http / / cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology

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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 678 PUMA sirna / * p53 p53 up-regulated modulator of apoptosispuma / hypoxia / reoxygenatioh / R PUMA sirna si-puma PUMA H / R RT-PCR Western 50 nmol / L si-puma H / R PUMA MTT lactate dehydrogenaseldh si-puma H / R 6 h LDH P < 0 01 Annexin V-FITC / PI si-puma caspase-3 H / R 6h P < 0 01 RT-PCR H / R 6 h si-puma Bax Bcl-2 P < 0 05 PUMA sirna H / R PUMA H / R / p53 / RNA Q753 R PUMA-targeting sirna Protects Rat Cardiomyocyte from Hypoxia / Reoxygenation-induced Apoptosis 1 FU Jing-Jing 2 LUO Da-Ya 1 WAN Fu-Sheng 1 * 1 Department of Biochemistry and Molecular BiologyBasic Medical College of Nanchang UniversityNanchang China 2 Department of Clinical LaboratoryChildren's Hospital of Jiangxi ProvinceNanchang China Abstract To study the effect of p53 up-regulated modulator of apoptosis PUMA in hypoxia / reoxygenation H / R mediated rat cardiomyocyte injurysirna targeted PUMA was designed and transfected into primary cultured rat cardiomyocytes in parallel with a scramble sirna Optimized dose of PUMA-specific sirna was 50 nmol / L as determined by RT-PCR and Western blotting The MTT assay and lactate dehydrogenase LDH activity measurement showed that the cell survival was enhanced and LDH release was reduced after the transfection of PUMA sirna P < 0 01compared with the control PUMA sirna could also down-regulate caspase-3 activity and decrease apoptosis rate P < 0 01 from the results of annexin V apoptosis assay and PI-stained with flow cytometry Down-regulation of Bax and up-regulation of Bcl-2 were detected in RT-PCR P < 0 05 These results indicated that PUMA sirna could protect cardiomyocytes from apoptosis and could be considered for the therapy for cardiac ischemia / reperfusion injury No No 2010JZY0237 * Tel Fax wanfs01@ 163 com Received January Accepted March Supported by National Natural Science Foundation of China No and Natural Science Foundation of Jiangxi Province No 2010JZY0237 * Corresponding author Tel Fax wanfs01@ 163 com

2 Key words p53 up-regulated modulator of apoptosis PUMA hypoxia / reoxygenation cardiomyocytes apoptosis RNA interference p53 p53 upregulated modulator of of apoptosispuma Bcl-2 scramble sirna si-scr BH3-only 5'-CGUGUGACCACUGGCAUUC-3' 5'-GA Ad-PUMA AUGCCAGUGGUCACACG-3' 5'-UUCU Ad-p PUMA CGUUCGGAGAA-3' sirna PUMA BLAST 3' dtdt PUMA sirna 3 ~ 6 / sir-ribo TM Transfection Control ischemia / reperfusion injury IRI Cy3 sirna si-cy3 7 8 PUMA d 9 60% Lipofectamine TM 2000 IRI IRI sirna 24 h IRI H / R / 11 hypoxia / reoxygenationh / R PUMA 3 d 95% N 2 5% CO 2 10 H / R 95% IRI PUMA sirna PUMA 5 h 95% O 2 5% CO 2 H / R PUMA CO 2 H / R 5% CO h IRI 1 5 si-puma Lipofectamine TM 2000 Trizol Invitrogen si-cy3 DMEM Opti-MEM Gibco Fermentas PUMA Abcam β-actin Bcl-2 20 nmol / L 50 nmol / L 100 nmol / L si-cy3 18 h PBS Bax Santa Cruz HRP 1 ~ nm MTT Sigma Annexin Ⅴ-FIFC / PI 600 Caspase-3 = Sparage Dawley 0 ~ sirna PUMA sirna si-puma CCGAACGUGUCACGU-3' 5'-ACGUGACA 1 4 H / R N 2 5% CO 2 37 si-cy3 RT-PCR PUMA mrna si-puma / 100% RT-PCR si-puma si-puma GenBank PUMA cdna si-puma 24 h AY H / R RT-PCR

