Wnt/β-catenin信号通路对肺腺癌A549细胞肿瘤干细胞特性作用研究
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摘要
目的：不同浓度氯化锂(LiCl)作用A549细胞或利用小干扰RNA(siRNA)技术干扰A549细胞P-catenin的表达从而激活或抑制Wnt/β-catenin信号通路,观察细胞增殖、克隆形成、迁徙及耐药能力等肿瘤干细胞特性的改变,并且观察肺癌干细胞标记物OCT-4的表达变化,从而探讨Wnt/β-catenin信号通路对肺腺癌A549细胞肿瘤干细胞特性的作用并初步探讨其作用机制。
方法：A549细胞经不用浓度的LiCl作用不同时间,选定最佳条件,观察其对Wnt/β-catenin通路的激活作用；β-catenin干扰质粒转染A549细胞,观察其对细胞内Wnt/p-catenin信号通路的抑制作用；观察指标：噻唑蓝(MTT)法检测细胞增殖能力、流式细胞术进行细胞周期分析、克隆形成实验观察细胞克隆形成率、Millicell小室迁徙实验测定细胞的迁徙能力、药物敏感实验观察细胞耐药能力的改变、实时(real time)PCR法及western blot蛋白印迹法检测干细胞标记OCT-4基因及蛋白水平表达的改变,研究Wnt/β-catenin信号通路激活与抑制对上述指标的作用。
结果：经预试验确定LiCl的最佳作用浓度为10mmol/L,最佳作用时间为24 h。A549细胞经10mmol/L LiCl作用24 h后,与对照组相比,蛋白印迹实验结果显示P-catenin的蛋白水平表达增加(1.240±1.220 vs 0.987±0.903,P<0.05),免疫组化显示P-catenin的表达出现胞浆内积聚及向胞核内转移的现象,real time PCR结果发现Wnt/β-catenin信号通路的靶基因cyclin D1表达上调(0.023±0.006 vs 0.007±0.003, P<0.05), Wnt/p-catenin信号通路激活；Wnt/β-catenin信号通路激活后,A549细胞的增殖能力增强,增殖率为32.24%,G0-G1期细胞明显减少[(63.3±3.205)%vs(79.81±1.5)%,P<0.01],S期细胞增多[(32.866±3.631)%vs(19.513±2.508)%,P<0.01],克隆形成率增加[(22.875±2.115)%vs(11.455±4.274)%,P<0.01],穿过Millicell小室底膜的细胞数明显增多(52.5±4.041 vs 27.250±2.500,P<0.01),对顺铂的耐药能力在所检测药物浓度均有不同程度的增强,OCT-4基因(0.073±0.012 vs 0.017±0.006,P<0.01)及蛋白(0.920±0.182 vs 0.335±0.064,P<0.01)表达水平均上调；β-catenin干扰质粒转染A549细胞后,real timePCR结果发现β-catenin mRNA表达明显下降(0.002±0.001 vs 0.023±0.002,P<0.01),同时Wnt/β-catenin信号通路的靶基因cyclin D1 mRNA表达下调(0.005±0.002 vs 0.042±0.004,P<0.05),此信号通路被抑制；Wnt/β-catenin信号通路抑制后,细胞的增殖能力降低,增殖抑制率为20.29%,G0-G1期细胞明显增多[(86.4±2.6)%vs(73.8±0.9)%,P<0.01],S期及G2-M期细胞减少,克隆形成率降低[(31.6±7.7)%vs(46.9±7.3)%,P<0.05],穿过Millicell小室底膜的细胞数明显减少(16.0±3.8 vs 32.7±3.1,P<0.01),对顺铂的耐药能力降低,OCT-4基因[(0.016±0.011)vs(0.043±0.032),P<0.05]及蛋白[(0.28±0.078)vs(0.555±0.088),P<0.05]表达水平均下调。
结论：A549细胞经10 mmol/L LiCl作用24 h,可激活细胞内Wnt/β-catenin信号通路；Wnt/β-catenin信号通路的激活可促进A549细胞由G1期向S期转换,从而促进细胞增殖,同时克隆形成、迁徙及耐药能力等肿瘤干细胞特性增强,肺癌干细胞标记OCT-4的基因及蛋白表达均增加；β-catenin干扰质粒转染A549细胞可抑制细胞内Wnt/β-catenin信号传导途径；Wnt/β-catenin信号通路的抑制可使细胞发生G0-G1期阻滞,抑制细胞增长,同时克隆形成、迁徙及耐药等肿瘤干细胞特性受抑制,肺癌干细胞标记物OCT-4的基因及蛋白表达均降低。以Wnt/β-catenin信号传导途径为靶点,抑制此通路在肺腺癌中的活化及其靶基因的表达,可能成为肺癌分子靶向治疗并有可能是针对肺癌干细胞治疗的新靶点。
Objective:To study the effects of Wnt/β-catenin signaling pathway in lung adenocarcinoma A549 cells.
