miR-26a regulates mouse hepatocyte proliferation via directly targeting the 3’ untranslated region of CCND2 and CCNE2

Jian Zhou, Wei-Qiang Ju, Xiao-Peng Yuan, Xiao-Feng Zhu, Dong-Ping Wang and Xiao-Shun HeGuangzhou, China

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miR-26a regulates mouse hepatocyte proliferation via directly targeting the 3’ untranslated region of CCND2 and CCNE2

Jian Zhou, Wei-Qiang Ju, Xiao-Peng Yuan, Xiao-Feng Zhu, Dong-Ping Wang and Xiao-Shun He
Guangzhou, China

Author Affiliations: Organ Transplant Center, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China (Zhou J, Ju WQ, Yuan XP, Zhu XF, Wang DP and He XS)

? 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

Published online May 21, 2015.

BACKGROUND: The deficiency of liver regeneration needs to be addressed in the fields of liver surgery, split liver transplantation and living donor liver transplantation. Researches of microRNAs would broaden our understandings on the mechanisms of various diseases. Our previous research confirmed that miR-26a regulated liver regeneration in mice; however, the relationship between miR-26a and its target, directly or indirectly, remains unclear. Therefore, the present study further investigated the mechanism of miR-26a in regulating mouse hepatocyte proliferation.

METHODS: An established mouse liver cell line, Nctc-1469, was transfected with Ad5-miR-26a-EGFP, Ad5-anti-miR-26a-EGFP or Ad5-EGFP vector. Cell proliferation was assessed by MTS, cell apoptosis and cell cycle by flow cytometry, and gene expression by Western blotting and quantitative real-time PCR. Dual-luciferase reporter assays were used to test targets of miR-26a.

RESULTS: Compared with the Ad5-EGFP group, Ad5-antimiR-26a-EGFP down-regulated miR-26a and increased proliferation of hepatocytes, with more cells entering the G1 phase of cell cycle (82.70%±1.45% vs 75.80%±3.92%), and decreased apoptosis (5.50%±0.35% vs 6.73%±0.42%). CCND2 and CCNE2 were the direct targeted genes of miR-26a. miR-26a downregulation up-regulated CCND2 and CCNE2 expressions and down-regulated p53 expression in Nctc-1469 cells. On the contrary, miR-26a over-expression showed the opposite results.

CONCLUSIONS: miR-26a regulated mouse hepatocyte proliferation by directly targeting the 3’ untranslated regions of cyclin D2/cyclin E2; miR-26a also regulated p53-mediated apoptosis. Our data suggested that miR-26a may be a promising regulator in liver regeneration.

(Hepatobiliary Pancreat Dis Int 2016;15:65-72)

KEY WORDS:microRNA; miR-26a; gene expression; hepatocyte; proliferation; regulation

Introduction

The healthy adult liver has enormous regenerative capacity. Under normal circumstances, adult liver cells are quiescent, and divide only one to two times a year in mice. Nevertheless, adult hepatocytes have the capacity to divide many times in response to partial hepatectomy. After 70% partial hepatectomy, hepatocytes immediately enter and progress the cell cycle by a highly synchronized method, so liver regeneration complete liver reconstruction in 7 to 10 days after 70% partial hepatectomy in rodents.[1-6]Therefore, the mice are often used as experimental models to study mechanisms of liver regeneration. Although miRNAs have been displayed to post-transcriptionally regulate gene expression that orchestrate cell proliferation in various biological events, such as cancer, their roles in liver regeneration are still unclear.

miRNAs regulate a plenty of biological events, including cell differentiation, proliferation, apoptosis, metabolism and even carcinogenesis.[7-11]It was reported that miR-26a was involved in numerous cell activities,[12-14]and especially presented an anti-proliferative property in human hepatocellular carcinoma.[13]Another study suggested that members of the miR-26a family inhibited tumorigenesis in B lymphoma cells.[15]Our previous study showed that miR-26a regulates liver regeneration after 70% partial hepatectomy in mice during liverregeneration,[16]but the mechanism is not completely clear. Hence it is important to study the role of miR-26a and its direct or indirect target genes in liver disease. The present study aimed to further elucidate the mechanism of miR-26a in the proliferation of mouse hepatocyte.

