612 612 ~ 6 2012-5 - 4 9 14 http / /www. cnki. net /kcms /detail /34. 1086. R. 20120504. 0914. 006. html sirna β-arrestin2 230032 doi 10. 3969 /j. issn. 1001-1978. 2012. 05. 006 A 1001-1978 2012 05-0612 - 05 R-332 R322. 47 R329. 25 R575. 202. 2 RNA small interfering RNA sirna ECM HSC β-arrestin2 hepatic stellate cell T6HSC-T6 sirna ECM β-arrestin2 Lipofectamine HSC-T6 2 HSC - Western HSCs blot β-arrestin2 Western blot Bcl-2 Bax sirna HSC-T6 β-arrestin2 HSC ECM β-arrestin2 mrna 70% ± 1. 76% 68. 43% ± 2. 88% P < 0. 01 HSC 3 Bcl-2 32. 58% ± 3. 46% P < 0. 01 Bax 38. 00% ± 3. 72% P < 0. 01 β-arrestin2 sirna HSC-T6 37. 5% HSC β-arrestin sirna β-arrestin2 HSC-T6 β-arrestin2 Bcl-2 Bax GPCR β-arrestin HSC-T6 4-5 β-arrestin2 RNA β-arrestin2 Bcl-2 Bax β-arrestin2 2011-12 - 23 2012-02 - 21 No 30973543 81173075 No 20113420120002 No KJ2012A153 No 090413108 No 2010SQRL079 1988 - E-mail sy0012006@ 126. com 1981 - E-mail sunwyi51@ hotmail. com 1960 - E-mail wwei@ ahmu. edu. cn extracellular matrixecm 1 hepatic stellate cells HSC β-arrestins G G protein coupled receptorgpcr 6-7 β-arrestin2 8-9 β-arrestin2 HSC β-arrestin2 RNAsmall interfering RNAsiRNA HSC-T6 β-arrestin2 Bcl-2 Bax HSC sirna β-arrestin2 HSC 1 1. 1 HSC-T6 1. 2 Lipofectamine 2000 Invitrogen β-arrestin2 Santa cruz Bcl-2 Bax β-actin MBI
613 Fermentas IgG H + min sirna Lipofectamine 2000 L 20 min sirna- polyvinylidene fluoride PVDF Millipore SuperSignal West Femto Kit 6 h Pierce 4 sirna 2. 3 RT-PCR β-arrestin2 mrna RT-PCR TRizol RNA Invitrogen DMEM PCR β-arrestin2 5'-CCACGTCACCAA- FBS GIBco RT- CAATTCTG 5'-TTGGTGTCTTCGTGCTTGAG- PCR Fermentas 3' GAPDH 5'-TCAAGAAGGTGGT- 1. 3 Olympus IX-71 Beckman FC500 SpotAdvanced DYY-10 Image-Pro plus 2. 4 Western blot β-arrestin2 Bcl-2 Bax PCR Biorad My Cycler 2 2. 1 HSC-T6 HSC-T6 PAGE 200 ma 10% DMEM 37 5% CO 2 80% 0. 25% 24 h 2 h ECL HSC-T6 5 10 5 / 6 Image-Pro plus 2. 2 sirna HSC-T6 2. 2. 1 β-arrestin2 sirna PBS β-arrestin2 4 sirna sirna-1 5'-AGCGUGACUUUGUGGAU- CATT-3' 5'-UGAUCCACAAAGUCACG- CUTT-3' sirna-2 5'-GACCGGAAAGUGUU- UGUGATT-3' 5'-UCACAAACACUUUCCG- GUCTT-3' sirna-3 5'-GACCGACUGCUGA- AGAAGUTT-3' 5'-ACUUCUUCAGCAGUC- GGUCTT-3' sirna-4 5'-UCGAGCCUUCU- GUGCCAAATT-3' 5'-UUUGGCACAGAAG- GCUCGATT-3' 2. 2. 2 3 1 arrestin2 mrna mrna DMEM 70% ± 1. 76% 69% ± 1. 93% β- arrestin2 2 sirna1 sirna2 sirna4 mrna scrambled RNA β- arrestin2 3 sirna β-arrestin2 R1 R2 R3 R4 β-arrestin2 sirna P > 0. 05 Fig 1 β-arrestin2 GAAGCAG-3' 5'-AGGTGGAAGAATGG- GAGTTG-3' PCR 94 5 min 94 30 s 58 30 s 72 30 s 72 10 min 4 10 min 30 PCR 2% Lowry 20 μl SDS- PVDF 2 h β-arrestin2 Bcl- 2 Bax 4 2. 5 AnnexinV /PI 3 2. 6 SPSS 17. 0 x 珋 ± s 3 3. 1 sirna β-arrestin2 β-arrestin2 RT-PCR sirna3 HSC-T6 β- sirna3 β-arrestin2 β-arrestin2 mrna 3. 2 sirna β-arrestin2 HSC-T6 β-arres- 2. 2. 3 HSC-T6 tin2 Western blot 80% Lipo- HSC-T6 sirna2 sirna3 β-arres- 29. 00% ± 1. 68% fectamine 2000 sirna sirna tin2 20 μmol L - 1-20 5 68. 43% ± 2. 88%
614 β-arrestin2 β-arrestin2 β-arrestin2 Bcl-2 32. 58% ± 3. 46% mrna Fig 2 P < 0. 01 Bax 38% ± 3. 72% P < 0. 01 Bcl-2 Bax Fig 1 Transcriptional expression of the β-arrestin2 in the stable HSC-T6 cells transfected with β-arrestin2 sirna M Marker C Blank control NC Negative control R1 sirna1 R2 sirna2 R3 sirna3 R4 sirna 4 Each sample was tested in triplicate. Densitometry values in the histograms were expressed as -fold change relative to the blank controlwhich was assigned a value of 1. ** P < 0. 