TGF-β1反义寡核苷酸对人横纹肌肉瘤细胞生长分化的影响
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摘要
横纹肌肉瘤(rhabdomyosarcoma,RMS)是一种好发于儿童的恶性软组织肿瘤,尽管能表达生肌调节因子(MRFs),如MyoD1,但不会终末分化成骨骼肌。有研究报道RMS细胞可以产生多种生长因子,并可能在RMS细胞生长、分化中起着重要调控作用。转化生长因子-β1(transforming growth factor-β1,TGF-β1)是一种能调节各种细胞生长与分化的多功能细胞生长因子,TGF-β1信号通路中某一成分缺失,其抗增殖作用丧失可诱发上皮性肿瘤。然而,TGF-β1作为一种肌分化抑制因子,在RMS细胞生长与分化中的作用机制尚不清楚。为了探讨TGF-β1在RMS中的作用,本课题组曾用RT-PCR和Western blot检测了横纹肌肉瘤RD细胞株和人成肌细胞(Myoblasts)中TGF β1/Smad信号通路各成分mRAN和蛋白水平表达,结果显示,TGF-β1、TGFβR Ⅰ、TGFβR Ⅱ、Smad2和Smad4在RD细胞中的表达较成肌细胞增高,表明RD细胞可以作为研究TGF-β1在RMS中作用的理想模式。目前关于TGF-β1在RMS中的作用机制罕见报道。本研究是通过这一实验模式,进一步探讨TGF-β1信号转导在PMS细胞生长与分化中的作用。
反义寡核苷酸(ASON)技术能有效封闭靶基因的表达,常用来探讨某些生长因子对细胞的调节作用。有研究报道用ASON阻断TGF-β1
Rhabdomyosarcomas (RMS), the most common soft tissue sarcomas in childhood, although expressing some myogenic regulatory factors( MRFs), such as MyoDl, RMS cells undergoes very limited muscle differentiation and fail into terminal skeletal muscle differentiation. Some research reported that RMS cells can express growth factors which are likely to be involved in the growth and differentiation of tumor cells. Transforming growth factor ( TGF)-p is a multifunctional cytokine that regulate growth and differentiation of various cell types. TGF β1 is a potent growth inhibitor, with tumor-suppressing activity ,cancers are often refractile to this growth inhibition because of genetic loss of TGF- β1 signaling components. However, TGFβ1, a inhibitor of muscle differentiation , as well as an autocrine product of RMS cells, maybe regulate the tumor cells growth and differentiation by some way which has not yet been investigated. To study the role of TGF- β1 in the human rhabdomyosarcoma cell line, RD, we have made an attempt to establish a framework for the expression of components of TGFβ1/Smad signaling pathway in RD cells compared with the normal
myoblasts by RT-PCR and Western blot. We have shown that RD cells displayed higher expression of TGF-PU TGFpR I, TGFpR IK Smad2and Smad4 at both the mRNA and protein levels than myoblasts, and suggest that the RD cells line is a suitable model to study the role of TGF- ￡ 1 signalling in RMS. To date, little is known of TGF-13 1 action in RMS cells. In the present study, we use this model system to investigate whether the autocrine TGF- P 1 loop is responsible for cell growth and differentiation in RD cell.Antisense oligodeoxyribonucletide designed to inhibit target gene expression would be useful in assessing possible roles of some growth factors in cellular regulation. Indeed, antisense oligodeoxyribonucletide have been used to target TGF- 3 1 in attempt to evaluate its involvement in the formation and development of tumor. However, the effects of oligodeoxyribonucletide on TGF- P 1 production was not determined, this is very important, as will be demonstrated in our study, because oligodeoxyribonucletide treatment can lead to non-specific biological effects. In this research, we designed different oligodeoxyribonucletide to target TGF- P 1 mRNA, and choose one of best antisense oligodeoxyribonucletide, which specifically inhibits the production of TGF- & 1, transfer to the human rhabdomyosarcoma cell line, RD, to investigate the involvement of TGF- P 1 in cell growth and differentiation. The summarized results as follows:1. Immunohistochemical technique(S-P) and RT-PCR were used todetect the expression of TGF-pK TGFpR I, TGFPRIK Smad2 and Smad4 inrhabdomyosarcoma and normal skeletal muscles. Overexpression of TGFpi,Smad2, and Smad4 in RMS were 58.6% (41/70) , 70% (49/70) and 75.7%(53/70) respectively. The highexpression of above proteins in normal
