ORIGINAL ARTICLE: HEPATOLOGY
miR-222 Overexpression May Contribute to Liver Fibrosis in Biliary Atresia by Targeting PPP2R2A
Rui Dong, yYijie Zheng, Gong Chen, Rui Zhao, Zhijian Zhou, and Shan Zheng
ABSTRACT Objective: Biliary atresia (BA) is a devastating liver disease in infants. Progressive hepatic fibrosis is often observed in postoperative patients with BA even after a successful Kasai portoenterostomy procedure. MicroRNA222 (miRNA) has been linked to the activation of stellate cells and the progression of liver fibrosis. Methods: In this study, the miR-222 expression profile in BA and infants with anicteric choledochal cyst (CC) was determined. The functional effect of miR-222 inhibition on the growth of the human hepatic stellate cell line LX-2 was also evaluated. The downstream signaling pathways and target of miR-222 were determined by coupling gene expression profiling and pathway analysis and by in silico prediction, respectively. In addition, we demonstrated miR-222 overexpression in patients with BA compared with choledochal cyst controls. Results: Inhibition of miR-222 in the LX-2 cell line significantly decreased cell proliferation. We also identified protein phosphatase 2A subunit B as a target of miR-222. The downstream signaling pathway, Akt, was also influenced by miR-222. A consistent reduction of Akt phosphorylation and Ki67 in the LX-2 line was shown following miR-222 suppression. Conclusions: Our results show that miR-222 overexpression is common in BA and contributes to LX-2 cell proliferation by targeting protein phosphatase 2A subunit B and Akt signaling. Key Words: Akt signaling, biliary atresia, cell proliferation, miR-222, protein phosphatase 2A subunit B
(JPGN 2015;60: 84–90)
Received June 11, 2014; accepted September 15, 2014. From the Department of Pediatric Surgery, Children’s Hospital, Fudan University, and Key Laboratory of Neonatal Disease, Ministry of Health, and the yDepartment of Immunology, Shanghai Medical College, Fudan University, and Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai, China. Address correspondence and reprint requests to Shan Zheng, Department of Pediatric Surgery, Children’s Hospital, Fudan University, and Key Laboratory of Neonatal Disease, Ministry of Health, 399 Wan Yuan Road, Shanghai 201102, China (e-mail: [email protected]). Drs Dong and Zheng contributed equally to the article and are co-first authors. Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.jpgn.org). This study received financial support from the National Natural Science Foundation of China (nos. 30973139, 81370472, and 81300517), and the Science Foundation of Shanghai (nos. 11JC1401300 and 13ZR1451800). The authors report no conflicts of interest. Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000573
B
iliary atresia (BA) is the most common cause of neonatal cholestasis. This devastating disease of extrahepatic bile ducts is characterized by periductular inflammation and fibrosis. BA is associated with the progressive obliteration of bile ducts during the first few weeks of life (1,2). Even after successful Kasai portoenterostomy, progressive hepatic fibrosis is persistent in postoperative patients with BA (3). A novel family of noncoding RNAs (19–25 nucleotides in length), referred to as microRNAs (miRNAs), has been found to provide additional control in the complicated regulation of gene expression (4–6). It has been shown that miRNAs regulate more than one-third of all human genes (7). By regulating multiple target genes, miRNAs profoundly control a wide variety of signaling pathways. There is now overwhelming evidence that miRNAs regulate a variety of biological processes, including cell development, proliferation, differentiation, and apoptosis. Although attempts have been made to identify important genes and pathways that contribute to the tumorigenesis of BA, knowledge of genomic aberrations associated with noncoding genes, such as miRNAs, and their contributions to BA are limited. We previously found that miR-222 may modulate liver fibrosis in a murine model of BA (8). In the present study, we detected the expression miR-222. Furthermore, we determined a functional role for miR-222 in the proliferation of LX-2 cells, which was in agreement with the associated clinicopathological features. Our studies showed that Akt signaling was the major pathway influenced by miR-222 following miR-222 inhibition. Using a computational algorithm, we predicted the protein phosphatase 2A subunit B (PPP2R2A) gene as a cellular target, which is a component of protein phosphatase 2A (PP2A). Hepatic lobule localization of positive immunostaining for pAkt (phosphorylated RAC-a-serine/threonine-protein kinase) Ki67, and PPP2R2A was assessed by immunohistochemistry (IHC). Our novel findings highlight a new pathway for the regulation of Akt signaling in BA and provide an important target for the diagnosis of patients with BA.
