| Objective:To investigate the regulation of JAK2 tyrosine kinase inhibitor ruxolitinib on JAK2V617 F positive myeloproliferative neoplasmss(MPN)cells with extracellular matrix metalloproteinase(MMP),and to explore the influence of ruxolitnib with JAK2?MMP-2 and MMP-9 in JAK2V617 F mutation positive human erythroleukemia HEL cell line.The purpose of this study was to investigate the effect of ruxolitinib on the proliferation,migration,induction and inhibition of JAK2,MMP-2,MMP-9 protein and gene expression in HEL cells and primary culture cells,and to provide theoretical and experimental evidence for the clinical treatment in patients of myeloproliferative neoplasms by ruxolitinib.Methods:1 40 cases of newly diagnosed JAK2V617 F positive MPN patients in the Fist Hospital of Baoding between January 2012 and December 2015 were selected.13 cases of PV,14 cases of ET and 13 cases of PMF.15 cases of healthy volunteers were enrolled as controls.The median age in group MPN and healthy volunteers was 59(32~72)and 55(36~78)years.The study was conducted in accordance with the " blood disease diagnosis and treatment standard".All patients provided written informed consent.This study was approved by Baoding First Hospital Ethics Committee.2 Experimental groups in cellsThe HEL cells with JAK2V617 F mutation positive were divided into ruxolitnib treated group and control group.The control group was treated with10% NBS RPMI1640.The ruxolitinib treatment group was cultured in 10%neonatal bovine serum RPMI1640 medium containing different concentrations of ruxolitinib.The concentration of ruxolitnib were(0 nmol/L,50 nmol/L,100 nmol/L,250 nmol/L,500 nmol/L,1000 nmol/L).3 Real-time quantitative PCR was used to detect the mutation burden of JAK2V617 F,the m RNA expression of MMP-2and MMP-9in MPN patients.4 The expression of p-JAK2,MMP-2 and MMP-9 protein was detected by immunohistochemistry in pathological tissues of patients bone marrow.5 Cell counting Kit(CCK-8)was used to observe the inhibitory effect of ruxolitinib on HEL cell proliferation6 18 samples of bone marrow in patients were selected and treated with ruxolitinib 24 h in vitro.CD34 + cells expression levels were detected by flow cytometry.7 Cell migration ability after treated with ruxolitinib 24 h was tested by transwell chambers.8 The protein expression of JAK2? MMP-2and MMP-9 in HEL cells and in MPN patients were applied to examineby Western blot.Results:1 The JAK2V617 F mutation burden were detected between 26.8% and75.4% by the fluorescence quantitative polymerase chain reaction(Q-PCR).The result of Q-PCR: The relative expression of JAK2 m RNA in control group and treated groups by ruxolitinib in different concentration(0 nmol/L,50 nmol/L,100 nmol/L,250 nmol/L,500 nmol/L,1000 nmol/L)for 48 h groups were(0.431±0.043)?(0.362±0.032)?(0.254±0.031)?(0.181±0.022)?(0.143±0.022)?(0.143±0.022).The MMP-2 m RNA relative expression(relative grey absorption)were(0.397±0.031)?(0.362±0.030)?(0.235±0.023)?(0.188±0.018)?(0.155±0.010)?(0.098±0.009).The MMP-9 m RNA relative expression(relative grey absorption)were(0.321±0.032)?(0.252±0.025)?(0.245±0.024)?(0.215±0.025)?(0.155±0.018)?(0.098±0.009).The?-actin m RNA expression levels were no significant difference in different groups.The JAK2 ? MMP-2 and MMP-9 m RNA expression of MPN cells in ruxolitinib-treated groups had gently descent tendency compare with control groups(P<0.05).With the increase of concentration of ruxolitnib,the m RNA expression levels of JAK2,MMP-2 and MMP-9was decreased.There was a significant negative correlation between the two groups(P <0.05).The test results also show that the m RNA expression levels of JAK2,MMP-2 and MMP-9 could down-regulate after ruxolitnib in MPN cells.2 The 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].The expression levels of p-JAK2,MMP-2 and MMP-9 were significantly decreased.3 There was divided into JAK2V617F/JAK2?50% and JAK2V617F/JAK2 < 50% in the newly diagnosed group.The results showed that: The expression levels of p-JAK2,MMP-2 and MMP-9 in the JAK2V617F/JAK2?50% were more higher than in the JAK2V617F/JAK2 <50% group [(85.56± 20.56)% vs(72.84±17.57)%,P<0.05;(56.87±23.32)%vs(41.20±17.21)%,P<0.05;(41.20±17.21)% vs(47.38±17.36)%,P<0.05].4 The result of CCK-8 showed that: The study on the inhibition rate of HEL cells for treatment of different concentrations of ruxolitinib(0nmol/l,50nmol/l,100 nmol/l,250 nmol/l,500 nmol/l,1000 nmol/L),treated for 24 h were(76.10±3.09)% ?