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Fas siRNA reduces apoptotic cell death of allogeneic-transplanted hepatocytes in mouse spleen
Fas siRNA Reduces Apoptotic Cell Death of AllogeneicTransplanted Hepatocytes in Mouse Spleen J. Wang, W. Li, J. Min, Q. Ou, and J. Chen
ABSTRACT Background. Synthetic siRNAs 21 to 23 nucleotides in length silence gene expression posttranscriptionally, and RNA interference targeting Fas protects mice from fulminant hepatitis. Fas-mediated apoptosis has also been implied in the mechanism of hepatocyte apoptosis upon allogenic hepatocyte transplantation (HTx), and blockade of Fas and Fas ligand interaction successfully promotes the repopulation of allogenic liver cells in recipient spleens. In the present study, we further investigated the protective effects of Fas silencing on allogeneic hepatocytes transplanted into mouse spleens. Materials and Methods. Hepatocytes were isolated from BALB/c mice and mock transfected or transfected with Fas siRNA or GFP siRNA (n ⫽ 8/group). The expression of Fas was examined by RT-PCR and flow cytometric analysis. Forty-eight hours later, the cells were transplanted into spleens of allogenic B6 mice. Spleens were harvested on day 21 after transplantation. Apoptosis was assessed by TUNEL assay, survival of hepatocytes by alanine transaminase (ALT) assay. Results. Fas siRNA transfection reduced Fas expression on hepatocytes at both mRNA and protein levels (P ⬍ .05). Upon transplanting into recipient spleens, hepatocytes transfected with Fas siRNA demonstrated a lower percentage of apoptosis detected by TUNEL (6 ⫾ 3% in Fas siRNA group vs 12 ⫾ 5% in GFP siRNA group and 10 ⫾ 3% in mock transfected group; P ⬍ .05), and increased survival as determined by ALT assay (38.2 ⫾ 10.6 IU/g in Fas siRNA group vs 21.3 ⫾ 8.4 IU/g in GFP siRNA group and 18.5 ⫾ 5.9 IU/g in mock-transfected animals). Conclusion. Fas silencing by RNA interference reduces apoptosis and increases survival of allogenic transplanted hepatocytes, and thus holds promise to inhibit acute rejection after hepatocyte transplantation.
S
YNTHETIC siRNAs 21 to 23 nucleotides in length silence gene expression posttranscriptionally,1 and our previous study showed that RNA interference targeting Fas protected mice from fulminant hepatitis.2 Fasmediated apoptosis has also been implied in the mechanism of hepatocyte apoptosis upon allogenic hepatocyte transplantation (HTx),3 and blockade of Fas and Fas ligand interactions promotes the repopulation of allogenic liver cells in recipient spleens.4 In the present study, we investigated the protective effects of Fas silencing on allogeneic hepatocytes transplanted into mouse spleens. 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00438-X 1594
MATERIALS AND METHODS Preparation of siRNAs Fas and control GFP siRNAs were synthesized using 2⬘-O-ACERNA phosphoramidites (Dharmacon Research, Lafayette, Calif). The sense and anti-sense strands of siRNAs were:
From the Department of Surgery, Sun-Yat-Sen Memorial Hospital of Sun-Yat-Sen University of Medical Science, Guangzhou, China. Address reprint requests to Jie Wang, Department of Surgery, Sun-Yat-Sen Memorial Hospital, 107 Yanjiang West Road, Guangzhou 510120, P.R. China. E-mail: [email protected] © 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 1594-1595 (2003)
FAS
SIRNA
REDUCES APOPTOTIC CELL DEATH
Fas, 5⬘-P.GUGCAAGUGCAAACCAGACdTdT-3⬘ (sense), 5⬘-P.GUCUGGUUUGCACUUGCACdTdT-3⬘ (antisense); GFP, 5⬘-P.GGCUACGUCCAGGAGCGCACC-3⬘ (sense), 5⬘-P.UGCGCUCCUGGACGUAGCCUU-3⬘ (antisense). RNAs were deprotected and annealed according to the manufacturer’s instruction.
Transfection of siRNAs Hepatocytes isolated from donor BALB/c mice were transfected with oligofectamine according to the manufacturer’s protocol in the presence or absence of 1 nmol siRNA duplex. After overnight incubation, cells were washed and transplanted. Expression of Fas on hepatocytes at both mRNA and protein levels was detected using RT-PCR and flow cytometric analysis.
Hepatocyte Transplantation Hepatocytes transfected with Fas siRNA, control GFP siRNA, or mock-transfected (n ⫽ 8/group) were transplanted into spleens of B6 mice as described before.4 Spleens were harvested on day 21 after transplantation for histological evaluation and apoptosis assay. Apoptosis of transplanted hepatocytes was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL, Boehringer Mannheim GmbH, Mannheim, Germany). The number of TUNEL-positive hepatocytes exhibiting apoptotic morphology was expressed as a percentage. At least 1000 transplanted cells were counted. Additionally, the survival of transplanted cells in recipient spleens was evaluated by examining the splenic level of alanine transaminase (ALT), a specific enzyme produced by hepatocytes, using an ALT assay kit (Boehringer Mannheim GmbH, Mannheim, Germany). The amount of ALT in recipient splenic tissue was expressed in units of ALT per gram of splenic tissue (IU/g).
