siRNA抑制syk基因对外周T细胞淋巴瘤细胞增殖与凋亡的影响
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摘要
目的探讨siRNA抑制脾酪氨酸激酶(syk)基因后对外周T细胞淋巴瘤细胞株HUT-78细胞增殖与凋亡的影响。方法针对syk基因特异靶点设计3条siRNA(siRNA-1、siRNA-2和siRNA-3),采用电穿孔法转染外周T细胞淋巴瘤细胞株HUT-78,同时设Mock组和siRNA-NC组,转染48 h后,分别用RT-PCR和Western blot技术检测syk mRNA和蛋白的表达水平,筛选出最有效的syk siRNA;syk siRNA转染HUT-78后分别用软琼脂克隆形成实验检测syk下调后HUT-78细胞的克隆形成能力,MTT法检测干扰24、48、72 h后细胞增殖情况,流式细胞术检测syk下调后HUT-78细胞的凋亡情况。结果 RT-PCR和Western blot结果显示,与Mock组和siRNA-NC组相比,3条siRNA均能有效降低HUT-78细胞中syk mRNA和蛋白的表达,其中syk siRNA-1抑制效果最明显。克隆形成实验显示,下调syk基因表达后HUT-78细胞的克隆形成能力与Mock组相比明显下降(P<0.05);MTT实验结果显示,下调syk基因表达后HUT-78细胞的增殖能力与Mock组相比显著下降(P<0.05);流式细胞术结果显示,下调syk基因表达后HUT-78细胞的凋亡比例明显高于Mock组(P<0.05)。结论 siRNA下调syk基因表达后可抑制外周T细胞淋巴瘤细胞的增殖、克隆形成,促进凋亡,推测syk基因在外周T细胞淋巴瘤的发生发展中发挥重要作用,有可能成为外周T细胞淋巴瘤基因治疗的新靶点。
Objective To investigate the influence of down-regulation of syk via siRNA on the proliferation and apoptosis of human peripheral T-cell lymphoma HUT-78 cells. Methods 3 siRNAs targeting syk were designed,synthetized,and transfected into HUT-78 cells via electroporation. Cells transfected with negative control siRNA and untreated cells were used as controls. 48 h after transfection,the expression of syk mRNA and protein were examined by RT-PCR and Western blot respectively,and the most effective syk siRNA was screened out. The proliferation of cells after 24 h,48 h and 72 h interference and colony formation ability were examined by MTT and soft agar assay. Flow cytometry was used to detect the cell apoptosis. Results 3 siRNAs notably down-regulated syk expression at mRNA and protein levels,and the inhibitory effect of siRNA-1 was the most obvious. The colony formation of syk siRNA-1 group was significantly decreased compared with control groups after down-regulationof syk expression(P < 0. 05). The results of MTT test showed that the proliferation ability of syk siRNA-1 group was significantly decreased compared with control groups(P < 0. 05). Flow cytometry results showed that the apoptosis ratio of syk siRNA-1 group was about 37%,obviously higher than control groups(P < 0. 05). Conclusion The down-regulation of syk expression suppressed the cell proliferation and colony formation ability,and promoted the cell apoptosis of peripheral T-cell lymphoma cells. The results showed that syk gene may play a very important role in the occurrence and development of peripheral T-cell lymphoma,and it may be a novel target for gene therapy of peripheral T-cell lymphoma.
Objective To investigate the influence of down-regulation of syk via siRNA on the proliferation and apoptosis of human peripheral T-cell lymphoma HUT-78 cells. Methods 3 siRNAs targeting syk were designed,synthetized,and transfected into HUT-78 cells via electroporation. Cells transfected with negative control siRNA and untreated cells were used as controls. 48 h after transfection,the expression of syk mRNA and protein were examined by RT-PCR and Western blot respectively,and the most effective syk siRNA was screened out. The proliferation of cells after 24 h,48 h and 72 h interference and colony formation ability were examined by MTT and soft agar assay. Flow cytometry was used to detect the cell apoptosis. Results 3 siRNAs notably down-regulated syk expression at mRNA and protein levels,and the inhibitory effect of siRNA-1 was the most obvious. The colony formation of syk siRNA-1 group was significantly decreased compared with control groups after down-regulationof syk expression(P < 0. 05). The results of MTT test showed that the proliferation ability of syk siRNA-1 group was significantly decreased compared with control groups(P < 0. 05). Flow cytometry results showed that the apoptosis ratio of syk siRNA-1 group was about 37%,obviously higher than control groups(P < 0. 05). Conclusion The down-regulation of syk expression suppressed the cell proliferation and colony formation ability,and promoted the cell apoptosis of peripheral T-cell lymphoma cells. The results showed that syk gene may play a very important role in the occurrence and development of peripheral T-cell lymphoma,and it may be a novel target for gene therapy of peripheral T-cell lymphoma.
