 50, 100, 250, 500, 1 000 nmol/L) . The cell viability was detected by CCK-8 test; cell migration ability was tested by transwell chambers. The mRNA expression levels of JAK2, MMP-2 and MMP-9 were detected by real-time-PCR. The protein expression levels of p-JAK2, MMP-2 and MMP-9 were detected by Western blot. Results: ①The expression levels of p-JAK2, MMP-2 and MMP-9 in the newly diagnosed group were significantly higher than control group respectively [ (78.56±24.55) % vs (41.59±17.29) %, P<0.05; (48.25±18.74) % vs (22.79±13.89) %, P<0.05; (53.29±19.28) % vs (15.56±14.96) %, P<0.05]. Spearman correlation analysis showed the positive correlation of MMP-2 and MMP-9 protein expression levels with JAK2V617F mutation (r=0.526, P=0.001; r=0.543, P=0.001) . ②The proliferation of HEL cells was inhibited by different concentrations of ruxolitinib in time and dose dependent manner. ③Cell migration test showed the number of cells leaked to the low chamber in MPN patients bone marrow cells and HEL cells treated with 5 nmol/L of ruxolitinib group were significantly lower than that without ruxolitinib treatment after 24 h [ (154.7±27.5) vs (320.3±67.3) , t=13.47, P<0.05; (70.7±10.5) vs (135.3±16.7) , t=13.89, P<0.05]. The mRNA and protein expression levels of JAK2, MMP-2 and MMP-9 decreased with the increased concentration of ruxolitinib. Conclusion: Ruxolitinib inhibits MPN cell migration and expression of MMP-2 and MMP-9 via JAK2 signal pathway.

Keywords: Cell migration assays; Matrix Metalloproteinase 9; Matrix metalloproteinase 2; Myeloproliferative neoplasms; Ruxolitinib.

图1. 免疫组织化学染色法检测骨髓增殖性肿瘤（MPN）患者（A）和对照组（B）骨髓组织磷酸化JAK激酶2（p-JAK2）、基质金属蛋白酶2（MMP-2）、MMP-9表达（高倍镜）

图2. Western blot检测不同浓度Ruxolitinib对HEL细胞p-JAK2、MMP-2、MMP-9表达的影响

1~6分别为0、50、100、250、500、1 000 nmol/L Ruxolitinib 组。p-JAK2：磷酸化Janus 激酶2；MMP-2：基质金属蛋白酶2；MMP-9、基质金属蛋白酶9