METHODS Patients The study group consisted of 13 infants with BA and 11 infants with anicteric choledochal cyst (CC). Infants diagnosed as having BA or CC were included in the study based on clinical, cholangiogram, and histological findings. The control group comprised patients with CC with normal liver function and no history of immune-mediated diseases. Liver biopsy tissues were obtained from patients with type III BA at the time of surgery (Kasai). Study participants were prospectively recruited from the Children’s Hospital of Fudan University. Clinical data for infants with BA were obtained retrospectively from clinical files (Table 1). The ethics committee at the Children’s Hospital of Fudan University approved
84 JPGN Volume 60, Number 1, January 2015 Copyright 2014 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
JPGN
Volume 60, Number 1, January 2015
Contribution of miR-222 in Liver Fibrosis in Biliary Atresia
TABLE 1. Distribution of study subjects and the liver function tests in infants with BA and CC
Age, days Male Female Diagnosis type ALP, IU/L ALT, IU/L AST, IU/L DBIL, mmol/L TBIL, mmol/L GGT, IU/L TBA, mmol/L
BA
CC
P
70.6 3.9 7 6 III1 685.0 72.1 131.1 21.8 187.0 28.8 118.4 9.2 146.0 12.5 585.3 101.0 114.3 9.2
95.9 8.5 6 5 I2 132.9 23.9 25.0 1.9 17.9 2.6 3.1 0.4 10.1 0.9 27.3 3.9 4.2 0.6
0.28 N/A N/A N/A
miR-222 Overexpression May Contribute to Liver Fibrosis in Biliary Atresia by Targeting PPP2R2A
Rui Dong, yYijie Zheng, Gong Chen, Rui Zhao, Zhijian Zhou, and Shan Zheng
ABSTRACT Objective: Biliary atresia (BA) is a devastating liver disease in infants. Progressive hepatic fibrosis is often observed in postoperative patients with BA even after a successful Kasai portoenterostomy procedure. MicroRNA222 (miRNA) has been linked to the activation of stellate cells and the progression of liver fibrosis. Methods: In this study, the miR-222 expression profile in BA and infants with anicteric choledochal cyst (CC) was determined. The functional effect of miR-222 inhibition on the growth of the human hepatic stellate cell line LX-2 was also evaluated. The downstream signaling pathways and target of miR-222 were determined by coupling gene expression profiling and pathway analysis and by in silico prediction, respectively. In addition, we demonstrated miR-222 overexpression in patients with BA compared with choledochal cyst controls. Results: Inhibition of miR-222 in the LX-2 cell line significantly decreased cell proliferation. We also identified protein phosphatase 2A subunit B as a target of miR-222. The downstream signaling pathway, Akt, was also influenced by miR-222. A consistent reduction of Akt phosphorylation and Ki67 in the LX-2 line was shown following miR-222 suppression. Conclusions: Our results show that miR-222 overexpression is common in BA and contributes to LX-2 cell proliferation by targeting protein phosphatase 2A subunit B and Akt signaling. Key Words: Akt signaling, biliary atresia, cell proliferation, miR-222, protein phosphatase 2A subunit B
(JPGN 2015;60: 84–90)
Received June 11, 2014; accepted September 15, 2014. From the Department of Pediatric Surgery, Children’s Hospital, Fudan University, and Key Laboratory of Neonatal Disease, Ministry of Health, and the yDepartment of Immunology, Shanghai Medical College, Fudan University, and Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai, China. Address correspondence and reprint requests to Shan Zheng, Department of Pediatric Surgery, Children’s Hospital, Fudan University, and Key Laboratory of Neonatal Disease, Ministry of Health, 399 Wan Yuan Road, Shanghai 201102, China (e-mail: [email protected]). Drs Dong and Zheng contributed equally to the article and are co-first authors. Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.jpgn.org). This study received financial support from the National Natural Science Foundation of China (nos. 30973139, 81370472, and 81300517), and the Science Foundation of Shanghai (nos. 11JC1401300 and 13ZR1451800). The authors report no conflicts of interest. Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000573