Methods:After A549 cells were stimulated with Lithium Chloride (LiCl) or transfected withβ-catenin interference plasmids, real time PCR was employed to detect the expression ofβ-catenin, cyclin D1 (a target gene of Wnt/β-catenin pathway) and OCT-4. Western blot was used to measure the expression of P-catenin and the stem cell marker OCT-4. A MTT cell proliferation assay was performed to study the proliferating capacity. Flow cytometry was used for cell cycle analysis. Clone formation and Millicell chamber experiments were performed to evaluate the clone formation and migration capacities of cells. Drug resistance experiments were performed to study the drug resistance capacity.
Results:Stimulating A549 cells with 10 mmol/L Lithium Chloride (LiCl) for 24 h resulted in accumulation ofβ-catenin and upregulation of a typical Wnt target gene cyclin D 1(0.023±0.006 vs 0.007±0.003, P<0.05). This stimulation also significantly enhanced proliferation, clone formation[(22.875±2.115)% vs (11.455±4.274)%, P<0.01], migration (52.5±4.041 vs 27.250±2.500, P< 0.01) and drug resistance abilities in A549 cells. Moreover, the upregulation of OCT-4, a stem cell marker, was observed through real time PCR and western blot. In a reverse approach, Wnt signaling pathway was inhibited by knocking down the expression ofβ-catenin using RNA interference technology. This inhibition resulted in down-regulation of the Wnt target gene cyclin D1 (0.005±0.002 vs 0.042±0.004, P<0.05) as well as the proliferation, clone formation [(31.6±7.7)% vs (46.9±7.3)%, P<0.05], migration (16.0±3.8 vs 32.7±3.1, P<0.01) and drug resistance abilities. Meanwhile, the expression of OCT-4 was reduced after the inhibition of Wnt/β-catenin signaling.
Conclusions:Wnt signaling plays an important role in lung cancer stem cell properties and the expression of OCT-4, which may be a new target for lung cancer therapy.
