let-7/LIN28通路相关基因多态性与乳腺癌发生易感性的关联研究
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摘要
既往研究证实let-7可以转录后抑制LIN28基因的表达,而LIN28又能够阻断let-7成熟通路。let-7和LIN28以及二者组成的双重负反馈调控通路的异常与恶性肿瘤的发生存在关联。本课题主要研究let-7/LIN28通路相关基因胚系变异对不同个体生理状态下该通路水平的影响,以及这种影响是否与乳腺癌发生风险存在相关。通过计算机分析以及荧光报告实验,我们发现一个位于let-7在LIN28 mRNA上结合序列附近的单核苷酸多态性位点(SNP)rs3811463能够改变LIN28 mRNA的局部二级结构并影响let-7对LIN28的调控作用,导致let-7/LIN28通路水平的异常。通过对正常乳腺组织内let-7、LIN28mRNA、LIN28蛋白水平的分析,我们发现let-7在携带rs3811463少见等位基因(rs3811463-C)的个体正常乳腺组织内的水平显著高于携带野生型rs3811463等位基因(rs3811463-T)的个体正常乳腺组织内的水平而LIN28蛋白的表达则呈相反的趋势。基于此,我们推测le-7/LIN28通路相关的SNP可能会影响该通路的水平且潜在地改变不同个体罹患肿瘤的风险。接下来,通过病例对照研究,我们着重研究了let-7及LIN28基因常见SNP与乳腺癌的关系。在第一个病例对照研究(n=2,300)中,我们发现LIN28基因上的两个SNP位点与乳腺癌的发生显著相关。这两个位点分别为rs3811463与rs6697410,均位于LIN28基因的3’非编码区(3'UTR).rs3811463的C等位基因能增加乳腺癌的发病风险(OR为1.25,P=0.0091),而rs6697410的少见等位基因G则能降低乳腺癌的发病风险(OR为0.76,P=0.0086)。二期于独立人群中进行的病例对照研究(n=1,156)证实了最初的结果。两个研究(n=3,456)的合并P值为8.0×10-5(rs3811463)和7.0×10-4(rs6697410)。通过软琼脂实验,我们进一步揭示了高水平的LIN28蛋白能促进正常乳腺上皮细胞(MCF 10A)的转化。总体上,本课题发现let-7/LIN28通路相关基因的胚系变异能引起不同个体体内该通路水平的变化,这种变化在汉族女性中可导致乳腺癌发生易感性的差异。
Previous studies have shown that let-7 post-transcriptionally represses the expression of LIN28, and LIN28, in turn, blocks the maturation of let-7, forming a double-negative feedback loop. In this study, we investigated the effect of germline genetic variants on regulating the homeostatic status of the let-7/LIN28 loop and the influence of these variations on breast cancer risk. We demonstrated that the single nucleotide polymorphism (SNP), rs3811463, located near the binding site of let-7 in LIN28 may lead to a differential regulation of let-7 binding to LIN28 by altering the local mRNA secondary structure, thus disturbing the feedback loop between these two factors. Moreover, normal breast tissue harboring the rs3811463-C allele had significantly lower let-7 levels and higher LIN28 protein levels relative to the normal breast tissue harboring the wild-type T allele. Because the in vitro and ex vivo experiments consistently suggested that LIN28 modified breast cancer susceptibility, we subsequently evaluated the relationship between common SNPs found in LIN28 and breast cancer in a stepwise manner. The first hospital-based association study (n= 2,300) demonstrated that two SNPs (including rs3811463) significantly associated with breast cancer risk. The C allele of the rs3811463 SNP corresponded to an increased risk with an odds ratio (OR) of 1.25 (P=0.0091), which was successfully replicated in another independent study (n=1,156) with community-based controls. The combined P-value of the two studies was 8.0×10-5. Furthermore, a soft agar assay indicated that LIN28 was able to transform MCF-10A cells, which is a normal breast epithelial cell line. Taken together, our study demonstrated that host genetic variation disturbing the regulation of the let-7/LIN28 double-negative feedback loop can alter breast cancer risk.