skeletal muscles were 28% (7/25), 24% (6/25) and 28% (7/25) with significant difference ( P <0.01) . Expression of TpR I , TpR II were detected in most of RMS and all of normal skeletal muscles without significant difference ( PX).O1) The mRNA expression of TGF(31, TpR I , TPR II, Smad2 and Smad4 in RD cell line is higher than that of the myoblasts. There is no significant relationship among the expression of above proteins, histological grade and prognosis of tumor (PO.05) except for expression of TGFpl protein in grade I and II. There exists an autocrine pathway in RMS, suggest that TGF- 1 may play a complex role in development and progression of rhabdomyosarcoma.2. To observe the biological properties of primary human embryonic skeletal myoblasts cultured in different medium with RT-PCR, flow cytometry (FCM) and rate of myotube formation (RMF), and to investigate the effect of transforming growth factor- P 1 (TGF- P 1) to the proliferation and differentiation of myoblast. Results showed that the primary myoblasts cultured in DM (DMEM supplemented with 3% fetal bovine serum), in contrast with the control group in GM (DMEM supplemented with 10% fetal bovine serum), increased the rate of myotube formation (RMF), more myofilament formed and more myoblasts exited S phase. The expression of Myogenin mRNA was increased obviously than that of the control group, the change of MyoDl and Myf5 mRNA was not as well as Myogenin, These data indicate that different culture medium induce diverse biological function of myoblasts, and provide a means of study role of TGFpi in RMS cell and myoblast. With TGF- P 1 (^2ng/ml) cultured for 6 days, all differentiation index of myoblasts above is decreased ,and more cells of experimental group
entered the phase of DNA duplication, however, with TGF- P 1 (^ lng/ml) cultured, exert little effect on growth and differentiation of myoblast. All of theses suggested that weather or not TGF- 0 lin RMS exert growth inhibitory effect in a concentration-dependent manner.3. To establish a credible way of transfer TGF-pi antisense oligonucleotide (TGF- P 1ASON) to RD cells, and provide a possible method of research in thepathogenic role of TGF-01 on rhabdomyosarcoma. TGF-piASON were synthesized, makered by 6-carboxy-iluorescein ( 6-FAM) and combined to LipoGenTM to prepare Lip- ASON complex. To analysis the property, dose of complex, and transfer time, results showed that the transfer efficiency is close to relation with all of these. The mean fluorescent index by FCM combined the transfer rates by manual counting can more objectively inspect transfer efficiency. Proper dose of Lip- ASON complex can improve transfer efficiency to maximum and with least of poison. The TGF-{$1 mRNA and protein of transferred RD cells are investigated by RT-PCR and ELISA, showed that TGF-fll ASON2 (3* gggggtacggcgggagccs' ) maybe is the best antisense to TGF-B1 mRNA, provide a means of research the role of TGF-61 in the RMS in the next part.4. To investigate the effect of TGF-61 ASON on the growth and differentiation of RD cells by MTT, FCM, RT-PCR, Immunofluorescent and electron microscope, and to study the mechanism of it by Western blot combined with RT-PCR to explore changes of Smads and MRFs. Results showed that TGF-81ASON could induce RD cells exit cell cycle, and higher protein expression of Myosin and a-Sarcomeric Actin than that of control group. It suggested that TGF- ￡ 1ASON could inhibit growth of RD cells and
induce differentiation of RD cell. Using RT-PCR with sequence-specific primers and Western blot with specific antibody, we have shown that TGF- P 1 ASON could reduce the expression of Smad2 and Smad4, and increase the expression of some MRFs, such as myogenin and MyoDl. These study suggested that TGF- P 1ASON broken off TGF- P 1 signaling pathway, stopped uniting Smads with MRFs, and induced RMS cells' cytoskeletons protein expression more, such as Myosin and a-Sarcomeric Actin.In summary, there is TGF- P 1 autocrine loop in RD cells, on the use of ASON transfer ,showed that TGF- 3 1ASON could inhibit growth of RD cells and induce differentiation of RD cell. In this study, we provide an application of ASON transfer to the research of growth factors of cells, conformed that TGF-J31 inhibit muscle differentiation of RD cells, and the result maybe from Smads cooperating with MRFs to inhibit muscle transcription of cytoskeletons gene.
