sirna ECM hepatic stellate cells HSC HSC ECM HSC HSCs β-arrestins G coupled receptor GPCR β-arrestin GPCR RNA small interfering RNA sirna

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1 ~ http / /www. cnki. net /kcms /detail / R html sirna β-arrestin doi /j. issn A R-332 R R R 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% % ± 2. 88% P < HSC 3 Bcl % ± 3. 46% P < Bax % ± 3. 72% P < β-arrestin2 sirna HSC-T % 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-arrestin No No No KJ2012A153 No No 2010SQRL sy @ 126. com sunwyi51@ hotmail. com 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 HSC-T Lipofectamine 2000 Invitrogen β-arrestin2 Santa cruz Bcl-2 Bax β-actin MBI

2 613 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 Olympus IX-71 Beckman FC500 SpotAdvanced DYY-10 Image-Pro plus 2. 4 Western blot β-arrestin2 Bcl-2 Bax PCR Biorad My Cycler HSC-T6 HSC-T6 PAGE 200 ma 10% DMEM 37 5% CO 2 80% 0. 25% 24 h 2 h ECL HSC-T / 6 Image-Pro plus 2. 2 sirna HSC-T 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' 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 > Fig 1 β-arrestin2 GAAGCAG-3' 5'-AGGTGGAAGAATGG- GAGTTG-3' PCR 94 5 min s s s min 4 10 min 30 PCR 2% Lowry 20 μl SDS- PVDF 2 h β-arrestin2 Bcl- 2 Bax AnnexinV /PI SPSS x 珋 ± s 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 HSC-T6 tin2 Western blot 80% Lipo- HSC-T6 sirna2 sirna3 β-arres % ± 1. 68% fectamine 2000 sirna sirna tin2 20 μmol L % ± 2. 88%

3 614 β-arrestin2 β-arrestin2 β-arrestin2 Bcl % ± 3. 46% mrna Fig 2 P < Bax 38% ± 3. 72% P < 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 < 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 < 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 < ** P < vs blank control sirna β-arrestin2 HSC Bcl-2 Bax Fig 3 sirna3 HSC-T sirna β-arrestin2 HSC-T6 3. 5% 4. 7% sirna β- arrestin % Fig 4 4 HSC ECM HSC HSC Bcl-2 Bcl-2 Bax Bcl- 2 Bcl-2 Bax Bcl-2 Bax Bcl-2 /Bax Arrestins 48 ~ 55 ku

4 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 restin 4 β-arrestins β- arrestin1 β-arrestin2 Arrestin 2 Arrestin 3 β-arrestins GPCRs GPCRs GPCRs 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 CD T β-arrestins Bcl DEN β- arrestin2 Bcl-xL Bax p53 p53 J. Biochim Biophys Acta 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 Lek J Sun W YWei W. Signal transduction in hepatic stellate cells and possible drug targets J. Chin Pharmacol Bull Qu 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 Sci Natarajan KBerk B C. Crosstalk coregulation mechanisms of G protein-coupled receptors and receptor tyrosine kinases J. Methods Mol Biol Kovacs J JHara M RDavenport C Let al. Arrestin development emerging roles for beta-arrestins in developmental signaling. Dev Cell Mol Cancer Res McLennan G PKiss AMiyatake Met al. Kappa opioids promote the proliferation of astrocytes via Gbetagamma and beta-arrestin 2-dependent MAPK-mediated pathwaysj. J Neurochem Sun XZhang YWang Jet al. Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt / GSK3ss pathways. Traffic Yu F SYang J SYu C Set al. Safrole induces apoptosis in hu-

5 616 man oral cancer HSC-3 cells J. J Dent Res Saile 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 Invest Spampanato 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 Oncol Li XYe HCai Let al. Millimeter wave radiation induces apoptosis via affecting the ratio of Bax /Bcl-2 in SW1353 human chondrosarcoma cells J. Oncol Rep Wagener B MMarjon N ARevankar C Met al. Adaptor protein-2 interaction with arrestin regulates GPCR recycling and apoptosis J. Traffic Kocan MPfleger K D. Detection of GPCR /beta-arrestin interactions in live cells using bioluminescence resonance energy transfer technology J. Methods Mol Biol Miyatake 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 Neurochem Ma LPei G. Beta-arrestin signaling and regulation of transcription J. J Cell Sci Pt Sun XZhang YWang Jet al. Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt / GSK3ss pathways J. Biochim Biophys Acta Shi YFeng YKang Jet al. Critical regulation of CD4 + T cell survival and autoimmunity by beta-arrestin 1J. Nat Immunol Khan M SDevaraj HDevaraj N. Chrysin abrogates early hepatocarcinogenesis and induces apoptosis in N-nitrosodiethylamine-induced preneoplastic nodules in ratsj. Toxicol Appl Pharmacol Cai QGuo YXiao Bet al. Epstein-Barr Virus Nuclear Antigen 3C Stabilizes Gemin3 to Block p53-mediated Apoptosis J. PLoS Pathog e Boulaiz 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 Sci Boularan CScott M GBourougaa Ket al. beta-arrestin 2 oligomerization controls the Mdm2-dependent inhibition of p53j. Proc Natl Acad Sci USA 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 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 < and % ± 2. 88% P < as compared with the control group. Bcl-2 expression was also inhibited by % ± 3. 46% P < while Bax expression was increased by % ± 3. 72% P < 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