干扰β-catenin表达对淋巴瘤细胞增殖、凋亡及cleaved caspase 3蛋白表达水平的影响
|
摘要
目的探讨干扰β-连环蛋白(β-catenin)表达对淋巴瘤细胞增殖、凋亡及活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase 3)表达水平的影响。方法以淋巴瘤细胞Raji为研究对象,采用β-catenin小干扰RNA(siRNA)转染细胞作为β-catenin siRNA组,以阴性对照control siRNA转染细胞作为siRNA control组,以不转染的细胞作为对照组。分别采用实时逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)检测β-catenin mRNA和β-catenin蛋白表达水平,噻唑蓝(MTT)法检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测cleaved caspase 3、c-myc、细胞周期蛋白D1(cyclin D1)的表达水平。结果 siRNA control组和对照组的β-catenin mRNA和β-catenin蛋白表达水平、细胞存活率、细胞凋亡率及cleaved caspase 3、c-myc、cyclin D1蛋白表达水平比较,差异均无统计学意义(P﹥0.05)。β-catenin siRNA组的β-catenin mRNA和β-catenin蛋白表达水平、细胞存活率及c-myc、cyclin D1蛋白表达水平均低于对照组,细胞凋亡率和cleaved caspase 3蛋白表达水平均高于对照组,差异均有统计学意义(P﹤0.05)。结论干扰β-catenin的表达可能通过影响cyclin D1、c-myc、cleaved caspase 3蛋白的表达,抑制淋巴瘤细胞增殖,促进淋巴瘤细胞凋亡。
Objective To investigate the influence of interfering β-catenin expression on the proliferation, apoptosis and expression of cleaved cysteinyl aspartate specific proteinase 3(cleaved caspase 3) in lymphoma cells. Method Lymphoma cell Raji was used in the study, cells transfected with β-catenin small interfering RNA(β-catenin siRNA) and negative control sequences(siRNA control) were named β-catenin siRNA group and siRNA control group, the non-transfected cells served as control group. The levels of β-catenin m RNA and β-catenin protein were detected by real time reverse transcription-polymerase chain reaction(RT-PCR) and Western blot, cell proliferation was determined by methylthiazolyldiphenyl-tetrazolium bromide(MTT), apoptosis was assessed by flow cytometry, the expression levels of cleaved caspase3, c-myc and cyclin D1 were detected by Western blot. Result The levels of β-catenin mRNA and β-catenin protein, cell survival rate, apoptosis rate, and the expression levels of cleaved caspase 3, c-myc and cyclin D1 proteins in siRNA control group were not significantly different from those in the control group(P>0.05). Compared with the control group, the levels of β-catenin m RNA and β-catenin protein in β-catenin siRNA group were significantly lower, as well as the cell survival rate and the expression of c-myc and cyclin D1 proteins were significantly lower, while the rate of apoptosis and the expression of cleaved caspase 3 protein were significantly higher, indicating statistically significant differences(P<0.05).Conclusion Interfering the expression of β-catenin can inhibit the proliferation and apoptosis of lymphoma cells through adjusting the expression of cyclin D1, c-myc, and cleaved caspase 3 proteins.
Objective To investigate the influence of interfering β-catenin expression on the proliferation, apoptosis and expression of cleaved cysteinyl aspartate specific proteinase 3(cleaved caspase 3) in lymphoma cells. Method Lymphoma cell Raji was used in the study, cells transfected with β-catenin small interfering RNA(β-catenin siRNA) and negative control sequences(siRNA control) were named β-catenin siRNA group and siRNA control group, the non-transfected cells served as control group. The levels of β-catenin m RNA and β-catenin protein were detected by real time reverse transcription-polymerase chain reaction(RT-PCR) and Western blot, cell proliferation was determined by methylthiazolyldiphenyl-tetrazolium bromide(MTT), apoptosis was assessed by flow cytometry, the expression levels of cleaved caspase3, c-myc and cyclin D1 were detected by Western blot. Result The levels of β-catenin mRNA and β-catenin protein, cell survival rate, apoptosis rate, and the expression levels of cleaved caspase 3, c-myc and cyclin D1 proteins in siRNA control group were not significantly different from those in the control group(P>0.05). Compared with the control group, the levels of β-catenin m RNA and β-catenin protein in β-catenin siRNA group were significantly lower, as well as the cell survival rate and the expression of c-myc and cyclin D1 proteins were significantly lower, while the rate of apoptosis and the expression of cleaved caspase 3 protein were significantly higher, indicating statistically significant differences(P<0.05).Conclusion Interfering the expression of β-catenin can inhibit the proliferation and apoptosis of lymphoma cells through adjusting the expression of cyclin D1, c-myc, and cleaved caspase 3 proteins.
引文
[1] Yahalom J, Illidge T, Specht L, et al. Modern radiation therapy for extranodal lymphomas:field and dose guidelines from the International Lymphoma Radiation Oncology Group[J]. Int J Radiat Oncol Biol Phys, 2015, 92(1):11-31.