Methods

Vector construction

At first, anti- or pri-miR-26a sequences were individually introduced into a pShuttle-IRES-hrGFP-1 vector (Agilent Technologies, USA). After linearization with PmeI and pAdWasy-1 (Agilent Technologies, USA), the pShuttle-IRES-hrGFP-1 vector was recombined into a pAdEasy-IRES-hrGFP-1 vector. Then, a 293AD cell line (Cell Biolabs, San Diego, CA, USA)[17]was transfected with the pAdEasy-IRES-hrGFP-1 vector, and liquid supernatant including viral particles was isolated and collected. The viral particles including Ad5-anti-miR-26a-EGFP or Ad5-miR-26a-EGFP vector were established.

Cell culture and transient transfection

An established mouse liver cell line, Nctc-1469 (ATCC, Virginia, USA),[18]was obtained from the SuJi biotech company (Guangzhou, China). The Nctc-1469 cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) under a humidified atmosphere containing 5% CO2at 37 ℃. Transfections with Ad5-anti-miR-26a-EGFP (2.5×1010IU/mL), Ad5-miR-26a-EGFP (2.12×1010IU/mL) or Ad5-EGFP (4.5× 1010IU/mL) were conducted using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. All experiments repeated three times independently.

Transfection efficiency assessment

The Ad5-miR-26a-EGFP vector was diluted to different concentrations of 2.12×1010IU/mL, 2.12×108IU/mL, and 2.12×106IU/mL with PBS, respectively. Similarly, the Ad5-anti-miR-26a-EGFP vector was diluted into 2.5×1010IU/mL, 2.5×108IU/mL and 2.5×106IU/mL, respectively. Each vector was transfected to Nctc-1469 cells. Three days later, the cells were collected, and miR-26a expression was tested by qRT-PCR. All experiments repeated three times independently.

Cell proliferation by MTS assay

The Nctc-1469 cells were transfected with Ad5-miR-26a-EGFP, Ad5-anti-miR-26a-EGFP or Ad5-EGFP in the 24-well plates, and re-seeded in 96-well plates at a density of 1000 cells per well at 48 hours after transfection. At the indicated time points (24, 48, 72, 96, 120 hours) after re-seeding in 96-well plates, 10 μL MTS was added to the culture medium, and incubated for 4 hours. The absorbance at 490 nm of each sample was recorded by microplate reader (Thermo Fisher Scientific, USA). All experiments repeated three times independently.

Cell cycle analysis by flow cytometry

The Nctc-1469 cells were cultured in 6-well plates, at a density of 2×106cells per well, were transfected with Ad5-miR-26a-EGFP, Ad5-anti-miR-26a-EGFP or Ad5-EGFP. After 72 hours, the cells were collected and fixed with 70% ethanol for 30 minutes, and then washed with ice-cold PBS twice. The cells were spun down and re-suspended using RNase-containing PBS (1:100 in dilution) on ice before staining with propidium iodide and analyzed using a flow cytometer (FACSCalibur, BD, USA). All experiments repeated three times independently.

Cell apoptosis analysis by flow cytometry

The Nctc-1469 cells were cultured in 6-well plates, and were transfected with Ad5-miR-26a-EGFP, Ad5-antimiR-26a-EGFP or Ad5-EGFP. At 48, 72 and 120 hours after transfection, the cells were collected for apoptotic analysis by flow cytometry analysis. The annexin V detection kit was used to detect apoptotic cells. Data acquisition and analysis were performed using a FACSCalibur Cytometer (BD, USA). For each analysis, 1×105cells were scanned. All experiments repeated three times independently.