反义寡核苷酸(ASON)技术能有效封闭靶基因的表达,常用来探讨某些生长因子对细胞的调节作用。有研究报道用ASON阻断TGF-β1
Rhabdomyosarcomas (RMS), the most common soft tissue sarcomas in childhood, although expressing some myogenic regulatory factors( MRFs), such as MyoDl, RMS cells undergoes very limited muscle differentiation and fail into terminal skeletal muscle differentiation. Some research reported that RMS cells can express growth factors which are likely to be involved in the growth and differentiation of tumor cells. Transforming growth factor ( TGF)-p is a multifunctional cytokine that regulate growth and differentiation of various cell types. TGF β1 is a potent growth inhibitor, with tumor-suppressing activity ,cancers are often refractile to this growth inhibition because of genetic loss of TGF- β1 signaling components. However, TGFβ1, a inhibitor of muscle differentiation , as well as an autocrine product of RMS cells, maybe regulate the tumor cells growth and differentiation by some way which has not yet been investigated. To study the role of TGF- β1 in the human rhabdomyosarcoma cell line, RD, we have made an attempt to establish a framework for the expression of components of TGFβ1/Smad signaling pathway in RD cells compared with the normal
myoblasts by RT-PCR and Western blot. We have shown that RD cells displayed higher expression of TGF-PU TGFpR I, TGFpR IK Smad2and Smad4 at both the mRNA and protein levels than myoblasts, and suggest that the RD cells line is a suitable model to study the role of TGF- ￡ 1 signalling in RMS. To date, little is known of TGF-13 1 action in RMS cells. In the present study, we use this model system to investigate whether the autocrine TGF- P 1 loop is responsible for cell growth and differentiation in RD cell.Antisense oligodeoxyribonucletide designed to inhibit target gene expression would be useful in assessing possible roles of some growth factors in cellular regulation. Indeed, antisense oligodeoxyribonucletide have been used to target TGF- 3 1 in attempt to evaluate its involvement in the formation and development of tumor. However, the effects of oligodeoxyribonucletide on TGF- P 1 production was not determined, this is very important, as will be demonstrated in our study, because oligodeoxyribonucletide treatment can lead to non-specific biological effects. In this research, we designed different oligodeoxyribonucletide to target TGF- P 1 mRNA, and choose one of best antisense oligodeoxyribonucletide, which specifically inhibits the production of TGF- & 1, transfer to the human rhabdomyosarcoma cell line, RD, to investigate the involvement of TGF- P 1 in cell growth and differentiation. The summarized results as follows:1. Immunohistochemical technique(S-P) and RT-PCR were used todetect the expression of TGF-pK TGFpR I, TGFPRIK Smad2 and Smad4 inrhabdomyosarcoma and normal skeletal muscles. Overexpression of TGFpi,Smad2, and Smad4 in RMS were 58.6% (41/70) , 70% (49/70) and 75.7%(53/70) respectively. The highexpression of above proteins in normal
skeletal muscles were 28% (7/25), 24% (6/25) and 28% (7/25) with significant difference ( P <0.01) . Expression of TpR I , TpR II were detected in most of RMS and all of normal skeletal muscles without significant difference ( PX).O1) The mRNA expression of TGF(31, TpR I , TPR II, Smad2 and Smad4 in RD cell line is higher than that of the myoblasts. There is no significant relationship among the expression of above proteins, histological grade and prognosis of tumor (PO.05) except for expression of TGFpl protein in grade I and II. There exists an autocrine pathway in RMS, suggest that TGF- 1 may play a complex role in development and progression of rhabdomyosarcoma.2. To observe the biological properties of primary human embryonic skeletal myoblasts cultured in different medium with RT-PCR, flow cytometry (FCM) and rate of myotube formation (RMF), and to