3 7 PUMA sirna / 673 PUMA mrna RNA 1 2H / R 6 h RNA GAPDH RT-PCR H / R 3si-SCR Lipo-fectamine TM nmol / L GAPDH Table 1 PCR 5 μl scramble sirna si-scr 24 h 2% H / R 4si-PUMA Lipofectamine TM nmol / L si-puma 24 h GAPDH A H / R Table 1 The primer sequences for semi-quantitative RT-PCR 1 7 RT-PCR RNA Eppendorf PUMA Bax Bcl-2 PUMA Bax Bcl-2 A / A GAPDH Gene Primer sequences Length of product / bp PUMA Bax Bcl-2 GAPDH Forward 5'-AGC GGC GGA GAC AAG AA-3' Reverse 5'-CAA GTC CGT ATC TCC ATC AGT-3' Forward 5'-CAT CCA GGA TCG AGC AGA-3' Reverse 5'-CAA AGT AGA AGA GGG CAA CC-3' Forward 5'-GGG ATA CTG GAG ATG AAG ACT-3' Reverse 5'CCA CCG AAC TCA AAG AAG G-3' Forward 5'-GCA AGT TCA ACG GCA CAG-3' Reverse 5'-AGG TGG AAG AAT GGG AGT TGC T-3' Western Bradford SDS-PAGE 1 12 caspase-3 PVDF ECL β-actin A 1 13 A / A β-actin 1 9 MTT 96 t P < / 2 well μl MTT 5 mg / ml si-puma μl 37 4 h 150 μl si-cy3 18 h DMSO 10 min 490 nm si-cy3 = / Fig 1A 100% 1 10 LDH 200 μl LDH Beckman PUMA PUMA P < PBS nmol / L 100 nmol / L PUMA annexinv PI 15 min Becton Dickinson Facscallbar caspase-3 x 珋 ± s SPSS nmol / L si-cy3 47 2% 50 nmol / L 100 nmol / L si-cy3 90 2% 95 6% Fig 1B Fig 2A si- 100 nmol / L GAPDH

4 Fig 2 PUMA expression in cardiomyocytes transfected with various concentrations of si-puma detected using RT-PCR Various concentrations of PUMA targeted sirna were transfected into primary cultured rat cardiomyocytes using cationic liposome Lipofectamine 2000 TM the 0 nmol / L si-puma group was transfected with Lipofectamine2000 TM only The expression of PUMA mrna was detected using RT-PCR All results were normalized to the value obtained for GAPDH mrna expression A Confirmation of the correct RT-PCR products using 2% agarose gel electrophoresis B Quantitation of RT-PCR results by scanning densitometry of DNA M DNA marker 1 0 nmol / L si-puma group 2 20 nmol / L si-puma group 3 50 nmol / L si-puma group nmol / L si-puma group Values are expressed as x 珋 ± sn = 6 * P < 0 01 vs 0 nmol / L group Fig 2B 50 Table 2 Effects of extrogenous interference of PUMA on nmol / L PUMA cardiomyocyte survival rateand LDH leakage si-cy3 si-puma Group Survive rate % LDH in medium U / L 50 nmol / L sirna Control 95 2 ± 2 75 H / R 6 h 60 7 ± 6 84 * 152 ± ± 107 * si-scr 61 3 ± 5 77 * & ± 113 * & 2 2 si-puma H / R PUMA si-puma 79 3 ± 4 29 # 802 ± 55 # P < 0 01 vs Control group & P > 0 05 vs H / R 6 h group Fig 3 P < 0 01 vs H / R 6 h group x 珋 ± sn = 6 PUMA mrna H / R PUMA mrna H / R 6 h P > 0 05 H / R 6 h si-puma PUMA mrna 2 4 si-puma H / R P < 0 01 si-scr H / R 6h Annexin V-FITC / PI PUMA mrna P > Fig nmol / L si-puma H / R 3 57 ± 0 71 H / R PUMA si-puma 2 3 si-puma ± 4 02 % H / R 6h ± 5 02 % MTT P < 0 01 si-scr LDH Table ± 5 13 % H / R 6h LDH P > 0 05 H / R LDH 2 5 si-puma H / R caspase-3 H / R 6h si-puma caspase-3 1 LDH P < 0 01 si-caspase-3 Fig 5 H / R SCR LDH caspase ± 0 34