(66.04±3.14)% ?(60.06±2.71)% ?(59.27±2.86)% ?(57.86±2.32)%;treated for 48 h were(70.14±3.39)%?(59.71±3.34)%?(52.80±2.61)%?(43.30±2.96)%(38.56±2.15)%;treated for 72 h were(55.83±3.43)% ?(47.63±3.12)% ?(36.43±2.98)% ?(31.05±2.78)% ?(14.48±2.86)%.The proliferative activity of HEL cells could obviously inhibited by ruxolitnib with 24 h,48h,72 h groups.The difference between the groups was statistically significant(P <0.05).With the increase of time and concentration,the inhibition rate increased(P <0.05).5 Effects of ruxolitinib on Bone Marrow CD34 + Cells in JAK2V617 F Positive MPN Patients: The proportion of CD34 + cells in 18 newly diagnosed MPN patients was higher than that in the control group [(0.29 ± 0.13)% vs(0.23 ± 0.05)%,t = 0.286,P <0.001],The level of CD34 + cells in MPN patients treated with 250 nmol / L ruxolitinib group was lower than that in theuntreated group [(0.29 ± 0.13)% vs(0.14 ± 0.07)%,P <0.001].The expression of CD34 + cells in 250nmol/l Ruxolitinib treated group PV(5 patients),ET(6cases)and PMF(7 patients)was lower than that in the untreated group [(0.18± 0.05)vs(0.26±0.05)?(0.19 ± 0.05)vs(0.25 ± 0.05)?(0.14 ± 0.08)vs(0.35± 0.08),P <0.05.]6 Transwell migration assay was used to observe the effect of ruxolitinib on migration of MPN primary cells and HEL cells: The number of MPN primary cells treated with 5 nmol / L ruxolitinib was significantly lower than that of group without ruxolitinib after 24 h(154.7 ± 27.5 vs 320.3 ± 67.3,t =13.47,P <0.01).The number of HEL cells treated with 5 nmol / L Ruxolitinib was less than that of the ruxolitinib control group(70.7 ± 10.5 vs 135.3 ± 16.7,t = 13.89,P <0.01).(CCK 8 showed no significant difference between the control group and the ruxolitinib group in the 24 h group,and the concentration of ruxolitinib was 5 nmol / L.)7 The result of Western blot: The relative expression level of p-JAK2protein(relative grey absorption)in HEL cells treated with ruxolitinib at different concentrations(0nmol/l,50 nmol/l,100 nmol/l,250 nmol/l,500nmol/l,1000 nmol/L)for 48 h groups were(0.12 ± 0.12),(1.21 ± 0.10),(1.12 ±0.09),(0.98 ± 0.07),(0.45 ± 0.04)?(0.15 ± 0.01).The MMP-2 protein expression level of were(0.53±0.04)?(0.42±0.03)?(0.31±0.03)?(0.27±0.02)?(0.23±0.01)?(0.19±0.01).The MMP-9 protein expression level of were(0.78±0.06)?(0.65±0.04)?(0.54±0.04)?(0.30±0.02)?(0.27±0.02)?(0.23±0.01).Compared with the control group,the expression of p-JAK2,MMP-2 and MMP-9 in HEL cells was significantly inhibited by ruxolitinib group(P <0.05).However,there was no significant difference in ?-actin levels in different groups.The protein expression levels of p-JAK2,MMP-2and MMP-9 decreased with the prolongation of concentration.There is a significant negative correlation between them.There was significant statistical significance(P <0.05).The expression levels of p-JAK2,MMP-2 and MMP-9(relative grey absorption)in MPN primary cells treated with 250 nmol / L ruxolitinib was less than that of the untreated with ruxolitinib controlgroup(0.83±0.16vs1.55±0.34,0.72±0.13vs1.42±0.31,0.68±0.13vs1.33±0.29,P <0.01).The results showed that the expression levels of p-JAK2,MMP-2 and MMP-9 could decrease after ruxolitinib the in MPN cells.Conclusions:1 The expression levels of p-JAK2,MMP-2 and MMP-9 in the JAK2V617 F positive MPN patients group were more higher than those in the control group.Further,during the course of ruxolitinib the levels of expression on p-JAK2,MMP-2 and MMP-9 was significantly decreased in MPN patients.2 Ruxolitnib significantly inhibited the proliferation of HEL cells in vitro,and the HEL cell viability decreased significantly after the increase of the concentration of ruxolitinib.3 Ruxolitnib can inhibit the expression level of CD34 + in MPN cells.4 The expression levels of JAK2,MMP-2,MMP-9 m RNA and p-JAK2,MMP-2 and MMP-9 protein in HEL cells were down-regulated by ruxolitinib in vitro and decreased with the increase of ruxolitinib concentration.5 Ruxolitinib can inhibit the migration of primary culture of human MPN cellsand HEL cells. |
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