方法：A549细胞经不用浓度的LiCl作用不同时间,选定最佳条件,观察其对Wnt/β-catenin通路的激活作用；β-catenin干扰质粒转染A549细胞,观察其对细胞内Wnt/p-catenin信号通路的抑制作用；观察指标：噻唑蓝(MTT)法检测细胞增殖能力、流式细胞术进行细胞周期分析、克隆形成实验观察细胞克隆形成率、Millicell小室迁徙实验测定细胞的迁徙能力、药物敏感实验观察细胞耐药能力的改变、实时(real time)PCR法及western blot蛋白印迹法检测干细胞标记OCT-4基因及蛋白水平表达的改变,研究Wnt/β-catenin信号通路激活与抑制对上述指标的作用。
结果：经预试验确定LiCl的最佳作用浓度为10mmol/L,最佳作用时间为24 h。A549细胞经10mmol/L LiCl作用24 h后,与对照组相比,蛋白印迹实验结果显示P-catenin的蛋白水平表达增加(1.240±1.220 vs 0.987±0.903,P<0.05),免疫组化显示P-catenin的表达出现胞浆内积聚及向胞核内转移的现象,real time PCR结果发现Wnt/β-catenin信号通路的靶基因cyclin D1表达上调(0.023±0.006 vs 0.007±0.003, P<0.05), Wnt/p-catenin信号通路激活；Wnt/β-catenin信号通路激活后,A549细胞的增殖能力增强,增殖率为32.24%,G0-G1期细胞明显减少[(63.3±3.205)%vs(79.81±1.5)%,P<0.01],S期细胞增多[(32.866±3.631)%vs(19.513±2.508)%,P<0.01],克隆形成率增加[(22.875±2.115)%vs(11.455±4.274)%,P<0.01],穿过Millicell小室底膜的细胞数明显增多(52.5±4.041 vs 27.250±2.500,P<0.01),对顺铂的耐药能力在所检测药物浓度均有不同程度的增强,OCT-4基因(0.073±0.012 vs 0.017±0.006,P<0.01)及蛋白(0.920±0.182 vs 0.335±0.064,P<0.01)表达水平均上调；β-catenin干扰质粒转染A549细胞后,real timePCR结果发现β-catenin mRNA表达明显下降(0.002±0.001 vs 0.023±0.002,P<0.01),同时Wnt/β-catenin信号通路的靶基因cyclin D1 mRNA表达下调(0.005±0.002 vs 0.042±0.004,P<0.05),此信号通路被抑制；Wnt/β-catenin信号通路抑制后,细胞的增殖能力降低,增殖抑制率为20.29%,G0-G1期细胞明显增多[(86.4±2.6)%vs(73.8±0.9)%,P<0.01],S期及G2-M期细胞减少,克隆形成率降低[(31.6±7.7)%vs(46.9±7.3)%,P<0.05],穿过Millicell小室底膜的细胞数明显减少(16.0±3.8 vs 32.7±3.1,P<0.01),对顺铂的耐药能力降低,OCT-4基因[(0.016±0.011)vs(0.043±0.032),P<0.05]及蛋白[(0.28±0.078)vs(0.555±0.088),P<0.05]表达水平均下调。
结论：A549细胞经10 mmol/L LiCl作用24 h,可激活细胞内Wnt/β-catenin信号通路；Wnt/β-catenin信号通路的激活可促进A549细胞由G1期向S期转换,从而促进细胞增殖,同时克隆形成、迁徙及耐药能力等肿瘤干细胞特性增强,肺癌干细胞标记OCT-4的基因及蛋白表达均增加；β-catenin干扰质粒转染A549细胞可抑制细胞内Wnt/β-catenin信号传导途径；Wnt/β-catenin信号通路的抑制可使细胞发生G0-G1期阻滞,抑制细胞增长,同时克隆形成、迁徙及耐药等肿瘤干细胞特性受抑制,肺癌干细胞标记物OCT-4的基因及蛋白表达均降低。以Wnt/β-catenin信号传导途径为靶点,抑制此通路在肺腺癌中的活化及其靶基因的表达,可能成为肺癌分子靶向治疗并有可能是针对肺癌干细胞治疗的新靶点。
Objective:To study the effects of Wnt/β-catenin signaling pathway in lung adenocarcinoma A549 cells.
Methods:After A549 cells were stimulated with Lithium Chloride (LiCl) or transfected withβ-catenin interference plasmids, real time PCR was employed to detect the expression ofβ-catenin, cyclin D1 (a target gene of Wnt/β-catenin pathway) and OCT-4. Western blot was used to measure the expression of P-catenin and the stem cell marker OCT-4. A MTT cell proliferation assay was performed to study the proliferating capacity. Flow cytometry was used for cell cycle analysis. Clone formation and Millicell chamber experiments were performed to evaluate the clone formation and migration capacities of cells. Drug resistance experiments were performed to study the drug resistance capacity.