Western blotting analysis

Cell samples were homogenized in lysis buffer (Promega, USA), incubated for 30 minutes on ice, then centrifuged for 15 minutes at 14 000×g. All buffers were treated with a protease inhibitor cocktail (Konchem, China). Equal amounts of protein were separated on 12%-15% SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The antibodies included anti-p53 (Santa cruz, USA), and anti-GAPDH (Kangcheng, China). Immunoblots were developed using anti-rabbit-HRP secondary antibodies (Dako, CA, USA), followed by detection with immobilon Western chemilimunescent HRP substrate (Millipore, USA). GAPDH was used as a referenced gene. All experiments repeated three times independently.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from prepared liver cells with Trizol (Invitrogen, Carlsbad, CA, USA). Reagent and cDNA were synthesized according to the manufacturer’ s protocol (MBI Fermentas). qRT-PCR was performedusing a standard SYBR Green PCR Master Mix (Toyobo, Osaka, Japan), and PCR-specific amplification was performed in the Applied Biosystems (ABI7500) real-time PCR machine. The expression of genes (miR-26a, U6, p53, 18s rRNA) in all groups were calculated with the 2-ΔΔCtmethod.[19]The primers used are listed in Table. The 18s rRNA was used as referenced gene of p53, and the U6 was used as referenced gene of miR-26a. First, we compared the gene expression of the Ad5-EGFP group in mRNA level with that of the control group (without transfected). Second, we compared the gene expressions of the Ad5-miR-26a-EGFP group and Ad5-anti-miR-26a-EGFP group to that of the Ad5-EGFP group, respectively. All experiments repeated three times independently.

Dual-luciferase reporter assays

For the miRNA screen, a 293AD cell line (Cell Biolabs, San Diego, CA, USA) was seeded in 96-well plates at a density of 5000 cells per well. After 24 hours, the cells were transiently transfected with 5 ng of pRL-CMV (renilla luciferase reporter), 50 ng of either p-LUC or p-LUC-CCND2 UTR or p-LUC-CCNE2 UTR (firefly luciferase reporter), and 5 pmol of miRNA mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Firefly and renilla luciferase activities were measured 36 hours after transfection using a dual-luciferase reporter assay kit (Promega, Madison, WI, USA). Firefly luciferase activity was normalized using renilla luciferase activity. The pRL-CMV renilla luciferase reporter and small RNAs were simultaneously introduced into 293AD cells. These cells were collected at 36 hours after transfection and luciferase activity was assayed as above. All experiments repeated three times independently.

Statistical analysis

Table. Primers used in reverse transcription and quantitative realtime PCR

All data were expressed as mean±standard deviation, with at least three independent experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA). Statistical significance was assumed as P< 0.05.

Results

Transfection efficiency and dose-dependent relationship of vector in vitro

To evaluate the availability of vectors, and whether their effects were in a dose-dependent manner, we measured the miR-26a expression in Nctc-1469 mouse liver cells transfected with Ad5-EGFP, Ad5-miR-26a-EGFP or Ad5-anti-miR-26a-EGFP (Fig. 1A-C). The miR-26a expression was obviously lower after transfection with 2.5× 1010IU/mL (A1) or 2.5×108IU/mL (A2) in the Ad5-antimiR-26a-EGFP group than in the control group (cells without treatment) (0.99±0.10 vs 1.57±0.09, P<0.001; 1.35±0.14 vs 1.57±0.09, P<0.05). A significant difference in subgroup A1 was also observed in comparison with the Ad5-EGFP group (0.99±0.10 vs 1.46±0.14, P<0.001) (Fig. 1D). In contrast, a higher miR-26a expression was observed after transfection with 2.12×1010IU/mL (M1) and 2.12×108IU/mL (M2) of the Ad5-miR-26a-EGFP group compared with the control group (3.05±0.15 vs 1.57±0.09, P<0.001; 2.14±0.08 vs 1.57±0.09, P<0.001). And furthermore, similar results were shown in subgroup M1 and M2 compared with the Ad5-EGFP group (3.05±0.15 vs 1.46±0.14, P<0.001; 2.14±0.08 vs 1.46±0.14,

P<0.001) (Fig. 1E). In addition, no significant difference was seen between the Ad5-EGFP and control groups. The final transfection concentration was 2.5×1010IU/mL in the Ad5-anti-miR-26a-EGFP group and 2.12×1010IU/mL in the Ad5-miR-26a-EGFP group.

