Lin28B介导肝癌细胞中HBx相关Let-7沉默
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摘要
目的:Let-7在多种肿瘤包括肝癌中低表达,扮演抑癌基因角色。我们的前期研究提示HBx(人乙肝病毒X蛋白)在肝癌细胞中显著下调Let-7表达,但内在机制有待进一步揭示。Lin28B能负调控Let-7的转录后加工成熟,这促使我们验证其是否介导了肝癌细胞中HBx相关的Let-7低表达。方法:qRT-PCR检测c-Myc和Lin28B特异siRNA转染HepG2-HBx细胞(HBx稳定转染的HepG2细胞)前后Let-7的表达变化,同时在肝癌细胞和或组织中验证HBx和Lin28B的表达相关性。最后,利用细胞周期及增殖实验检测Lin28B对HepG2细胞的生物学影响。结果:Lin28B高表达于HBx稳定/瞬时转染的HepG2肝癌细胞及HBV阳性的肝癌和肝硬化组织。Lin28B介导了HBx在HepG2肝癌细胞中对Let-7的抑制。Lin28B特异siRNAs可阻滞细胞周期进展进而抑制HepG2细胞生长。结论:HBx在肝癌细胞中通过诱导Lin28B表达而抑制Let-7。Lin28B可能成为治疗HBV相关肝癌的新靶点。
Objective: To determine whether HBx acts through Lin28 B to repress Let-7. The Let-7 microRNA( miRNA) family is frequently downregulated in multiple human tumors,including hepatocellular carcinoma( HCC).Our previous report demonstrated that hepatitis B virus X protein( HBx) suppressed Let-7 expression in HepG2 hepatoma cells. However,the underlying mechanisms were not elucidated. Lin28 B was known to negatively regulate the maturation of Let-7. Methods: Real-time reverse-transcription polymerase chain reaction( qRT-PCR) was performed to examine Let-7 expression before and after treatment with c-Myc-and Lin28B-specific siRNAs in HepG2 cells transfected with HBx. mRNA and protein analyses were employed to determine the correlation of HBx and Lin28 B in HCC tissues and cells. Cell cycle and proliferation assays were performed to delineate the consequences of Lin28 B silence in HepG2 cells expressing HBx. Results: Lin28 B was overexpressed in HBx-transfected cells and HBV-infected liver tissues. Lin28 B mediated the repression of Let-7 in HepG2 cells. Reduced expression of Lin28 B inhibited the growth and cell cycle progression of HepG2 cells by derepressing Let-7. Conclusion: Lin28 B indcution is responsible for the Let-7 repression in hepatoma cells expressing HBx. Lin28 B has the potential to be a novel molecular target in the treatment of HBV + HCC.
Objective: To determine whether HBx acts through Lin28 B to repress Let-7. The Let-7 microRNA( miRNA) family is frequently downregulated in multiple human tumors,including hepatocellular carcinoma( HCC).Our previous report demonstrated that hepatitis B virus X protein( HBx) suppressed Let-7 expression in HepG2 hepatoma cells. However,the underlying mechanisms were not elucidated. Lin28 B was known to negatively regulate the maturation of Let-7. Methods: Real-time reverse-transcription polymerase chain reaction( qRT-PCR) was performed to examine Let-7 expression before and after treatment with c-Myc-and Lin28B-specific siRNAs in HepG2 cells transfected with HBx. mRNA and protein analyses were employed to determine the correlation of HBx and Lin28 B in HCC tissues and cells. Cell cycle and proliferation assays were performed to delineate the consequences of Lin28 B silence in HepG2 cells expressing HBx. Results: Lin28 B was overexpressed in HBx-transfected cells and HBV-infected liver tissues. Lin28 B mediated the repression of Let-7 in HepG2 cells. Reduced expression of Lin28 B inhibited the growth and cell cycle progression of HepG2 cells by derepressing Let-7. Conclusion: Lin28 B indcution is responsible for the Let-7 repression in hepatoma cells expressing HBx. Lin28 B has the potential to be a novel molecular target in the treatment of HBV + HCC.
引文
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[2]Barh D,Malhotra R,Ravi B,et al.MicroRNA Let-7:An emerging next-generation cancer therapeutic[J].Curr Oncol,2010,17(1):70-80.
[3]Wang Y,Lu Y,Toh ST,et al.Lethal-7 is down-regulated by the hepatitis b virus x protein and targets signal transducer and activator of transcription 3[J].Journal of Hepatology,2010,53(1):57
[4]Wu G,Yu F,Xiao Z,et al.Hepatitis b virus x protein downregulates expression of the mir-16 family in malignant hepatocytes in vitro[J].Br J Cancer,2011,105(1):146-153.
[5]Zeng L,Yu J,Huang T,et al.Differential combinatorial regulatory network analysis related to venous metastasis of hepatocellular carcinoma[J].BMC Genomics,2012,13(Suppl 8):S14.
[6]Viswanathan SR,Daley GQ.Lin28:A microrna regulator with a macro role[J].Cell,2010,140(4):445-449.
[7]Chang TC,Zeitels LR,Hwang HW,et al.Lin-28b transactivation is necessary for myc-mediated Let-7 repression and proliferation[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(9):3384-3389.
[8]Feitelson MA,Lee J.Hepatitis B virus integration,fragile sites,and hepatocarcinogenesis[J].Cancer Letters,2007,252(2):157-170.
[9]Moss EG,Lee RC,Ambros V.The cold shock domain protein lin-28 controls developmental timing in c.Elegans and is regulated by the lin-4 rna[J].Cell,1997,88(5):637-646.
[10]Guo Y,Chen Y,Ito H,et al.Identification and characterization of lin-28 homolog b(lin28b)in human hepatocellular carcinoma[J].Gene,2006,38(4):51-61.
[11]Viswanathan SR,Powers JT,Einhorn W,et al.Lin28 promotes transformation and is associated with advanced human malignancies[J].Nature Genetics,2009,41(7):843-848.
[12]Pang M,Wu G,Hou X,et al.Lin28b promotes colon cancer migration and recurrence[J].Plo S One,2014,9(10):e109169.
[13]Ji J,Wang XW.A yin-yang balancing act of the lin28/Let-7link in tumorigenesis[J].Journal of Hepatology,2010,53(5):974-975.
[14]Nguyen LH,Robinton DA,Seligson MT,et al.Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models[J].Cancer Cell,2014,26(2):248-261.
[15]Yang X,Lin X,Zhong X,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):9463-9472.
[16]Clippinger AJ,Gearhart TL,Bouchard MJ.Hepatitis B virus x protein modulates apoptosis in primary rat hepatocytes by regulating both nf-kappab and the mitochondrial permeability transition pore[J].Journal of Virology,2009,83(10):4718-4731.
[17]Shih WL,Kuo ML,Chuang SE,et al.Hepatitis b virus x protein inhibits transforming growth factor-beta-induced apoptosis through the activation of phosphatidylinositol 3-kinase pathway[J].J Biol Chem,2000,275(33):25858-25864.
[18]Marusawa H,Matsuzawa S,Welsh K,et al.Hbxip functions as a cofactor of survivin in apoptosis suppression[J].EMBO J,2003,22(11):2729-2740.
[19]Wang XW,Gibson MK,Vermeulen W,et al.Abrogation of p53-induced apoptosis by the hepatitis b virus x gene[J].Cancer Research,1995,55(24):6012-6016.
[20]Terradillos O,Pollicino T,Lecoeur H,et al.p53-independent apoptotic effects of the hepatitis b virus hbx protein in vivo and in vitro[J].Oncogene,1998,17(16):2115-2123.