Results:Stimulating A549 cells with 10 mmol/L Lithium Chloride (LiCl) for 24 h resulted in accumulation ofβ-catenin and upregulation of a typical Wnt target gene cyclin D 1(0.023±0.006 vs 0.007±0.003, P<0.05). This stimulation also significantly enhanced proliferation, clone formation[(22.875±2.115)% vs (11.455±4.274)%, P<0.01], migration (52.5±4.041 vs 27.250±2.500, P< 0.01) and drug resistance abilities in A549 cells. Moreover, the upregulation of OCT-4, a stem cell marker, was observed through real time PCR and western blot. In a reverse approach, Wnt signaling pathway was inhibited by knocking down the expression ofβ-catenin using RNA interference technology. This inhibition resulted in down-regulation of the Wnt target gene cyclin D1 (0.005±0.002 vs 0.042±0.004, P<0.05) as well as the proliferation, clone formation [(31.6±7.7)% vs (46.9±7.3)%, P<0.05], migration (16.0±3.8 vs 32.7±3.1, P<0.01) and drug resistance abilities. Meanwhile, the expression of OCT-4 was reduced after the inhibition of Wnt/β-catenin signaling.
Conclusions:Wnt signaling plays an important role in lung cancer stem cell properties and the expression of OCT-4, which may be a new target for lung cancer therapy.
引文
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[1]Bansal N, Banerjee D. Tumor initiating cells[J].2009,10(2):192-196.
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[4]Eramo a, Lotti F, Setteet G, et al. Identification and expansion of the tumorigenic lung cancer stem cell population[J]. Cell Death and Differentiation,2008,15(3): 504-514.
[5]Singh SK, Clarke ID, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res,2003,63(18):5821-5828.
[6]Collins AT, Berry PA, Hyde C, et al. Prospective identification of tumorigenic prostate cancer stem cells[J]. Cancer Res,2005,65 (23):10946-10951.
[7]O'Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[J]. Nature,2007,445(7123): 106-110.
[8]Al-Hajj M, Clarke MF. Self-renewal and solid tumor stem cells[J].Oncogene,2004, 23(43):7274-7282.
[9]Dean M, Fojo T, Bates S, et al. Tumour stem cells and drug resistance[J]. Nat Rev Cancer,2005,5(4):275-284.
[10]Lou H, Dean M. Targeted therapy for cancer stem cells:the patched pathway and ABC transporters[J]. Oncogene,2007,26(9):1357-1360.
[11]Hadnagy A, Gaboury L, Beaulieu R, et al. SP analysis may be used to identify cancer stem cell populations[J]. Exp Cell Res,2006,312(19):3701-3710.
[12]Nelson WJ, Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways[J]. Science,2004,303(5663):1483-1487.
[13]Reya T, Clevers H. Wnt signaling in stem cells and cancer[J]. Nature, 2005,434(7035):843-850.
[14]Pinto D, Gregorieff A, Begthel H, et al. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium [J]. Genes Dev,2003,17(14):1709-1713.
[15]Duncan AW, Rattis FM, DiMascio LN, et al. Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance[J]. Nature Immunol,2005,6(3): 314-322.
[16]Zhu AJ, Watt FM. β-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion[J]. Development,1999, (126):2285-2298.
[17]Muroyama Y, Kondoh H, Takada S. Wnt proteins promote neuronal differentiation in neural stem cell culture[J]. Biochem Biophys Res Commun,2004, 313(4):915-921.
[18]He B, Barg RN, You L, et al. Wnt signaling in stem cells and non-small-cell lung cancer[J]. Clin Lung Cancer,2005,7(1):54-60.
[19]Ovring IM, Clever HC. Molecular causes of colon cancer[J]. Eur Clin Invest, 2002,32(6):448-457.
[20]Musgrove EA. Wnt signalling via the epidermal growth factor receptor:a role in breast cancer[J]. Breast Cancer Res,2004,6(2):65-68.
[21]Maelandsmo GM, Holm R, Nesland JM, et al. Reduced beta-catenin expression in the cytoplasm of advanced-stage superficial spreading malignant melanoma[J]. Clin Cancer Res,2003,9(9):3383-3388.
[22]Khan NI, Bradstock KF, Bendall LJ, et al. Activation of Wnt/beta-catenin pathway mediates growth and survival in B-cell progenitor acute lymphoblastic leukaemia[J]. Br J Haematol,2007,138(3):338-348.