investigate the effect of transforming growth factor- P 1 (TGF- P 1) to the proliferation and differentiation of myoblast. Results showed that the primary myoblasts cultured in DM (DMEM supplemented with 3% fetal bovine serum), in contrast with the control group in GM (DMEM supplemented with 10% fetal bovine serum), increased the rate of myotube formation (RMF), more myofilament formed and more myoblasts exited S phase. The expression of Myogenin mRNA was increased obviously than that of the control group, the change of MyoDl and Myf5 mRNA was not as well as Myogenin, These data indicate that different culture medium induce diverse biological function of myoblasts, and provide a means of study role of TGFpi in RMS cell and myoblast. With TGF- P 1 (^2ng/ml) cultured for 6 days, all differentiation index of myoblasts above is decreased ,and more cells of experimental group
entered the phase of DNA duplication, however, with TGF- P 1 (^ lng/ml) cultured, exert little effect on growth and differentiation of myoblast. All of theses suggested that weather or not TGF- 0 lin RMS exert growth inhibitory effect in a concentration-dependent manner.3. To establish a credible way of transfer TGF-pi antisense oligonucleotide (TGF- P 1ASON) to RD cells, and provide a possible method of research in thepathogenic role of TGF-01 on rhabdomyosarcoma. TGF-piASON were synthesized, makered by 6-carboxy-iluorescein ( 6-FAM) and combined to LipoGenTM to prepare Lip- ASON complex. To analysis the property, dose of complex, and transfer time, results showed that the transfer efficiency is close to relation with all of these. The mean fluorescent index by FCM combined the transfer rates by manual counting can more objectively inspect transfer efficiency. Proper dose of Lip- ASON complex can improve transfer efficiency to maximum and with least of poison. The TGF-{$1 mRNA and protein of transferred RD cells are investigated by RT-PCR and ELISA, showed that TGF-fll ASON2 (3* gggggtacggcgggagccs' ) maybe is the best antisense to TGF-B1 mRNA, provide a means of research the role of TGF-61 in the RMS in the next part.4. To investigate the effect of TGF-61 ASON on the growth and differentiation of RD cells by MTT, FCM, RT-PCR, Immunofluorescent and electron microscope, and to study the mechanism of it by Western blot combined with RT-PCR to explore changes of Smads and MRFs. Results showed that TGF-81ASON could induce RD cells exit cell cycle, and higher protein expression of Myosin and a-Sarcomeric Actin than that of control group. It suggested that TGF- ￡ 1ASON could inhibit growth of RD cells and
induce differentiation of RD cell. Using RT-PCR with sequence-specific primers and Western blot with specific antibody, we have shown that TGF- P 1 ASON could reduce the expression of Smad2 and Smad4, and increase the expression of some MRFs, such as myogenin and MyoDl. These study suggested that TGF- P 1ASON broken off TGF- P 1 signaling pathway, stopped uniting Smads with MRFs, and induced RMS cells' cytoskeletons protein expression more, such as Myosin and a-Sarcomeric Actin.In summary, there is TGF- P 1 autocrine loop in RD cells, on the use of ASON transfer ,showed that TGF- 3 1ASON could inhibit growth of RD cells and induce differentiation of RD cell. In this study, we provide an application of ASON transfer to the research of growth factors of cells, conformed that TGF-J31 inhibit muscle differentiation of RD cells, and the result maybe from Smads cooperating with MRFs to inhibit muscle transcription of cytoskeletons gene.
引文
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2. Lee C, Sintich SM, Mathews EP, et al. Transforming growth factor-beta in benign and malignant prostate. Prostate 1999; 39: 285-290.
3. Akhurst RJ, Balmain A. Genetic events and the role of TGF beta in epithelial tumour progression. J Pathol, 1999, 187: 82-90.