Previous studies have shown that let-7 post-transcriptionally represses the expression of LIN28, and LIN28, in turn, blocks the maturation of let-7, forming a double-negative feedback loop. In this study, we investigated the effect of germline genetic variants on regulating the homeostatic status of the let-7/LIN28 loop and the influence of these variations on breast cancer risk. We demonstrated that the single nucleotide polymorphism (SNP), rs3811463, located near the binding site of let-7 in LIN28 may lead to a differential regulation of let-7 binding to LIN28 by altering the local mRNA secondary structure, thus disturbing the feedback loop between these two factors. Moreover, normal breast tissue harboring the rs3811463-C allele had significantly lower let-7 levels and higher LIN28 protein levels relative to the normal breast tissue harboring the wild-type T allele. Because the in vitro and ex vivo experiments consistently suggested that LIN28 modified breast cancer susceptibility, we subsequently evaluated the relationship between common SNPs found in LIN28 and breast cancer in a stepwise manner. The first hospital-based association study (n= 2,300) demonstrated that two SNPs (including rs3811463) significantly associated with breast cancer risk. The C allele of the rs3811463 SNP corresponded to an increased risk with an odds ratio (OR) of 1.25 (P=0.0091), which was successfully replicated in another independent study (n=1,156) with community-based controls. The combined P-value of the two studies was 8.0×10-5. Furthermore, a soft agar assay indicated that LIN28 was able to transform MCF-10A cells, which is a normal breast epithelial cell line. Taken together, our study demonstrated that host genetic variation disturbing the regulation of the let-7/LIN28 double-negative feedback loop can alter breast cancer risk.
引文
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10. Sampson, V.B., et al., MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells [J]. Cancer Research,2007.67:p.9762-9770.
11. Lee, Y.S. and A. Dutta, The tumor suppressor microRNA let-7 represses the HMGA2 oncogene [J]. Genes & Development,2007.21(9): p.1025-1030.
12. Yu, F., et al., Iet-7 regulates self renewal and tumorigenicity of breast cancer cells [J]. Cell,2007.131(6):p.1109-1123.
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14. Yu, J.Y., et al., Induced pluripotent stem cell lines derived from human somatic cells [J]. Science,2007.318(5858):p.1917-1920.
15. Viswanathan, S.R., et al., Lin28 promotes transformation and is associated with advanced human malignancies [J]. Nature Genetics,2009. 41(7):p.843-848.
16. Rybak, A., et al., A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment [J]. Nature Cell Biology,2008.10(8):p.987-993.
17. Yang, X.J., et al., Double-Negative Feedback Loop between Reprogramming Factor LIN28 and microRNA let-7 Regulates Aldehyde Dehydrogenase 1-Positive Cancer Stem Cells [J]. Cancer Research,2010. 70(22):p.9463-9472.
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20. Heo, I., et al., Lin28 Mediates the Terminal Uridylation of let-7 Precursor MicroRNA [J]. Molecular Cell,2008.32(2):p.276-284.
21. Newman, M.A., J.M. Thomson, and S.M. Hammond, Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing [J]. Rna-a Publication of the Rna Society,2008.14(8):p. 1539-1549.
22. Iliopoulos, D., H.A. Hirsch, and K. Struhl, An Epigenetic Switch Involving NF-kappa B, Lin28, Let-7 MicroRNA, and IL6 Links Inflammation to Cell Transformation [J]. Cell,2009.139(4):p.693-706.
23. Dangi-Garimella, S., et al., Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7 [J]. Embo Journal,2009.28(4):p.347-358.
24. Gibbs, R.A., et al., The International HapMap Project [J]. Nature, 2003.426(6968):p.789-796.
25. Yu, K.D., et al., Functional polymorphisms, altered gene expression and genetic association link NRH:quinone oxidoreductase 2 to breast cancer with wild-type p53 [J]. Human Molecular Genetics,2009.18(13): p.2502-2517.
26. Yu, K.D., et al., A functional polymorphism in the promoter region of GSTM1 implies a complex role for GSTM1 in breast cancer [J]. Faseb Journal,2009.23(7):p.2274-2287.
27. Yin, W.J., et al., Clinicopathological features of the triple-negative tumors in Chinese breast cancer patients [J]. Breast Cancer Research and Treatment,2009.115(2):p.325-333.