Statistics All data were expressed as mean values ⫾ SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) and comparisons among groups performed by Bonferroni’s multiplecomparison t test. Significance level was set at P ⬍ 0.05.
RESULTS Fas Expression on Hepatocytes
Forty-eight hours after transfection, Fas mRNA expression in hepatocytes was reduced by Fas siRNA (0.81 ⫾ 0.27
1595 Table 1. Percentage of Apoptotic Transplanted Hepatocytes and Splenic ALT Levels Groups
TUNEL-Positive Hepatocytes (%)
Splenic ALT Levels (IU/g)
Fas siRNA GFP siRNA Mock-transfected
6 ⫾ 3* 12 ⫾ 5 10 ⫾ 3
38.2 ⫾ 10.6* 21.3 ⫾ 8.4 18.5 ⫾ 5.9
*P⬍.05 as compared to mock-transfected group.
times -actin) as compared with mock transfection (1.19 ⫾ 0.54 times -actin, P ⬍ .05) and control GFP siRNA transfection (1.43 ⫾ 0.72 times -actin, P ⬍ .05). Additionally, Fas siRNA transfection reduced the level of Fas receptor in hepatocytes (mean fluorescence intensity [MFI]: 11.6 ⫾ 5.4) determined by cytometric analysis, as compared with mock transfection (MFI: 33.2 ⫾ 8.3, P ⬍ .05) and control GFP siRNA transfection (MFI: 36.5 ⫾ 7.9, P ⬍ .05). Apoptosis and Survival of Transplanted Hepatocytes
As shown in Table 1, Fas siRNA transfection reduced the percentage of TUNEL-positive hepatocytes in the spleens of allogeneic recipients (P ⬍ .05), associated with a higher survival rate of allogeneic hepatocytes in recipient spleens as determined by ALT levels (P ⬍ .05). However, GFP siRNA transfection did not affect the apoptotic percentage of allogenic hepatocytes or ALT levels (P ⬎ .05). DISCUSSION
Fas silencing by RNA interference reduces apoptosis and increases survival of allogenic transplanted hepatocytes and thereby proffers the possibility to inhibit acute rejection after hepatocyte transplantation. REFERENCES 1. Hannon GJ: Nature 418:224, 2002 2. Song E, Lee SK, Wang J, et al: Nat Med 9:doi:10.1038/nm828, 2003 3. MacDonald G, Shi L, Velde CV, et al: J Exp Med 189:131, 1999 4. Song E, Chen J, Lutz J, et al: Transplant Proc 33:650, 2001
ABSTRACT Background. Synthetic siRNAs 21 to 23 nucleotides in length silence gene expression posttranscriptionally, and RNA interference targeting Fas protects mice from fulminant hepatitis. Fas-mediated apoptosis has also been implied in the mechanism of hepatocyte apoptosis upon allogenic hepatocyte transplantation (HTx), and blockade of Fas and Fas ligand interaction successfully promotes the repopulation of allogenic liver cells in recipient spleens. In the present study, we further investigated the protective effects of Fas silencing on allogeneic hepatocytes transplanted into mouse spleens. Materials and Methods. Hepatocytes were isolated from BALB/c mice and mock transfected or transfected with Fas siRNA or GFP siRNA (n ⫽ 8/group). The expression of Fas was examined by RT-PCR and flow cytometric analysis. Forty-eight hours later, the cells were transplanted into spleens of allogenic B6 mice. Spleens were harvested on day 21 after transplantation. Apoptosis was assessed by TUNEL assay, survival of hepatocytes by alanine transaminase (ALT) assay. Results. Fas siRNA transfection reduced Fas expression on hepatocytes at both mRNA and protein levels (P ⬍ .05). Upon transplanting into recipient spleens, hepatocytes transfected with Fas siRNA demonstrated a lower percentage of apoptosis detected by TUNEL (6 ⫾ 3% in Fas siRNA group vs 12 ⫾ 5% in GFP siRNA group and 10 ⫾ 3% in mock transfected group; P ⬍ .05), and increased survival as determined by ALT assay (38.2 ⫾ 10.6 IU/g in Fas siRNA group vs 21.3 ⫾ 8.4 IU/g in GFP siRNA group and 18.5 ⫾ 5.9 IU/g in mock-transfected animals). Conclusion. Fas silencing by RNA interference reduces apoptosis and increases survival of allogenic transplanted hepatocytes, and thus holds promise to inhibit acute rejection after hepatocyte transplantation.