5 7 PUMA sirna / 675 si-puma caspase-3 Bcl ± 0 25 H / R 6 h P < 0 01 si-scr H / R 6 h caspase-3 P > si-puma H / R 18 RNAi PUMA sirna PUMA Fig 6 H / PUMA R Bax mrna Bcl-2 mrna si-puma Bax si-puma Bcl-2 P < RNAi Fig 3 The effect of si-puma interference on PUMA mrna and protein expression Primary cultured rat cardiomyocytes were transiently transfected with the control scramble sirna si-scr or PUMA targeted sirna si-puma A RT-PCR product confirmation using 2% agarose gel electrophoresis B Western blot analysis showed PUMA and β-actin expression C Relative levels of PUMA expression assessed by densitometry All values were normalized to the value obtained for the loading control M DNA marker 1 Control group 2 H / R 6 h group 3 si-scr group 4 si- PUMA group Values are expressed as x 珋 ± sn = 6 * P < 0 01 vs control & P > 0 05 vs H / R 6 h group # P < 0 01 vs H / R 6 h group 0 05 si-scr H / R 6 h 19 P > 0 05 sirna RT- PCR si-puma si-puma PUMA p53 H / R6 h si-puma PUMA mrna 90% PUMA PUMA p53 up-regulated modulator of apoptosis Bax / Bak 8 17 PUMA H / R 60% si-scr PUMA mrna 12 ~ p53 14 PUMA si-puma PUMA H / R PUMA p53 p PUMA BH3 Toth 20 PUMA

6 Fig 1 Transfection efficiency of si-cy3 into cardiomyocytes 100 Primary cultured rat cardiomyocytes were transfected with various concentrations of Si-Cy3 using cationic liposome Lipofectamine2000 TM A Cardiomyocytes transfected with si-cy3 were observed using an optical microscope B The red fluorescence expressed by si-cy3 was observed under a fluorescence microscope Columns 123and 4 correspond to treatments of and 100 nmol / L si-cy3respectively Fig 4 Flow cytometric analysis of apoptosis in cardiomyocyte cells transfected with si-puma Primary cultured rat cardiomyocytes were transfected with the control scramble sirna si-scr or PUMA targeted sirna si-puma washed with 50 mmol / L cold PBS ph 7 6 suspended in 100 μl of binding buffer containing 5 μl of fluorescence-conjugated annexin V and 2 5 μl of PIand then incubated for 30 minutes at 37 The cells were then subjected to FACS analysis A Control group B H / R 6 h group C si-scr group D si-puma group Values are expressed as x 珋 ± sn = 6 PUMA / LDH 50% PUMA / Caspase-3 PUMA 2 Bcl-2 Bcl-2 Bax PUMA Bcl-2 mrna PUMA / Bax mrna Bax PUMA mrna Bcl-2 mrna / PUMA PUMA Bcl-2 H / R 6 h si-puma Bax