4. Moustakas A, Pardali K, Gaal A, et al. Mechanisms of TGF-beta signaling in regulation of cell growth and differentiation. Immunol Lett, 2002, 82: 85-91.
5. Battegay EJ. Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects. J Mol Med, 1995, 73: 333-346.
6. Pepper MS. Transforming growth factor-beta: vasculogenesis, angiogenesis, and vessel wall integrity. Cytokine Growth Factor Rev, 1997, 8: 21-43.
7. de Visser KE, Kast WM. Effects of TGF-beta on the immune system: implications for cancer immunotherapy. Leukemia, 1999, 13: 1188-1199.
8. Cui W, Fowlis DJ, Bryson S, et al. TGF betal inhibits the formation of benign skin tumors,. but enhances progression to invasive spindle carcinomas in transgenic mice. Cell, 1996, 86: 531-542.
9. Moses HL, Yang EY, Pietenpol JA. et al. TGF-beta stimulation and inhibition of cell proliferation: new mechanistic insights. Cell, 1990, 63: 245-247.
10. Olson EN, Steinberg E, Hu JS, et al. Regulation of myogenic differentiation by type beta transforming growth factor. J Cell Biol, 1986, 103: 1799-1805.
11. Massague J, Cheifetz S, Endo J. et al. Type β transforming growth factor is an inhibitor of myogenic differentiation. Proc Natl Acad Sci USA, 1986, 83: 8206-8210.
12. Florini J R, Roberts A B, Ewton D Z, et al. Transforming growth factor-β. A very potent inhibitor of myoblast-differentiation, identical to the differentiation inhibitor secreted by buffalo rat liver cells. J Biol Chem, 1986, 261: 16509-16513.
13. De Giovanni C, Melani C, Nanni P, et al. Redundancy of autocrine loops in human rhabdomyosarcoma cells: induction of differentiation by suramin. Br J Cancer, 1995, 72: 1224-1229.
14. Shapiro DN, Jones BG, Shapiro LH, et al. Antisens-mediated reduction in insulin-like growth factor-1 receptor expression suppresses the malignant phenotype of a human alveolar rhabdomyosareoma. J Clin Invest, 1994, 94: 1235-1242.
15. De Giovanni C, Landuzzi L, Frabetti F, et al. Antisens-epidermal growth factor receptor transfection impairs the proliferative ability of human rhabdomyosarcoma cells. Cancer Res, 1996, 56: 3898-3901.
16. Merlino G, Helman LJ. Rhabdomyosareoma: working out the pathways. Oncogene, 1999, 18: 5340-5348.
17. Bouche M, Canipari R, Melchirma R et al. TGF-β autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells. FASEB J, 2000, 14: 1147-1158.
18.王华,杨光华,步宏,等。转化生长因子β1及其受体在横纹肌肉瘤中的表达.临床与实验病理学杂志,2002,18:61-64.
19. Ingo T, Bernd D, Gunther H. Antisense therapy in oneology: new hope for an old idea. Lancet, 2001, 358: 4897-487.
20. Grooke ST. Potential roles of antisense technology in cancer chemopherapy. Oneogene, 2000, 19: 6651-6659.
21. Arias M, Sauer-Lehnen S, Treptau J, et al. Adenoviral expression of a transforming growth factor-β1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligatedrats. BMC Gastroenterol, 2003, 3: 29-38.
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23.赖日权,等.横纹肌肉瘤与胚胎横纹肌的形态学和免疫组织化学对比观察。中华病理学杂志,1993,22:265.
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44. Massague J, Cheifetz S, Endo J. et al. Type β transforming growth factor is an inhibitor of myogenic differentiation. Proc Natl Acad Sci USA, 1986,83:8206-8210.
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2. Lee C, Sintich SM, Mathews EP, et al. Transforming growth factor-beta in benign and malignant prostate. Prostate 1999; 39: 285-290.
3. Akhurst RJ, Balmain A. Genetic events and the role of TGF beta in epithelial tumour progression. J Pathol, 1999, 187: 82-90.
4. Moustakas A, Pardali K, Gaal A, et al. Mechanisms of TGF-beta signaling in regulation of cell growth and differentiation. Immunol Lett, 2002, 82: 85-91.