S
YNTHETIC siRNAs 21 to 23 nucleotides in length silence gene expression posttranscriptionally,1 and our previous study showed that RNA interference targeting Fas protected mice from fulminant hepatitis.2 Fasmediated apoptosis has also been implied in the mechanism of hepatocyte apoptosis upon allogenic hepatocyte transplantation (HTx),3 and blockade of Fas and Fas ligand interactions promotes the repopulation of allogenic liver cells in recipient spleens.4 In the present study, we investigated the protective effects of Fas silencing on allogeneic hepatocytes transplanted into mouse spleens. 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00438-X 1594
MATERIALS AND METHODS Preparation of siRNAs Fas and control GFP siRNAs were synthesized using 2⬘-O-ACERNA phosphoramidites (Dharmacon Research, Lafayette, Calif). The sense and anti-sense strands of siRNAs were:
From the Department of Surgery, Sun-Yat-Sen Memorial Hospital of Sun-Yat-Sen University of Medical Science, Guangzhou, China. Address reprint requests to Jie Wang, Department of Surgery, Sun-Yat-Sen Memorial Hospital, 107 Yanjiang West Road, Guangzhou 510120, P.R. China. E-mail: [email protected] © 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 1594-1595 (2003)
FAS
SIRNA
REDUCES APOPTOTIC CELL DEATH
Fas, 5⬘-P.GUGCAAGUGCAAACCAGACdTdT-3⬘ (sense), 5⬘-P.GUCUGGUUUGCACUUGCACdTdT-3⬘ (antisense); GFP, 5⬘-P.GGCUACGUCCAGGAGCGCACC-3⬘ (sense), 5⬘-P.UGCGCUCCUGGACGUAGCCUU-3⬘ (antisense). RNAs were deprotected and annealed according to the manufacturer’s instruction.
Transfection of siRNAs Hepatocytes isolated from donor BALB/c mice were transfected with oligofectamine according to the manufacturer’s protocol in the presence or absence of 1 nmol siRNA duplex. After overnight incubation, cells were washed and transplanted. Expression of Fas on hepatocytes at both mRNA and protein levels was detected using RT-PCR and flow cytometric analysis.
Hepatocyte Transplantation Hepatocytes transfected with Fas siRNA, control GFP siRNA, or mock-transfected (n ⫽ 8/group) were transplanted into spleens of B6 mice as described before.4 Spleens were harvested on day 21 after transplantation for histological evaluation and apoptosis assay. Apoptosis of transplanted hepatocytes was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL, Boehringer Mannheim GmbH, Mannheim, Germany). The number of TUNEL-positive hepatocytes exhibiting apoptotic morphology was expressed as a percentage. At least 1000 transplanted cells were counted. Additionally, the survival of transplanted cells in recipient spleens was evaluated by examining the splenic level of alanine transaminase (ALT), a specific enzyme produced by hepatocytes, using an ALT assay kit (Boehringer Mannheim GmbH, Mannheim, Germany). The amount of ALT in recipient splenic tissue was expressed in units of ALT per gram of splenic tissue (IU/g).
Statistics All data were expressed as mean values ⫾ SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) and comparisons among groups performed by Bonferroni’s multiplecomparison t test. Significance level was set at P ⬍ 0.05.
RESULTS Fas Expression on Hepatocytes
Forty-eight hours after transfection, Fas mRNA expression in hepatocytes was reduced by Fas siRNA (0.81 ⫾ 0.27
1595 Table 1. Percentage of Apoptotic Transplanted Hepatocytes and Splenic ALT Levels Groups
TUNEL-Positive Hepatocytes (%)
Splenic ALT Levels (IU/g)
Fas siRNA GFP siRNA Mock-transfected
6 ⫾ 3* 12 ⫾ 5 10 ⫾ 3
38.2 ⫾ 10.6* 21.3 ⫾ 8.4 18.5 ⫾ 5.9
*P⬍.05 as compared to mock-transfected group.
times -actin) as compared with mock transfection (1.19 ⫾ 0.54 times -actin, P ⬍ .05) and control GFP siRNA transfection (1.43 ⫾ 0.72 times -actin, P ⬍ .05). Additionally, Fas siRNA transfection reduced the level of Fas receptor in hepatocytes (mean fluorescence intensity [MFI]: 11.6 ⫾ 5.4) determined by cytometric analysis, as compared with mock transfection (MFI: 33.2 ⫾ 8.3, P ⬍ .05) and control GFP siRNA transfection (MFI: 36.5 ⫾ 7.9, P ⬍ .05). Apoptosis and Survival of Transplanted Hepatocytes
As shown in Table 1, Fas siRNA transfection reduced the percentage of TUNEL-positive hepatocytes in the spleens of allogeneic recipients (P ⬍ .05), associated with a higher survival rate of allogeneic hepatocytes in recipient spleens as determined by ALT levels (P ⬍ .05). However, GFP siRNA transfection did not affect the apoptotic percentage of allogenic hepatocytes or ALT levels (P ⬎ .05). DISCUSSION
Fas silencing by RNA interference reduces apoptosis and increases survival of allogenic transplanted hepatocytes and thereby proffers the possibility to inhibit acute rejection after hepatocyte transplantation. REFERENCES 1. Hannon GJ: Nature 418:224, 2002 2. Song E, Lee SK, Wang J, et al: Nat Med 9:doi:10.1038/nm828, 2003 3. MacDonald G, Shi L, Velde CV, et al: J Exp Med 189:131, 1999 4. Song E, Chen J, Lutz J, et al: Transplant Proc 33:650, 2001