miR-26a modulates Nctc-1469 mouse liver cell growth

To assess the effect of miR-26a on mouse Nctc-1469 liver cell growth, an MTS assay was used as described in the method section. The MTS assays were performed at 24-hour intervals. Anti-miR-26a expression markedly enhanced Nctc-1469 liver cell growth at 72 hours compared with Ad5-EGFP (P<0.01, Fig. 2A); on the contrary, miR-26a over-expression significantly inhibited Nctc-1469 liver cell growth compared with Ad5-EGFP, especially at 72 hours (P<0.001, Fig. 2A). Cell cycle analysis showed that the percentage of cells in the G1 phase of cell cycle in the Ad5-anti-miR-26a-EGFP, Ad5-miR-26a-EGFP, Ad5-EGFP and control groups (without transfection) were 82.70% ± 1.45%, 67.90%±2.62%, 75.80%±3.92%, and 75.60%±3.58%, respectively, suggesting that more cells reentered G1 phase under treatment of anti-miR-26a, or were prevent-ed from entering G1 phase by miR-26a over-expression (Fig. 2B). In addition, no significant difference was observed between the Ad5-EGFP and control groups.

miR-26a modulates CCND2 and CCNE2 by directly binding their 3’ UTR

Fig. 1. The in vitro transfection efficiency. A-C: The Nctc-1469 cells were transfected with Ad5-EGFP, Ad5-miR-26a-EGFP or Ad5-antimiR-26a-EGFP; D: Expression of miR-26a after transfection with different concentrations of Ad5-anti-miR-26a-EGFP, Ad5-EGFP, or no transfection (control); E: Expression of miR-26a after transfection with different concentrations of Ad5-miR-26a-EGFP, Ad5-EGFP, or no transfection (control). *: P<0.05, ***: P<0.001.

Fig. 2. miR-26a modulates mouse liver cell growth. A: Cell proliferation was examined by MTS assay at the indicated time points, and the absorbance of MTS by each sample was recorded at 490 nm after staining; B: Cells transfected with Ad5-EGFP, Ad5-miR-26a-EGFP or Ad5-anti-miR-26a-EGFP and control group were analyzed for cell cycle analysis by flow cytometry. All data were obtained from at least three independent experiments and were shown as the mean±SD. *: P<0.05, **: P<0.01, ***: P<0.001, compared with Ad5-EGFP.

To study the mechanism through which miR-26a modulates cell cycle of proliferative phase of mouse liver cells, we analyzed putative targets of miR-26a using algorithms including miRanda, Targetscan and PicTar.[20-22]The predicted targets of miR-26a include cyclin D2 (CCND2), cyclin E2 (CCNE2), cyclin E1 (CCNE1) and cyclin-depen-dent kinase 6 (CDK6), all of which play important roles in cell cycle.[23]We constructed full-length fragments of the CCND2, CCNE2, CCNE1 and CDK6 mRNA 3’ UTR (miR-26a binding site mutants and wild-type), and immediately inserted them to the downstream of the luciferase reporter gene. The miR-26a mimic or control RNA was cotransfected with different luciferase-3’ UTR constructs into 293AD cells. The results suggested that luciferase activity had significant difference between the normal control group and miR-26a mimics group in CCND2 wild-type constructs (P<0.01), but no difference in CCND2 3’ UTR mutant constructs (Fig. 3A). Likely, luciferase activity had a significant difference between normal control group and miR-26a mimics group in CCNE2 wild-type constructs (P<0.05), but no difference in CCNE2 3’ UTR mutant 1 constructs or mutant 2 or mutant 1 and 2 (Fig. 3B). Conversely, luciferase activity had no difference between the normal control group and miR-26a mimics group in CCNE1 or CDK6 wild-type constructs or mutant constructs (Fig. 3C and D). These results suggested that miR-26a might regulate CCND2 and CCNE2 expression by directly binding target sites in the 3’ UTR.

miR-26a participates in p53 mediated apoptosis

Using flow cytometry, we demonstrated that treatment with Ad5-anti-miR-26a-EGFP for 72 hours significantly decreased apoptotic cells (5.50%±0.35%) compared with Ad5-EGFP treatment (6.73%±0.42%) (P<0.05). Conversely, a significant higher apoptotic rate was observed in the Ad5-miR-26a-EGFP group compared with the Ad5-EGFP group at 72 hours (P<0.05) (Fig. 4A-E). No statistical difference was observed between the control group (without transfection) and the Ad5-EGFP group at all time points. Next, to ascertain the cellular mechanisms underlying miR-26a-mediated apoptosis, both qRT-PCR and Western blotting were used to determine whether p53 network mediates the apoptosis or not. Interestingly, our results showed that p53 expression (in both mRNA and protein level) in the Ad5-anti-miR-26a-EGFP group was significantly decreased, while the p53 expression was significantly increased in the Ad5-miR-26a-EGFP group compared with the Ad5-EGFP group (Fig. 4F-G). Taken together, these results suggested that miR-26a might regulate apoptosis through the p53 network.