5. Battegay EJ. Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects. J Mol Med, 1995, 73: 333-346.
6. Pepper MS. Transforming growth factor-beta: vasculogenesis, angiogenesis, and vessel wall integrity. Cytokine Growth Factor Rev, 1997, 8: 21-43.
7. de Visser KE, Kast WM. Effects of TGF-beta on the immune system: implications for cancer immunotherapy. Leukemia, 1999, 13: 1188-1199.
8. Cui W, Fowlis DJ, Bryson S, et al. TGF betal inhibits the formation of benign skin tumors,. but enhances progression to invasive spindle carcinomas in transgenic mice. Cell, 1996, 86: 531-542.
9. Moses HL, Yang EY, Pietenpol JA. et al. TGF-beta stimulation and inhibition of cell proliferation: new mechanistic insights. Cell, 1990, 63: 245-247.
10. Olson EN, Steinberg E, Hu JS, et al. Regulation of myogenic differentiation by type beta transforming growth factor. J Cell Biol, 1986, 103: 1799-1805.
11. Massague J, Cheifetz S, Endo J. et al. Type β transforming growth factor is an inhibitor of myogenic differentiation. Proc Natl Acad Sci USA, 1986, 83: 8206-8210.
12. Florini J R, Roberts A B, Ewton D Z, et al. Transforming growth factor-β. A very potent inhibitor of myoblast-differentiation, identical to the differentiation inhibitor secreted by buffalo rat liver cells. J Biol Chem, 1986, 261: 16509-16513.
13. De Giovanni C, Melani C, Nanni P, et al. Redundancy of autocrine loops in human rhabdomyosarcoma cells: induction of differentiation by suramin. Br J Cancer, 1995, 72: 1224-1229.
14. Shapiro DN, Jones BG, Shapiro LH, et al. Antisens-mediated reduction in insulin-like growth factor-1 receptor expression suppresses the malignant phenotype of a human alveolar rhabdomyosareoma. J Clin Invest, 1994, 94: 1235-1242.
15. De Giovanni C, Landuzzi L, Frabetti F, et al. Antisens-epidermal growth factor receptor transfection impairs the proliferative ability of human rhabdomyosarcoma cells. Cancer Res, 1996, 56: 3898-3901.
16. Merlino G, Helman LJ. Rhabdomyosareoma: working out the pathways. Oncogene, 1999, 18: 5340-5348.
17. Bouche M, Canipari R, Melchirma R et al. TGF-β autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells. FASEB J, 2000, 14: 1147-1158.
18.王华,杨光华,步宏,等。转化生长因子β1及其受体在横纹肌肉瘤中的表达.临床与实验病理学杂志,2002,18:61-64.
19. Ingo T, Bernd D, Gunther H. Antisense therapy in oneology: new hope for an old idea. Lancet, 2001, 358: 4897-487.
20. Grooke ST. Potential roles of antisense technology in cancer chemopherapy. Oneogene, 2000, 19: 6651-6659.
21. Arias M, Sauer-Lehnen S, Treptau J, et al. Adenoviral expression of a transforming growth factor-β1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligatedrats. BMC Gastroenterol, 2003, 3: 29-38.
22. Hicks J and Flaitz C Rhabdomyosarcoma of the head and neck in children. Oral Oneology, 2002, 38: 450-459.
23.赖日权,等.横纹肌肉瘤与胚胎横纹肌的形态学和免疫组织化学对比观察。中华病理学杂志,1993,22:265.
24. Lollini PL, De Giovanni C, Landuzzi L, et al, Reduced metastatic ability of in vitro differentiated human rhabdomyosarcoma cells. Invasion Metastasis, 1991, 11: 116-124.
25. Rowley JA, Madlambayan G, Mooney DJ. Alginate hydrogels as synthetic extracelluar matrix materials. Biomaterials, 1999, 20: 45.
26. Celine BL, Rashmi K. Differentiation potential of primary myogenic cells derived from skeletal muscle of dystonia musculorum mice. Differentiation, 2002, 70: 247.
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