28. Zhu, T, et al., Oncogenic transformation of human mammary epithelial cells by autocrine human growth hormone [J]. Cancer Research, 2005.65(1):p.317-324.
29. Barrett, J.C., et al., Haploview:analysis and visualization of LD and haplotype maps [J]. Bioinformatics,2005.21(2):p.263-265.
30. Saunders, M.A., H. Liang, and W.H. Li, Human polymorphism at microRNAs and microRNA target sites [J]. Proceedings of the National Academy of Sciences of the United States of America,2007.104:p. 3300-3305.
31. Saetrom, P., et al., A Risk Variant in an miR-125b Binding Site in BMPR1B Is Associated with Breast Cancer Pathogenesis [J]. Cancer Research,2009.69(18):p.7459-7465.
32. Nicoloso, M.S., et al., Single-Nucleotide Polymorphisms Inside MicroRNA Target Sites Influence Tumor Susceptibility [J]. Cancer Research,2010.70(7):p.2789-2798.
33. Mishra, P.J. and J.R. Bertino, MicroRNA polymorphisms:the future of pharmacogenomics, molecular epidemiology and individualized medicine [J]. Pharmacogenomics,2009.10(3):p.399-416.
34. Chin, L.J., et al., A SNP in a let-7 microRNA Complementary Site in the KRAS 3'Untranslated Region Increases Non-Small Cell Lung Cancer Risk [J]. Cancer Research,2008.68(20):p.8535-8540.
35. Stratton, M.R. and N. Rahman, The emerging landscape of breast cancer susceptibility [J]. Nature Genetics,2008.40(1):p.17-22.
36. Xie, X.H., et al., Systematic discovery of regulatory motifs in human promoters and 3'UTRs by comparison of several mammals [J]. Nature, 2005.434(7031):p.338-345.
37. Liang, D., et al., Genetic Variants in MicroRNA Biosynthesis Pathways and Binding Sites Modify Ovarian Cancer Risk, Survival, and Treatment Response [J]. Cancer Research,2010.70(23):p.9765-9776.
38. Ogino, S., et al., Standard mutation nomenclature in molecular diagnostics-Practical and educational challenges [J]. Journal of Molecular Diagnostics,2007.9(1):p.1-6.
1. Lagos-Quintana, M., et al., Identification of novel genes coding for small expressed RNAs [J]. Science,2001.294(5543):p.853-858.
2. Lau, N.C., et al., An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans [J]. Science,2001.294(5543): p.858-862.
3. Lee, R.C. and V. Ambros, An extensive class of small RNAs in Caenorhabditis elegans [J]. Science,2001.294(5543):p.862-864.
4. Selbach, M., et al., Widespread changes in protein synthesis induced by microRNAs [J]. Nature,2008.455(7209):p.58-63.
5. Baek, D., et al., The impact of microRNAs on protein output [J]. Nature,2008.455(7209):p.64-U38.
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10. Lim, L.P., et al., Vertebrate MicroRNA genes [J]. Science,2003. 299(5612):p.1540-1540.
11.Bartel, D.P., MicroRNAs:Genomics, biogenesis, mechanism, and function [J]. Cell,2004.116(2):p.281-297.
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31. Liang, D., et al., Genetic Variants in MicroRNA Biosynthesis Pathways and Binding Sites Modify Ovarian Cancer Risk, Survival, and Treatment Response [J]. Cancer Research,2010.70(23):p.9765-9776.
32. Nicoloso, M.S., et al., Single-Nucleotide Polymorphisms Inside MicroRNA Target Sites Influence Tumor Susceptibility [J]. Cancer Research,2010.70(7):p.2789-2798.
33. Saetrom, P., et al., A risk variant in an miR-125b binding site in BMPRIB is associated with breast cancer pathogenesis [J]. Cancer Res, 2009.69(18):p.7459-65.
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2. Yu, K.D., et al., Development and trends of surgical modalities for breast cancer in China:A review of 16-year data [J]. Annals of Surgical Oncology,2007.14(9):p.2502-2509.