Fig. 3. CCND2 and CCNE2 are direct target genes of miR-26a. A: Luciferase activity assays of wild-type and mutant CCND2 3’ UTR luciferase reporters after co-transfection with miR-26a mimics or normal control; B: Luciferase activity assays of wild-type and mutant CCNE2 3’ UTR luciferase reporters after co-transfection with miR-26a mimics or normal control; C: Luciferase activity assays of wildtype and mutant CCNE1 3’ UTR luciferase reporters after co-transfection with miR-26a mimics or normal control; D: Luciferase activity assays of wild-type and mutant CDK6 3’ UTR luciferase reporters after co-transfection with miR-26a mimics or normal control; *: P<0.05, **: P<0.01.

Fig. 4. miR-26a regulates the apoptosis of Nctc-1469 liver cells by participating in p53 network. A-E: The frequency of apoptosis cells in Nctc-1469 liver cells transfected with Ad5- EGFP, Ad5-miR-26a-EGFP, or Ad5-anti-miR-26a-EGFP was analyzed by flow cytometry. F: mRNA expression of p53 in Nctc-1469 liver cells was assessed by qRT-PCR. G: Protein expression of p53 in Nctc-1469 liver cells, with GAPDH used as loading control. *: P<0.05, **: P<0.01, ***: P<0.001.

Discussion

The study of miRNAs has opened a new era for further understanding of the mechanisms of various diseases. miRNAs are tiny endogenous RNAs that modulate expressions of hundreds of genes simultaneously. miRNAs orchestrate gene changes in gene networks and regulate cellular functions in both plants and animals. Although potential target genes of miR-26a can be predicted by bioinformatics analysis, the true target of miR-26a in the gene network may be different from the predicted target through the bioinformatics analysis; on the other hand we do not know how miR-26a accurately regulate its target on hepatocyte proliferation. We believe that these findings in the current study could provide a new vision to better understand how miR-26a modulates its target genes on hepatocyte proliferation.

Although it was reported that several miRNAs are involved in liver regeneration,[24-26]little is known about how miRNAs modulate hepatocyte proliferation and apoptosis during liver regeneration. Interestingly, some studies showed that miR-26a is down-regulated in nasopharyngeal carcinoma and breast cancer,[27, 28]indicating that it is a crucial miRNA in cell proliferation. What’s more, miR-26a could significantly inhibit cancer cell growth in human hepatocellular carcinoma.[13]Most importantly, our previous research confirmed that miR-26a expression could be greatly decreased through transfection with anti-expression of miR-26a virus carrier, which significantly increased liver regeneration in mice. On the contrary, expression of miR-26a obviously increased through transfection with over-expression of miR-26a virus carrier, which obviously inhibited liver regeneration in mice.[16]The present study further clarified the relative mechanism that miR-26a regulates mouse hepatocyte proliferation in vitro.

Liver regeneration is an immediate process after 70% partial hepatectomy. It consists of priming phase, pro-liferation/expansion phase and termination phase.[29]Undoubtedly, active cell cycle progression is necessary for hepatocyte proliferation during liver regeneration which is regulated by cyclin expression and activation of cyclindependant kinases (CDKs).[29]As shown in cell cycle analysis, miR-26a over-expression prevents hepatocytes from entering G1 phase. What is more, CCND2, CCNE1, CCNE2 and CDK6 predicted by algorithms analysis may be the target genes of miR-26a in regulating liver regeneration. In addition, as well known, cyclins D1, D2 and D3 play a key role in cell cycle machinery, and they positively regulate cell proliferation through binding to CDK4 and CDK6; these bindings led to the phosphorylation of reteineblastoma protein and G1/S transition of the cell.[30]On the other hand, CCNE2 and CCNE1 are actually required for normal proliferation of all mammalian cell types, especially for regulating transition of quiescent cells into the progression of cell cycle.[31]In this study, we confirmed that over-expression of miR-26a inhibited the expression of CCND2 and CCNE2, in contrast, miR-26a anti-expression caused increased expressions of CCND2 and CCNE2 in vitro, indicating that CCND2 and CCNE2 are potential target genes of miR-26a. Through dual-luciferase reporter assays, we further confirmed that miR-26a regulated mouse hepatocyte proliferation by directly targeting the 3’ untranslated region of CCND2/CCNE2. This finding provided a new perspective to better understand the relationship between miR-26a and its targets.

