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Expression and localization of sterile alpha motif domain containing 5 is associated with cell type and malignancy of biliary tree

View Article: PubMed Central - PubMed

ABSTRACT

Cholangiocarcinoma (CC) is a type of relatively rare neoplasm in adenocarcinoma. The characteristics of CCs as well as biliary epithelial cells are heterogeneous at the different portion of the biliary tree. There are two candidate stem/progenitor cells of the biliary tree, i.e., biliary tree stem/progenitor cell (BTSC) at the peribiliary gland (PBG) of large bile ducts and liver stem/progenitor cell (LPC) at the canals of Hering of peripheral small bile duct. Although previous reports suggest that intrahepatic CC (ICC) can arise from such stem/progenitor cells, the characteristic difference between BTSC and LPC in pathological process needs further investigation, and the etiology of CC remains poorly understood. Here we show that Sterile alpha motif domain containing 5 (SAMD5) is exclusively expressed in PBGs of large bile ducts in normal mice. Using a mouse model of cholestatic liver disease, we demonstrated that SAMD5 expression was upregulated in the large bile duct at the hepatic hilum, the extrahepatic bile duct and PBGs, but not in proliferating intrahepatic ductules, suggesting that SAMD5 is expressed in BTSC but not LPC. Intriguingly, human ICCs and extrahepatic CCs exhibited striking nuclear localization of SAMD5 while the normal hilar large bile duct displayed slight-to-moderate expression in cytoplasm. In vitro experiments using siRNA for SAMD5 revealed that SAMD5 expression was associated with the cell cycle regulation of CC cell lines. Conclusion: SAMD5 is a novel marker for PBG but not LPC in mice. In humans, the expression and location of SAMD5 could become a promising diagnostic marker for the cell type as well as malignancy of bile ducts and CCs.

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Relationship between SAMD5 expression and cell cycle in CC cell line.(A) Real-time RT-PCR analysis of SAMD5 mRNA in RBE cell line after 96 hours of knockdown using siRNA. n = 4 per each group. (B) Examination of RBE cell proliferation by WST-1 assay. n = 8 per each group. (C) Cell cycle analysis of RBE cell line by FACS. The knockdown of SAMD5 in RBE cell line resulted in significant increase of cell population at S and M/G2 phase compared to the control. n = 3 per each group. (D) Real-time RT-PCR analysis of SAMD5 mRNA in HuH28 cell line after 96 hours of overexpression. (E) Examination of HuH28 cell proliferation by WST-1 assay. n = 8 per each group. Data are mean ± standard error. *P <0.05; **P <0.01; ***P <0.001.
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pone.0175355.g006: Relationship between SAMD5 expression and cell cycle in CC cell line.(A) Real-time RT-PCR analysis of SAMD5 mRNA in RBE cell line after 96 hours of knockdown using siRNA. n = 4 per each group. (B) Examination of RBE cell proliferation by WST-1 assay. n = 8 per each group. (C) Cell cycle analysis of RBE cell line by FACS. The knockdown of SAMD5 in RBE cell line resulted in significant increase of cell population at S and M/G2 phase compared to the control. n = 3 per each group. (D) Real-time RT-PCR analysis of SAMD5 mRNA in HuH28 cell line after 96 hours of overexpression. (E) Examination of HuH28 cell proliferation by WST-1 assay. n = 8 per each group. Data are mean ± standard error. *P <0.05; **P <0.01; ***P <0.001.

Mentions: It has been reported previously that the SAM domain of SAMD4B regulates transcriptional activity of cell cycle-related genes such as AP-1, p53 and p21 [35]. Considering that SAMD5 was also localized in the nucleus of CCs, we hypothesized that SAMD5 might be involved in the cell cycle regulation of CCs. To address the hypothesis, we knocked down SAMD5 mRNA in RBE cell line using siRNAs. We tested three distinct siRNA sequences for SAMD5 and evaluated their knockdown efficiency by quantitative RT-PCR analysis 48 hours after lipofection. Among them, siRNA #1 worked most efficiently (S5A Fig) and decreased SAMD5 mRNA by 94% even 96 hours after lipofection (Fig 6A). To investigate the role of SAMD5 in the growth of RBE cell line, we performed WST-1 and FACS analysis 96 hours after knockdown of SAMD5. The WST-1 assay revealed that knockdown of SAMD5 accelerated the proliferation of RBE (Fig 6B and S5B Fig). Consistently, FACS analysis demonstrated that knockdown of SAMD5 significantly increased the cell population at S and G2/M phase compared to control siRNA (Fig 6C).