3. Walsh, T. and M.C. King, Ten genes for inherited breast cancer [J]. Cancer Cell,2007.11(2):p.103-105.
4. Esquela-Kerscher, A. and F.J. Slack, Oncomirs-microRNAs with a role in cancer [J]. Nature Reviews Cancer,2006.6(4):p.259-269.
5. Bartel, D.P., MicroRNAs:Genomics, biogenesis, mechanism, and function [J]. Cell,2004.116(2):p.281-297.
6. Pasquinelli, A.E., et al., Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA [J]. Nature,2000. 408(6808):p.86-89.
7. Reinhart, B.J., et al., The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans [J]. Nature,2000. 403(6772):p.901-906.
8. Bussing, I., F.J. Slack, and H. Grosshans, let-7 microRNAs in development, stem cells and cancer [J]. Trends in Molecular Medicine, 2008.14(9):p.400-409.
9. Johnson, S.M., et al., RAS is regulated by the let-7 MicroRNA family [J]. Cell,2005.120(5):p.635-647.
10. Sampson, V.B., et al., MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells [J]. Cancer Research,2007.67:p.9762-9770.
11. Lee, Y.S. and A. Dutta, The tumor suppressor microRNA let-7 represses the HMGA2 oncogene [J]. Genes & Development,2007.21(9): p.1025-1030.
12. Yu, F., et al., Iet-7 regulates self renewal and tumorigenicity of breast cancer cells [J]. Cell,2007.131(6):p.1109-1123.
13. Viswanathan, S.R. and G.Q. Daley, Lin28:A MicroRNA Regulator with a Macro Role [J]. Cell,2010.140(4):p.445-449.
14. Yu, J.Y., et al., Induced pluripotent stem cell lines derived from human somatic cells [J]. Science,2007.318(5858):p.1917-1920.
15. Viswanathan, S.R., et al., Lin28 promotes transformation and is associated with advanced human malignancies [J]. Nature Genetics,2009. 41(7):p.843-848.
16. Rybak, A., et al., A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment [J]. Nature Cell Biology,2008.10(8):p.987-993.
17. Yang, X.J., et al., Double-Negative Feedback Loop between Reprogramming Factor LIN28 and microRNA let-7 Regulates Aldehyde Dehydrogenase 1-Positive Cancer Stem Cells [J]. Cancer Research,2010. 70(22):p.9463-9472.
18. Viswanathan, S.R., G.Q. Daley, and R.I. Gregory, Selective blockade of MicroRNA processing by Lin28 [J]. Science,2008.320(5872):p. 97-100.
19. Hagan, J.P., E. Piskounova, and R.I. Gregory, Lin28 recruits the TUTase Zcchcll to inhibit let-7 maturation in mouse embryonic stem cells [J]. Nature Structural & Molecular Biology,2009.16(10):p. 1021-1025.
20. Heo, I., et al., Lin28 Mediates the Terminal Uridylation of let-7 Precursor MicroRNA [J]. Molecular Cell,2008.32(2):p.276-284.
21. Newman, M.A., J.M. Thomson, and S.M. Hammond, Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing [J]. Rna-a Publication of the Rna Society,2008.14(8):p. 1539-1549.
22. Iliopoulos, D., H.A. Hirsch, and K. Struhl, An Epigenetic Switch Involving NF-kappa B, Lin28, Let-7 MicroRNA, and IL6 Links Inflammation to Cell Transformation [J]. Cell,2009.139(4):p.693-706.
23. Dangi-Garimella, S., et al., Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7 [J]. Embo Journal,2009.28(4):p.347-358.
24. Gibbs, R.A., et al., The International HapMap Project [J]. Nature, 2003.426(6968):p.789-796.
25. Yu, K.D., et al., Functional polymorphisms, altered gene expression and genetic association link NRH:quinone oxidoreductase 2 to breast cancer with wild-type p53 [J]. Human Molecular Genetics,2009.18(13): p.2502-2517.
26. Yu, K.D., et al., A functional polymorphism in the promoter region of GSTM1 implies a complex role for GSTM1 in breast cancer [J]. Faseb Journal,2009.23(7):p.2274-2287.
27. Yin, W.J., et al., Clinicopathological features of the triple-negative tumors in Chinese breast cancer patients [J]. Breast Cancer Research and Treatment,2009.115(2):p.325-333.