Liver regeneration is known to involve multiple factors and pathways that result in both increased proliferation and decreased apoptosis of hepatocytes,[32, 33]while p53 network is a crucial regulator of both apoptosis and proliferation. It is well-known that p53 protein, as the “guardian of the genome”, plays a pivotal role in regulating multiple cellular processes, such as cell apoptosis.[34-38]The present study found that down-regulation of miR-26a reduced hepatocyte apoptosis in vitro. Importantly, miR-26a down-regulation resulted in the decrease of p53 protein expression. Therefore, we presume that miR-26a induces hepatocyte apoptosis by regulating the p53 signaling pathway. To test our hypothesis, we studied the effect of miR-26a over-expression on the p53 signaling pathway in Nctc-1469 cells. Our results showed that miR-26a over-expression increased cell apoptosis, and enhanced expression of p53 protein. Therefore, downregulation of miR-26a promotes hepatocyte proliferation by suppressing p53 mediated apoptosis. In addition, several studies[39-44]have confirmed that miR-26a is a potential target gene of p53, suggesting that a miR-26a-p53 positive feedback loop may exist in the proliferative phase of liver cells during liver regeneration. The balance between hepatocyte proliferation and apoptosis is crucial for appropriate remodeling of regenerated liver lobules and termination of liver regeneration.[29]

In conclusion, this study confirmed for the first time that miR-26a plays a crucial role in regulating proliferation of hepatocyte in vitro, and the mechanisms may be that miR-26a regulates mouse hepatocyte proliferation by directly targeting the 3’ untranslated region of CCND2/CCNE2 and by regulating p53-mediated apoptosis. The involvement of miR-26a in hepatocyte proliferation may lead to a potential therapeutic target in liver regeneration in the near future.

Contributors: ZJ proposed the study. ZJ, JWQ and HXS performed the research and wrote the first draft. YXP, ZXF and WDP collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. ZJ and JWQ contributed equally to this article. HXS is the guarantor.

隨著農(nóng)業(yè)供給側(cè)結(jié)構(gòu)性改革走向深入，一批特色農(nóng)產(chǎn)品區(qū)域品牌逐漸打響，五常大米、洛川蘋果、贛南臍橙、膠東白菜等都受到消費者的歡迎。但是這些品牌只在國內(nèi)有知名度，國際市場上卻屢屢受冷，比如四川泡菜就很有代表性。四川泡菜有很多優(yōu)勢：一是歷史悠久。二是有自然資源優(yōu)勢，產(chǎn)值很大。據(jù)顯示，2017年四川泡菜產(chǎn)值超330億元，約占全國比例70%。三是生產(chǎn)標準高，安全健康。為什么四川泡菜做得這么好，品牌在國外卻一直打不響？這是我國眾多優(yōu)質(zhì)農(nóng)產(chǎn)品遇到的普遍現(xiàn)象。農(nóng)產(chǎn)品要想“走出國門”，必須精心打造品牌。

Funding: This study was supported by grants from the Key Clinical Project from the Ministry of Health (159), the National Natural Science Foundation of China (30972951 and 81170448), Special Fund for Science Research by Ministry of Health (201002004), and the PhD Programs Foundation of Ministry of Education of China (20130171120076).

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received July 28, 2014

Accepted after revision March 2, 2015

Original Article / Liver

doi:10.1016/S1499-3872(15)60383-6

Corresponding Author:Xiao-Shun He, MD, PhD, Organ Transplant Center, the First Affiliated Hospital, Sun Yat-Sen University, No. 58 Zhongshan Er Road, Guangzhou 510080, China (Tel/Fax: +86-20-87306082; Email: gdtrc@126.com)