01 vs blank control. Fig 3 Effects of silencing HSCs β-arrestin2 gene on the expression of Bcl-2Bax C Blank control NC Negative control sirna3 sirna3 targeting β-arrestin2. Densitometry values in the histograms were expressed as -fold change relative to the blank controlwhich was assigned a value of 1. ** P < 0. 01 vs blank control. Fig 2 Protein level of the β-arrestin2 in the HSC-T6 cells transfected with β-arrestin2 sirna C Blank control NC Negative control R1 sirna1 R2 sirna2 R3 sirna3 R4 sirna 4 Each sample was tested in triplicate. Densitometry values in the histograms were expressed as -fold change relative to the blank controlwhich was assigned a value of 1. * P < 0. 05 ** P < 0. 01 vs blank control. 3. 3 sirna β-arrestin2 HSC Bcl-2 Bax Fig 3 sirna3 HSC-T6 3. 4 sirna β-arrestin2 HSC-T6 3. 5% 4. 7% sirna β- arrestin2 37. 5% Fig 4 4 HSC ECM HSC 10-11 HSC Bcl-2 Bcl-2 Bax Bcl- 2 Bcl-2 Bax Bcl-2 Bax Bcl-2 /Bax 12-13 Arrestins 48 ~ 55 ku
615 21-22 β-arrestin2 MDM2 β-arrestin2 p53 23 β-arrestin2 p53 sirna β-arrestin2 HSC Bcl-2 Bax β-arrestin2 AnnexinV /PI sirna β-arrestin2 HSC-T6 β-arrestin2 HSC sir- NA β- arrestin2 Fig 4 Effects of sirna β-arrestin2 on the apoptosis of HSC-T6 A blank control B negative control C HSC transfected with sirna β-arrestin2 Arrestins Arrestin 1 Ar- 1433-8. restin 4 β-arrestins β- arrestin1 β-arrestin2 Arrestin 2 Arrestin 3 β-arrestins GPCRs GPCRs GPCRs 14-16 β-arrestins β-arrestin2 β-arrestin1 β-arrestin2 pathways J 6Li T TAlemayehu MAziziyeh A Iet al. Beta-arrestin /Ral signaling regulates lysophosphatidic acid-mediated migration and in- β-arrestin2 GPCRs vasion of human breast tumor cells J 17-18 CD4 + 7 1064-77. T β-arrestins Bcl-2 19 20 DEN β- arrestin2 Bcl-xL Bax p53 p53 J. Biochim Biophys Acta20101800 9 912-8. Bcl-xl Bcl-2 Bax p53 Bax 9Wagener B MMarjon N ARevankar C Met al. Adaptor protein-2 interaction with arrestin regulates GPCR recycling and apop- Bcl-2 Bcl-xL tosis J 1Wirkowska APaczek L. Liver fibrosis and cirrhosis-choosen cytokinesgrowth factors and proteins. Part II J. Przegl Lek2011 68 4 228-30. 2. J. 200622 12 2Sun W YWei W. Signal transduction in hepatic stellate cells and possible drug targets J. Chin Pharmacol Bull200622 12 1433-8. 3Qu ZLou DPan Y. The role of IkappaBalpha in TNF-alpha-induced apoptosis in hepatic stellate cell line HSC-T6J. J Huazhong Univ Sci Technol Med Sci200727 4 407-10. 4Natarajan KBerk B C. Crosstalk coregulation mechanisms of G protein-coupled receptors and receptor tyrosine kinases J. Methods Mol Biol2006332 51-77. 5Kovacs J JHara M RDavenport C Let al. Arrestin development emerging roles for beta-arrestins in developmental signaling. Dev Cell200917 4 443-58.. Mol Cancer Res20097 7McLennan G PKiss AMiyatake Met al. Kappa opioids promote the proliferation of astrocytes via Gbetagamma and beta-arrestin 2-dependent MAPK-mediated pathwaysj. J Neurochem 2008107 6 1753-65. 8Sun XZhang YWang Jet al. Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt / GSK3ss pathways. Traffic200910 9 1286-300. 10Yu F SYang J SYu C Set al. Safrole induces apoptosis in hu-
616 man oral cancer HSC-3 cells J. J Dent Res201190 2 168-74. 11Saile BDiRocco PDudas Jet al. IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells HSC but DNA synthesis and proliferation in rat liver myofibroblasts rmf J. Lab Invest200484 8 1037-49. 12Spampanato CDe Maria SSarnataro Met al. Simvastatin inhibits cancer cell growth by inducing apoptosis correlated to activation of Bax and down-regulation of BCL-2 gene expression J. Int J Oncol2012 40 4 935-41. 13Li XYe HCai Let al. Millimeter wave radiation induces apoptosis via affecting the ratio of Bax /Bcl-2 in SW1353 human chondrosarcoma cells J. Oncol Rep2012 27 3 664-72. 