28. Zhu, T, et al., Oncogenic transformation of human mammary epithelial cells by autocrine human growth hormone [J]. Cancer Research, 2005.65(1):p.317-324.
29. Barrett, J.C., et al., Haploview:analysis and visualization of LD and haplotype maps [J]. Bioinformatics,2005.21(2):p.263-265.
30. Saunders, M.A., H. Liang, and W.H. Li, Human polymorphism at microRNAs and microRNA target sites [J]. Proceedings of the National Academy of Sciences of the United States of America,2007.104:p. 3300-3305.
31. Saetrom, P., et al., A Risk Variant in an miR-125b Binding Site in BMPR1B Is Associated with Breast Cancer Pathogenesis [J]. Cancer Research,2009.69(18):p.7459-7465.
32. Nicoloso, M.S., et al., Single-Nucleotide Polymorphisms Inside MicroRNA Target Sites Influence Tumor Susceptibility [J]. Cancer Research,2010.70(7):p.2789-2798.
33. Mishra, P.J. and J.R. Bertino, MicroRNA polymorphisms:the future of pharmacogenomics, molecular epidemiology and individualized medicine [J]. Pharmacogenomics,2009.10(3):p.399-416.
34. Chin, L.J., et al., A SNP in a let-7 microRNA Complementary Site in the KRAS 3'Untranslated Region Increases Non-Small Cell Lung Cancer Risk [J]. Cancer Research,2008.68(20):p.8535-8540.
35. Stratton, M.R. and N. Rahman, The emerging landscape of breast cancer susceptibility [J]. Nature Genetics,2008.40(1):p.17-22.
36. Xie, X.H., et al., Systematic discovery of regulatory motifs in human promoters and 3'UTRs by comparison of several mammals [J]. Nature, 2005.434(7031):p.338-345.
37. Liang, D., et al., Genetic Variants in MicroRNA Biosynthesis Pathways and Binding Sites Modify Ovarian Cancer Risk, Survival, and Treatment Response [J]. Cancer Research,2010.70(23):p.9765-9776.
38. Ogino, S., et al., Standard mutation nomenclature in molecular diagnostics-Practical and educational challenges [J]. Journal of Molecular Diagnostics,2007.9(1):p.1-6.
1. Lagos-Quintana, M., et al., Identification of novel genes coding for small expressed RNAs [J]. Science,2001.294(5543):p.853-858.
2. Lau, N.C., et al., An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans [J]. Science,2001.294(5543): p.858-862.
3. Lee, R.C. and V. Ambros, An extensive class of small RNAs in Caenorhabditis elegans [J]. Science,2001.294(5543):p.862-864.
4. Selbach, M., et al., Widespread changes in protein synthesis induced by microRNAs [J]. Nature,2008.455(7209):p.58-63.
5. Baek, D., et al., The impact of microRNAs on protein output [J]. Nature,2008.455(7209):p.64-U38.
6. Brennecke, J., et al., Principles of MicroRNA-target recognition [J]. Plos Biology,2005.3(3):p.404-418.
7. Bertino, J.R., D. Banerjee, and P.J. Mishra, Pharmacogenomics of microRNA:a miRSNP towards individualized therapy [J]. Pharmacogenomics,2007.8(12):p.1625-1627.
8. Ambros, V., MicroRNA pathways in flies and worms:Growth, death, fat, stress, and timing [J]. Cell,2003.113(6):p.673-676.
9. Lim, L.P., et al., The microRNAs of Caenorhabditis elegans [J]. Genes & Development,2003.17(8):p.991-1008.
10. Lim, L.P., et al., Vertebrate MicroRNA genes [J]. Science,2003. 299(5612):p.1540-1540.
11.Bartel, D.P., MicroRNAs:Genomics, biogenesis, mechanism, and function [J]. Cell,2004.116(2):p.281-297.
12. Esquela-Kerscher, A. and F.J. Slack, Oncomirs-microRNAs with a role in cancer [J]. Nature Reviews Cancer,2006.6(4):p.259-269.
13. Iorio, M.V., et al., MicroRNA gene expression deregulation in human breast cancer [J]. Cancer Res,2005.65(16):p.7065-70.
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