引文
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[13]Agarwal A,Tyner J W.RNAi screening of leukemia cells using electroporation[J].Methods Mol Biol,2016,1470:85-94.
[14]Fakhr E,Zare F,Teimoori-Toolabi L.Precise and efficient siRNA design:a key point in competent gene silencing[J].Cancer Gene Ther,2016,23(4):73-82.
[15]Wilcox R A,Sun D X,Novak A,et al.Inhibition of Syk protein tyrosine kinase induces apoptosis and blocks proliferation in T-cell non-Hodgkin lymphoma cell lines[J].Leukemia,2010,24(1):229-32.
[2]Savage K J.Peripheral T-cell lymphomas[J].Blood Rev,2007,21(4):201-16.
[3]李小秋.淋巴瘤病理研究新进展[J].中国癌症杂质,2014,24(10):721-6.
[4]Li X Q,Li G D,Gao Z F,et al.Distribution pattern of lymphoma subtypes in China:A nationwide multicenter study of 10 002 cases[J].J Diagn Concepts Pract,2012,11(2):111-5.
[5]Schmitz N,Trümper L,Ziepert M,et al.Treatment and prognosis of mature T-cell and NK-cell lymphoma:an analysis of patients with T-cell lymphoma treated in studies of the german high-grade nonhodgkin lymphoma study group[J].Blood,2010,116(18):3418-25.
[6]Coopman P J,Do M T,Barth M,et al.The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells[J].Nature,2000,406(6797):742-7.
[7]Layton T,Stalens C,Gunderson F,et al.Syk tyrosine kinase acts as a pancreatic adenocarcinoma tumor suppressor by regulating cellular growth and invasion[J].Am J Pathol,2009,175(6):2625-36.
[8]Hong J,Hu K,Yuan Y,et al.CHK1 targets spleen tyrosine kinase(L)for proteolysis in hepatocellular carcinoma[J].J Clin Invest,2012,122(6):2165-75.
[9]Ma J,Xing W,Coffey G,et al.Cerdulatinib,a novel dual SYK/JAK kinase inhibitor,has broad anti-tumor activity in both ABC and GCB types of diffuse large B cell lymphoma[J].Oncotarget,2015,6(41):43881-96.
[10]代群,詹鹤琴,曹立宇,等,Syk、PI3K、Akt在外周T细胞淋巴瘤中的表达及其意义[J].安徽医科大学学报,2014,49(8):1141-5.
[11]Friedberg J W,Sharman J,Sweetenham J,et al.Inhibition of Syk with fostamatinib disodium has chronic lympocytic leukemia[J].Blood,2010,115(13):2578-85.
[12]Feldman A L,Sun D X,Law M E,et al.Overexpression of Syk tyrosine kinase in peripheral T-cell lymphomas[J].Leukemia,2008,22(6):1139-43.
[13]Agarwal A,Tyner J W.RNAi screening of leukemia cells using electroporation[J].Methods Mol Biol,2016,1470:85-94.
[14]Fakhr E,Zare F,Teimoori-Toolabi L.Precise and efficient siRNA design:a key point in competent gene silencing[J].Cancer Gene Ther,2016,23(4):73-82.
[15]Wilcox R A,Sun D X,Novak A,et al.Inhibition of Syk protein tyrosine kinase induces apoptosis and blocks proliferation in T-cell non-Hodgkin lymphoma cell lines[J].Leukemia,2010,24(1):229-32.