[23]Ysebaert L, Chicanne G, Demur C, et al. Expression of beta-catenin by acute myeloid leukemia cells predicts enhanced clonogenic capacities and poor prognosis[J]. Leukemia,2006,20(7):1211-1216.
[24]Kawaguchi-Ihara N, Murohashi I, Nara N, et al.Promotion of the self-renewal capacity of human acute leukemia cells by Wnt3A[J]. Anticancer Res,2008,28(5A): 2701-2704.
[25]Thiago LS, Costa ES, Lopes DV, et al. The Wnt signaling pathway regulates Nalm-16 b-cell precursor acute lymphoblastic leukemic cell line survival and etoposide resistance[J]. Biomed Pharmacother,2010,64(1):63-72.
[26]Hu J, Dong A, Fernandez-Ruiz V, et al. Blockade of Wnt signaling inhibits angiogenesis and tumor growth inhepatocellular carcinoma. CancerRes,2009,69(17): 6951-6959.
[27]Yamashita T, Ji J, Budhu A, et al. EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features[J]. Gastroenterology, 2009,136(3):1012-1024.
[28]Yamashita T, Budhu A, Forgues M, et al. Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma. Cancer Res, 2007,67(22):10831-10839.
[29]Yang W, Yan HX, Chen L, et al. Wnt/beta-catenin signaling contributes to activation of normal and tumorigenic liver progenitor cells[J]. Cancer Res,2008, 68(11):4287-4295.
[30]Tepera SB, Mccrea PD, Rosen JM. A beta-catenin surviv-al signal is required for normal lobular development in the mammary and[J]. J Cell Sci,2003,116(Pt 6): 1137-1149.
[31]Liu BY, Mcdermott SP, Khwma SS, et al. The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells[J]. Proc Natl Acad Sci USA,2004,101(12):4158-4163.
[32]Li Y, Welm B, Podsypaina K, et al. Evidence that trans-genes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells[J]. Proc Natl Acad Sci USA,2003,100(26):15853-15858.
[33]Woodward WA, Chen MS, Behbod F, et al. WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells[J]. Proc Nat Acad Sci USA, 2007,104(2):618-623.
[34]Chen MS, Woodward WA, Behbod F, et al.Wnt/beta-catenin mediates radiationresistance of Scal+progenitors in an immortalized mammary gland cell line[J]. J Cell Sci,2007,120(Pt 3):468-477.
[35]Schulenburg A, Cech P, Herbacek I, et al. CD44-positive colorectal adenoma cells express thepotential stem cell markers musashi antigen (msil) andephrin B2 receptor (EphB2)[J]. Journal of PathologyJ Pathol,2007,213(2):152-160.
[36]Vera JM,Wielenga, Ron Smits, et al. Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway[J]. Am J Pathol,1999,154(2):515-523.
[37]Chien AJ, Moore EC, Lonsdorf AS, et al. Activated Wnt/beta-catenin signaling in melanoma is associated with decreased proliferation in patient tumors and a murine melanoma model[J]. Proc Natl Acad Sci U S A,2009,106(4):1193-1198.
[38]Weeraratna AT, Jiang Y, Hostetter G, et al. Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma[J]. Cancer Cell,2002,1(3):279-288.
[39]Da Forno PD, Pringle JH, Hutchinson P, et al. WNT5A expression increases during melanoma progression and correlates with outcome[J]. Clin Cancer Res,2008, 14(18):5825-5832.
[40]Malanchi I, Peinado H, Kassen D, et al. Cutaneous cancer stem cell maintenance is dependent onβ-catenin signalling [J]. Nature,2008,452(7187):650-653.
[41]Bisson I, Prowse DM. WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics. Cell Res,2009,19(6):683-697,
[42]Golestaneh N, Beauchamp E, Fallen S, et al. Wnt signaling promotes proliferation and sternness regulation of spermatogonial stem/progenitor cells. Reproduction, 2009,138(1):151-162.