SHH信号通路在骨髓增生异常综合征中的表达及SMO抑制剂对MUTZ-1细胞的作用研究

Expression of SHH signaling pathway in myelodysplastic syndrome and effect of SMO inhibitor on MUTZ-1 cells

摘要:

目的 探讨Sonic Hedgehog（SHH）信号通路相关基因在骨髓增生异常综合征（MDS）患者中的表达及Smoothened（SMO）抑制剂Jervine对MUTZ-1细胞的作用。方法 选取2016年6月—2018年3月于新疆医科大学第一附属医院血液病中心经骨髓细胞形态学、染色体R显带分析、荧光原位杂交、流式细胞术检查确诊的53例MDS患者。依据国际预后积分系统（IPSS）对患者预后分组，低危组2例，中危1组16例，中危2组21例，高危组14例。将低危组和中危1组归为相对低危组，中危2组和高危组归为相对高危组。选取同期该院25例缺铁性贫血患者作为正常对照组。采用实时荧光定量聚合酶链反应（qRT-PCR）检测3组患者的SHH、Patched-1、SMO和Gli-1 mRNA相对表达量；终浓度0 μmol/L、1 μmol/L、5 μmol/L、10 μmol/L Jervine作用于MUTZ-1细胞，孵育24 h；CCK-8法检测Jervine 0 μmol/L组、Jervine 1 μmol/L组、Jervine 5 μmol/L组、Jervine 10 μmol/L组MUTZ-1细胞的增殖率；Annexin V-FITC/PI双染色流式细胞术检测4组MUTZ-1细胞的凋亡率；qRT-PCR检测4组MUTZ-1细胞的SMO、Gli-1 mRNA相对表达量；Western blotting法检测4组MUTZ-1细胞SMO、Gli-1、Bcl-2、Caspase-3和Cyclin D1蛋白相对表达量。结果 3组患者的SHH、Patched-1 mRNA相对表达量比较，差异无统计学意义（P >0.05）；SMO、Gli-1 mRNA相对表达量比较，差异有统计学意义（P <0.05），相对高危组SMO和Gli-1 mRNA的相对表达量高于正常对照组和相对低危组（P <0.05）。SMO基因高、低表达组患者和Gli-1基因高、低表达组患者3年累积生存率比较，均差异有统计学意义（P <0.05）。Jervine 0 μmol/L组、Jervine 1 μmol/L组、Jervine 5 μmol/L组、Jervine 10 μmol/L组MUTZ-1细胞的增殖率比较，差异有统计学意义（P <0.05），MUTZ-1细胞增殖率与Jervine浓度呈负相关（r =-0.977，P =0.000），随Jervine浓度的升高而降低；Jervine 0 μmol/L组、Jervine 1 μmol/L组、Jervine 5 μmol/L组、Jervine 10 μmol/L组MUTZ-1细胞的凋亡率比较，差异有统计学意义（P <0.05），MUTZ-1细胞凋亡率与Jervine浓度呈正相关（r =0.997，P =0.000），随Jervine浓度的升高而增加。Jervine 0 μmol/L组、Jervine 1 μmol/L组、Jervine 5 μmol/L组、Jervine 10 μmol/L组MUTZ-1细胞的SMO、Gli-1 mRNA相对表达量和蛋白相对表达量比较，差异有统计学意义（P <0.05），Jervine 5 μmol/L组和Jervine 10 μmol/L组低于Jervine 0 μmol/L组（P <0.05）。Jervine 1 μmol/L组、Jervine 5 μmol/L组、Jervine 10 μmol/L组与Jervine 0 μmol/L组比较，Bcl-2、Cyclin D1蛋白相对表达量降低（P <0.05），Caspase-3蛋白相对表达量升高（P <0.05）。结论 MDS患者SHH信号通路异常激活，参与MDS的发生、发展，Jervine能有效抑制MUTZ-1细胞增殖，促进其凋亡。

Abstract:

Objective To observe the expression of Sonic Hedgehog (SHH) signaling pathway related genes in (MDS) of myelodysplastic syndrome and the inhibitory effect of SMO inhibitor Jervine on MUTZ-1 cells.Methods From June 2016 to March 2018, 53 patients with MDS in the Hematological Disease Center of the first affiliated Hospital of Xinjiang Medical University were diagnosed by bone marrow morphology, chromosome R-banding analysis, fluorescence in situ hybridization, and flow cytometry. According to the International Prognostic score system (IPSS), the patients were divided into low risk group (n = 2), medium risk group 1 (n = 21), and high risk group 2 (n = 14). The low risk group and middle risk group 1 were classified as relatively low risk group, and the middle risk group 2 and high risk group were classified as relatively high risk group. In the same period, 25 patients with iron deficiency anemia were selected as normal control group. The relative expressions of SHH, Patched-1, SMO, and Gli-1mRNA in three groups of patients were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). MUTZ-1 cells were treated with 0 μmol/L, 1 μmol/L, 5 μmol/L, and 10 μmol/ L Jervine for 24 hours to detect the proliferation rate of MUTZ-1 cells, the apoptosis rate was detected by AnnexinV-FITC/PI double staining flow cytometry, and the relative expression of SHH and Gli-1 mRNA in MUTZ-1 cells was detected by qRT-PCR. The relative expressions of SMO, Gli-1, Bcl-2, Caspase-3, and CyclinD1 proteins were detected by Western blotting.Results The relative expressions of SMO and Gli-1 mRNA in the relatively high risk group were higher than those in the normal control group and the relatively low risk group. The median survival time of patients with high expression of SMO and Gli-1 genes was shorter than that of patients with low expression. Jervine inhibited the expression of SMO and Gli-1 genes and proteins in MUTZ-1 cells. The proliferation rate of MUTZ-1 cells was negatively correlated with the concentration of Jervine (r = -0.977, P = 0.000) and decreased with the increase of Jervine concentration, while the apoptosis rate of MUTZ-1 cells was positively correlated with the concentration of Jervine (r = 0.997, P = 0.000) and increased with the increase of Jervine concentration. After the intervention of Jervine, the protein expression of target genes Bcl-2 and CyclinD1 was down-regulated, while the expression of Caspase-3 protein was increased.Conclusion SHH signal pathway is abnormally activated in patients with MDS, which is involved in the occurrence and development of MDS. SMO inhibitors can effectively inhibit the proliferation of MUTZ-1 cells and promote their apoptosis.