[2] Kochenderfer JN, Dudley ME, Kassim SH, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor[J]. J Clin Oncol, 2015, 33(6):540-549.
[3] Azzolin L, Panciera T, Soligo S, et al. YAP/TAZ incorporation in theβ-catenin destruction complex orchestrates the Wnt response[J]. Cell, 2014, 158(1):157-170.
[4] Huang J, Xiao D, Li G, et al. EphA2 promotes epithelialmesenchymal transition through the Wnt/β-catenin pathway in gastric cancer cells[J]. Oncogene, 2014, 33(21):2737-2747.
[5]李津,钟美佐.β-catenin、RIPK4、CDK8在套细胞淋巴瘤中的表达及临床意义[J].临床肿瘤学杂志, 2015, 20(3):233-237.
[6] Cai J, Maitra A, Anders RA, et al.β-Catenin destruction complex-independent regulation of Hippo-YAP signaling by APC in intestinal tumorigenesis[J]. Genes Dev, 2015, 29(14):1493-1506.
[7] Dudek H, Wong DH, Arvan R, et al. Knockdown ofβ-catenin with dicer-substrate siRNAs reduces liver tumor burden in vivo[J]. Mol Ther, 2014, 22(1):92-101.
[8] Janda CY, Dang LT, You C, et al. Surrogate Wnt agonists that phenocopy canonical Wnt andβ-catenin signalling[J].Nature, 2017, 545(7653):234-237.
[9] Deschene ER, Myung P, Rompolas P, et al.β-catenin activation regulates tissue growth non-cell autonomously in the hair stem cell niche[J]. Science, 2014, 343(6177):1353-1356.
[10] Koehler A, Schlupf J, Schneider M, et al. Loss of Xenopus cadherin-11 leads to increased Wnt/β-catenin signaling and up-regulation of target genes c-myc and cyclin D1 in neural crest[J]. Dev Biol, 2013, 383(1):132-145.
[11]秦贝贝,李亚青,李小丽,等. Wnt/β-catenin通路关键分子在NK/T细胞淋巴瘤组织中的表达及其临床意义[J].吉林大学学报(医学版), 2015, 41(2):230-234.
[12] Chen X, Meng J, Yue W, et al. Fibulin-3 suppresses Wnt/β-catenin signaling and lung cancer invasion[J]. Carcinogenesis, 2014, 35(8):1707-1716.
[13]赵海涛,张海涛. E-cadherin,β-catenin表达与胰腺癌预后的关系[J].实用癌症杂志, 2016, 31(6):899-900.
[14]魏国强,连世忠,郑绘霞. Wnt信号通路中糖原合成激酶3β和β-catenin在人脑胶质瘤中的表达及其意义[J].中国药物与临床, 2016, 16(9):1277-1279.
[15] Jin Y, Nie D, Li J, et al. Gas6/AXL signaling regulates selfrenewal of chronic myelogenous leukemia stem cells by stabilizingβ-catenin[J]. Clin Cancer Res, 2017, 23(11):2842-2855.
[16] Garapaty-Rao S, Nasveschuk C, Gagnon A, et al. Identification of EZH2 and EZH1 small molecule inhibitors with selective impact on diffuse large B cell lymphoma cell growth[J]. Chem Biol, 2013, 20(11):1329-1339.
[17] Jiang H, Zhao PJ, Su D, et al. Paris saponin I induces apoptosis via increasing the Bax/Bcl2 ratio and caspase3 expression in gefitinibresistant nonsmall cell lung cancer in vitro and in vivo[J]. Mol Med Rep, 2014, 9(6):2265-2272.
[18]张丹,吕亮,申兴斌,等.大肠癌中Survivin、NF-κB、I-κB及Caspase-3的表达及意义[J].中国老年学杂志, 2017,37(3):549-552.
[19] Ok CY, Xu-Monette ZY, Tzankov A, et al. Prevalence and clinical implications of cyclin D1 expression in diffuse large B-cell lymphoma(DLBCL)treated with immunochemotherapy:a report from the International DLBCL Rituximab-CHOP Consortium Program[J]. Cancer, 2014, 120(12):1818-1829.
[20]柴菲,陈振文,郗彦凤,等. c-myc在间变性大细胞淋巴瘤中的表达及其意义[J].白血病·淋巴瘤, 2015, 24(4):238-244.
[21] Huang F, Tong X, Fu L, et al. Knockdown of STAT3 by shRNA inhibits the growth of CAOV3 ovarian cancer cell line in vitro and in vivo.[J]. Acta Biochimica Et Biophysica Sinica, 2010, 40(6):519-525.
[22]崔艳香,王新花,陈勋.应用流式细胞术检测B细胞淋巴瘤患者CyclinD1和BCL-2的价值分析[J].中国实验血液学杂志, 2016, 24(1):98-101.
[23]杨顺娥,李迅,赵兵. NF-κB, c-myc和survivin在非霍奇金淋巴瘤中的表达及临床意义[J].肿瘤, 2009, 29(9):868-873.