B
iliary atresia (BA) is the most common cause of neonatal cholestasis. This devastating disease of extrahepatic bile ducts is characterized by periductular inflammation and fibrosis. BA is associated with the progressive obliteration of bile ducts during the first few weeks of life (1,2). Even after successful Kasai portoenterostomy, progressive hepatic fibrosis is persistent in postoperative patients with BA (3). A novel family of noncoding RNAs (19–25 nucleotides in length), referred to as microRNAs (miRNAs), has been found to provide additional control in the complicated regulation of gene expression (4–6). It has been shown that miRNAs regulate more than one-third of all human genes (7). By regulating multiple target genes, miRNAs profoundly control a wide variety of signaling pathways. There is now overwhelming evidence that miRNAs regulate a variety of biological processes, including cell development, proliferation, differentiation, and apoptosis. Although attempts have been made to identify important genes and pathways that contribute to the tumorigenesis of BA, knowledge of genomic aberrations associated with noncoding genes, such as miRNAs, and their contributions to BA are limited. We previously found that miR-222 may modulate liver fibrosis in a murine model of BA (8). In the present study, we detected the expression miR-222. Furthermore, we determined a functional role for miR-222 in the proliferation of LX-2 cells, which was in agreement with the associated clinicopathological features. Our studies showed that Akt signaling was the major pathway influenced by miR-222 following miR-222 inhibition. Using a computational algorithm, we predicted the protein phosphatase 2A subunit B (PPP2R2A) gene as a cellular target, which is a component of protein phosphatase 2A (PP2A). Hepatic lobule localization of positive immunostaining for pAkt (phosphorylated RAC-a-serine/threonine-protein kinase) Ki67, and PPP2R2A was assessed by immunohistochemistry (IHC). Our novel findings highlight a new pathway for the regulation of Akt signaling in BA and provide an important target for the diagnosis of patients with BA.
METHODS Patients The study group consisted of 13 infants with BA and 11 infants with anicteric choledochal cyst (CC). Infants diagnosed as having BA or CC were included in the study based on clinical, cholangiogram, and histological findings. The control group comprised patients with CC with normal liver function and no history of immune-mediated diseases. Liver biopsy tissues were obtained from patients with type III BA at the time of surgery (Kasai). Study participants were prospectively recruited from the Children’s Hospital of Fudan University. Clinical data for infants with BA were obtained retrospectively from clinical files (Table 1). The ethics committee at the Children’s Hospital of Fudan University approved
84 JPGN Volume 60, Number 1, January 2015 Copyright 2014 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
JPGN
Volume 60, Number 1, January 2015
Contribution of miR-222 in Liver Fibrosis in Biliary Atresia
TABLE 1. Distribution of study subjects and the liver function tests in infants with BA and CC
Age, days Male Female Diagnosis type ALP, IU/L ALT, IU/L AST, IU/L DBIL, mmol/L TBIL, mmol/L GGT, IU/L TBA, mmol/L
BA
CC
P
70.6 3.9 7 6 III1 685.0 72.1 131.1 21.8 187.0 28.8 118.4 9.2 146.0 12.5 585.3 101.0 114.3 9.2
95.9 8.5 6 5 I2 132.9 23.9 25.0 1.9 17.9 2.6 3.1 0.4 10.1 0.9 27.3 3.9 4.2 0.6
0.28 N/A N/A N/A