Expression and localization of sterile alpha motif domain containing 5 is associated with cell type and malignancy of biliary tree
Relationship between SAMD5 expression and cell cycle in CC cell line.(A) Real-time RT-PCR analysis of SAMD5 mRNA in RBE cell line after 96 hours of knockdown using siRNA. n = 4 per each group. (B) Examination of RBE cell proliferation by WST-1 assay. n = 8 per each group. (C) Cell cycle analysis of RBE cell line by FACS. The knockdown of SAMD5 in RBE cell line resulted in significant increase of cell population at S and M/G2 phase compared to the control. n = 3 per each group. (D) Real-time RT-PCR analysis of SAMD5 mRNA in HuH28 cell line after 96 hours of overexpression. (E) Examination of HuH28 cell proliferation by WST-1 assay. n = 8 per each group. Data are mean ± standard error. *P <0.05; **P <0.01; ***P <0.001.
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pone.0175355.g006: Relationship between SAMD5 expression and cell cycle in CC cell line.(A) Real-time RT-PCR analysis of SAMD5 mRNA in RBE cell line after 96 hours of knockdown using siRNA. n = 4 per each group. (B) Examination of RBE cell proliferation by WST-1 assay. n = 8 per each group. (C) Cell cycle analysis of RBE cell line by FACS. The knockdown of SAMD5 in RBE cell line resulted in significant increase of cell population at S and M/G2 phase compared to the control. n = 3 per each group. (D) Real-time RT-PCR analysis of SAMD5 mRNA in HuH28 cell line after 96 hours of overexpression. (E) Examination of HuH28 cell proliferation by WST-1 assay. n = 8 per each group. Data are mean ± standard error. *P <0.05; **P <0.01; ***P <0.001.
Mentions: It has been reported previously that the SAM domain of SAMD4B regulates transcriptional activity of cell cycle-related genes such as AP-1, p53 and p21 [35]. Considering that SAMD5 was also localized in the nucleus of CCs, we hypothesized that SAMD5 might be involved in the cell cycle regulation of CCs. To address the hypothesis, we knocked down SAMD5 mRNA in RBE cell line using siRNAs. We tested three distinct siRNA sequences for SAMD5 and evaluated their knockdown efficiency by quantitative RT-PCR analysis 48 hours after lipofection. Among them, siRNA #1 worked most efficiently (S5A Fig) and decreased SAMD5 mRNA by 94% even 96 hours after lipofection (Fig 6A). To investigate the role of SAMD5 in the growth of RBE cell line, we performed WST-1 and FACS analysis 96 hours after knockdown of SAMD5. The WST-1 assay revealed that knockdown of SAMD5 accelerated the proliferation of RBE (Fig 6B and S5B Fig). Consistently, FACS analysis demonstrated that knockdown of SAMD5 significantly increased the cell population at S and G2/M phase compared to control siRNA (Fig 6C).

View Article: PubMed Central - PubMed

ABSTRACT

Cholangiocarcinoma (CC) is a type of relatively rare neoplasm in adenocarcinoma. The characteristics of CCs as well as biliary epithelial cells are heterogeneous at the different portion of the biliary tree. There are two candidate stem/progenitor cells of the biliary tree, i.e., biliary tree stem/progenitor cell (BTSC) at the peribiliary gland (PBG) of large bile ducts and liver stem/progenitor cell (LPC) at the canals of Hering of peripheral small bile duct. Although previous reports suggest that intrahepatic CC (ICC) can arise from such stem/progenitor cells, the characteristic difference between BTSC and LPC in pathological process needs further investigation, and the etiology of CC remains poorly understood. Here we show that Sterile alpha motif domain containing 5 (SAMD5) is exclusively expressed in PBGs of large bile ducts in normal mice. Using a mouse model of cholestatic liver disease, we demonstrated that SAMD5 expression was upregulated in the large bile duct at the hepatic hilum, the extrahepatic bile duct and PBGs, but not in proliferating intrahepatic ductules, suggesting that SAMD5 is expressed in BTSC but not LPC. Intriguingly, human ICCs and extrahepatic CCs exhibited striking nuclear localization of SAMD5 while the normal hilar large bile duct displayed slight-to-moderate expression in cytoplasm. In vitro experiments using siRNA for SAMD5 revealed that SAMD5 expression was associated with the cell cycle regulation of CC cell lines. Conclusion: SAMD5 is a novel marker for PBG but not LPC in mice. In humans, the expression and location of SAMD5 could become a promising diagnostic marker for the cell type as well as malignancy of bile ducts and CCs.

No MeSH data available.


Related in: MedlinePlus