[2] Kochenderfer JN, Dudley ME, Kassim SH, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor[J]. J Clin Oncol, 2015, 33(6):540-549.
[3] Azzolin L, Panciera T, Soligo S, et al. YAP/TAZ incorporation in theβ-catenin destruction complex orchestrates the Wnt response[J]. Cell, 2014, 158(1):157-170.
[4] Huang J, Xiao D, Li G, et al. EphA2 promotes epithelialmesenchymal transition through the Wnt/β-catenin pathway in gastric cancer cells[J]. Oncogene, 2014, 33(21):2737-2747.
[5]李津,钟美佐.β-catenin、RIPK4、CDK8在套细胞淋巴瘤中的表达及临床意义[J].临床肿瘤学杂志, 2015, 20(3):233-237.
[6] Cai J, Maitra A, Anders RA, et al.β-Catenin destruction complex-independent regulation of Hippo-YAP signaling by APC in intestinal tumorigenesis[J]. Genes Dev, 2015, 29(14):1493-1506.
[7] Dudek H, Wong DH, Arvan R, et al. Knockdown ofβ-catenin with dicer-substrate siRNAs reduces liver tumor burden in vivo[J]. Mol Ther, 2014, 22(1):92-101.
[8] Janda CY, Dang LT, You C, et al. Surrogate Wnt agonists that phenocopy canonical Wnt andβ-catenin signalling[J].Nature, 2017, 545(7653):234-237.
[9] Deschene ER, Myung P, Rompolas P, et al.β-catenin activation regulates tissue growth non-cell autonomously in the hair stem cell niche[J]. Science, 2014, 343(6177):1353-1356.
[10] Koehler A, Schlupf J, Schneider M, et al. Loss of Xenopus cadherin-11 leads to increased Wnt/β-catenin signaling and up-regulation of target genes c-myc and cyclin D1 in neural crest[J]. Dev Biol, 2013, 383(1):132-145.
[11]秦贝贝,李亚青,李小丽,等. Wnt/β-catenin通路关键分子在NK/T细胞淋巴瘤组织中的表达及其临床意义[J].吉林大学学报(医学版), 2015, 41(2):230-234.
[12] Chen X, Meng J, Yue W, et al. Fibulin-3 suppresses Wnt/β-catenin signaling and lung cancer invasion[J]. Carcinogenesis, 2014, 35(8):1707-1716.
[13]赵海涛,张海涛. E-cadherin,β-catenin表达与胰腺癌预后的关系[J].实用癌症杂志, 2016, 31(6):899-900.
[14]魏国强,连世忠,郑绘霞. Wnt信号通路中糖原合成激酶3β和β-catenin在人脑胶质瘤中的表达及其意义[J].中国药物与临床, 2016, 16(9):1277-1279.
[15] Jin Y, Nie D, Li J, et al. Gas6/AXL signaling regulates selfrenewal of chronic myelogenous leukemia stem cells by stabilizingβ-catenin[J]. Clin Cancer Res, 2017, 23(11):2842-2855.
[16] Garapaty-Rao S, Nasveschuk C, Gagnon A, et al. Identification of EZH2 and EZH1 small molecule inhibitors with selective impact on diffuse large B cell lymphoma cell growth[J]. Chem Biol, 2013, 20(11):1329-1339.
[17] Jiang H, Zhao PJ, Su D, et al. Paris saponin I induces apoptosis via increasing the Bax/Bcl2 ratio and caspase3 expression in gefitinibresistant nonsmall cell lung cancer in vitro and in vivo[J]. Mol Med Rep, 2014, 9(6):2265-2272.
[18]张丹,吕亮,申兴斌,等.大肠癌中Survivin、NF-κB、I-κB及Caspase-3的表达及意义[J].中国老年学杂志, 2017,37(3):549-552.
[19] Ok CY, Xu-Monette ZY, Tzankov A, et al. Prevalence and clinical implications of cyclin D1 expression in diffuse large B-cell lymphoma(DLBCL)treated with immunochemotherapy:a report from the International DLBCL Rituximab-CHOP Consortium Program[J]. Cancer, 2014, 120(12):1818-1829.
[20]柴菲,陈振文,郗彦凤,等. c-myc在间变性大细胞淋巴瘤中的表达及其意义[J].白血病·淋巴瘤, 2015, 24(4):238-244.
[21] Huang F, Tong X, Fu L, et al. Knockdown of STAT3 by shRNA inhibits the growth of CAOV3 ovarian cancer cell line in vitro and in vivo.[J]. Acta Biochimica Et Biophysica Sinica, 2010, 40(6):519-525.
[22]崔艳香,王新花,陈勋.应用流式细胞术检测B细胞淋巴瘤患者CyclinD1和BCL-2的价值分析[J].中国实验血液学杂志, 2016, 24(1):98-101.
[23]杨顺娥,李迅,赵兵. NF-κB, c-myc和survivin在非霍奇金淋巴瘤中的表达及临床意义[J].肿瘤, 2009, 29(9):868-873.