14Wagener B MMarjon N ARevankar C Met al. Adaptor protein-2 interaction with arrestin regulates GPCR recycling and apoptosis J. Traffic200910 9 1286-300. 15Kocan MPfleger K D. Detection of GPCR /beta-arrestin interactions in live cells using bioluminescence resonance energy transfer technology J. Methods Mol Biol2009552 305-17. 16Miyatake MRubinstein T JMcLennan G Pet al. Inhibition of EGF-induced ERK / MAP kinase-mediated astrocyte proliferation by mu opioids integration of G protein and beta-arrestin 2-dependent pathways J. J Neurochem2009110 2 662-74. 17Ma LPei G. Beta-arrestin signaling and regulation of transcription J. J Cell Sci2007120 Pt 2 213-8. 18Sun XZhang YWang Jet al. Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt / GSK3ss pathways J. Biochim Biophys Acta20101800 9 912-8. 19Shi YFeng YKang Jet al. Critical regulation of CD4 + T cell survival and autoimmunity by beta-arrestin 1J. Nat Immunol 20078 8 817-24. 20Khan M SDevaraj HDevaraj N. Chrysin abrogates early hepatocarcinogenesis and induces apoptosis in N-nitrosodiethylamine-induced preneoplastic nodules in ratsj. Toxicol Appl Pharmacol 2011251 1 85-94. 21Cai QGuo YXiao Bet al. Epstein-Barr Virus Nuclear Antigen 3C Stabilizes Gemin3 to Block p53-mediated Apoptosis J. PLoS Pathog20117 12 e1002418. 22Boulaiz HAlvarez P JPrados Jet al. gef Gene Expression in MCF-7 Breast Cancer Cells is Associated with a Better Prognosis and Induction of Apoptosis by p53-mediated Signaling Pathway J. Int J Mol Sci201112 11 7445-58. 23Boularan CScott M GBourougaa Ket al. beta-arrestin 2 oligomerization controls the Mdm2-dependent inhibition of p53j. Proc Natl Acad Sci USA2007104 46 18061-6. Small interfering RNA targeting β-arrestin2 promoted apoptosis of hepatic stellate cells SONG YangSUN Wu-yiHU Shan-shanWANG Qing-tongWEI Wei Institute of Clinical PharmacologyAnhui Medical UniversityKey Laboratory of Anti-inflammatory and Immunopharmacology Ministry of EducationAnhui Engineering Technology Research Center of Anti-inflammatory and ImmunodrugsHefei 230032 China Abstract Aim To investigate the effect of synthetic small interfering RNA sirna targeting β-arrestin2 on the apoptosis of hepatic stellate cells HSC in vitro. Methods Synthetic sirna targeting β-arrestin2 was transfected into HSC-T6 cells by lipofectamine package. Negative sirna transfection and no transfection were used as negative and blank controlrespectively. After incubation with sirnatotal RNA and protein of HSC-T6 cells were extracted. The expression of β-arrestin2 gene and protein was detected by reverse transcription-polymerase chain reaction RT-PCR and Western blotting. HSC-T6 apoptosis was measured by flow cytometry. The expression of Bcl-2 and Bax were determined by Western blot. Results After HSC-T6 cells were transfected with β-arrestin2 sirna the level of β-arrestin2 mrna and protein expression was decresed by 70% ± 1. 76% P < 0. 01 and 68. 43% ± 2. 88% P < 0. 01 as compared with the control group. Bcl-2 expression was also inhibited by 32. 58% ± 3. 46% P < 0. 01 while Bax expression was increased by 38. 00% ± 3. 72% P < 0. 01. The apoptosis rate of HSC-T6 transfected with β-arrestin2 sir- NA was 37. 5% which was significantly higher than control. Conclusion Inhibition of β-arrestin2 by sir- NA may have a potential effect on prevention and treatment of hepatic fibrosis by promoting apoptosis of HSC. Key words hepatic stellate cells small interfering RNA β-arrestin2 apoptosis Bcl-2 Bax hepatic fibrosis