7 7 PUMA sirna / 677 Fig 5 The effect of extrogenous interference of PUMA on caspase-3 activity Primary cultured rat cardiomyocytes were transiently transfected with the control scramble sirna si-scr or PUMA targeted sirna si- PUMA Thirty-six hours after transfectionwhole cell lysates were preparedand caspase-3 activity was detected by spectrophotometry Each value was expressed as the ratio of caspase-3 activation level control levelwith the value of control level set to 1 1 Control group 2 H / R 6 h group 3 si-scr group 4 si-puma group Data is expressed as x 珋 ± sn = 6 * P < 0 01 vs control & P > 0 05 vs H / R 6 h group # P < 0 01 vs H / R 6 h group Fig 6 The effect of extrogenous interference of PUMA on mrna expression of apotosis-related molecules Primary cultured rat cardiomyocytes were transiently transfected with the control scramble sirna si-scr or PUMA targeted sirna si-puma A & B The mrna expression levels of Bax and Bcl-2 were detected using RT-PCRrespectively C Relative levels of Bax / Bcl-2 mrna assessed by densitometry 1 Control group 2 H / R 6 h group 3 si-scr group 4 si-puma group Values are expressed as x 珋 ± sn = 6 * P < 0 05vs control & P > 0 05 vs H / R 6 h group # P < 0 05 vs H / R 6 h group Bcl-2 PUMA Bcl-2 Bax PUMA si-puma 14 6% 9 8% 4 8% PUMA sirna PUMA sirna H / R PUMA H / R PUMA IRI PUMA IRI References 1 Yu JYue WWu Bet al PUMA sensitizes lung cancer cells to chemotherapeutic agents and irradiation J Clin Cancer Res Sun QSakaida TYue Wet al Chemosensitization of head and neck cancer cells by PUMAJ Mol Cancer Ther Pt Reimertz CK gel DRami Aet al Gene expression during ER stress-induced apoptosis in neurons induction of the BH3- only protein Bbc3 / PUMA and activation of the mitochondrial apoptosis pathway J J Cell Biol Wu BQiu WWang Pet al p53 independent induction of PUMA mediates intestinal apoptosis in response to ischaemiareperfusion J Gut p53 J Fu J JWan F S Promoting apoptotic effect of p53 up-regulated modulator of apoptosis J Chemi Life Nickson PToth AErhardt P PUMA is critical for neonatal cardiomyocyte apoptosis induced by endoplasmic reticulum stress J Cardiovasc Res Zhao Z QVelez D AWang N Pet al Progressively developed myocardial apoptotic cell death during late phase of reperfusion J Apoptosis Buja L M Myocardial ischemia and reperfusion injuryj Cardiovasc Pathol Yee K SVousden K H Contribution of membrane localization to the apoptotic activity of PUMAJ Apoptosis PUMA / J Fu J JDuan RLi Het al Study on PUMA-mediated hypoxia-reoxygenation-induced rat cardiomyocyte apoptosisj Chin Phamacol Bull

8 Jeffers J RParganas ELee Yet al Puma is an essential J Li HWan FS Yang mediator of p53-dependent and p53-independent apoptotic Wet al Effects of taurine on the apoptosis of cardiomyocyte and pathwaysj Cancer Cell its mechanismj J Nanchang Univ Nat Sci Gallenne TGautier FOliver Let al Bax activation by the BH3-only protein Puma promotes cell dependence on 12Yu JZhang LHwang Pet al PUMA induces the rapid antiapoptotic Bcl-2 family membersj J Cell Biol apoptosis of colorectal cancer cells Mol Cell Nakano KVousden K PUMAa novel proapoptotic geneis induced by p53j Mol Cell Han J Flemington C Houghton A Bet al Expression of Bbc3 a pro-apoptotic BH3-only geneis regulated by diverse cell death and suvival signalsj Proc Natl Acad Sci USA sirna NFBD1 18Yu JZhang L PUMAa potent killer with or without p53j Oncogene Suppl 1 S RNA VEGF J Zhang S YGe Y LLuo D Yet al Small interfering RNAs directed against VEGF gene inhibit proliferation of breast cancer cells in vitro and in vivoj Chin J Biochem Mol Biol HeLa J 20Toth AJeffers J RNickson Pet al Targeted deletion of Puma Bu Y QYang Z MLan Het al Effects of sirnamediated NFBD1 silencing on cellular growth and apoptosis in during ischemia reperfusion J Am J Physiol Heart Circ attenuates cardiomyocyte death and improves cardiac function HeLa cellsj Chin J Biochem Mol Biol Physiol H