Secretin stimulates biliary cell proliferation by regulating expression of microRNA 125b and microRNA let7a in mice

Abstract

Background & aims: Proliferating cholangiocytes secrete and respond to neuroendocrine hormones, including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice.

Methods: Cholestasis was induced in secretin knockout (Sct(-/-)) and wild-type (control) mice by bile duct ligation (BDL). At days 3 and 7 after BDL, control and Sct(-/-) mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected. Immunohistochemical, immunoblot, luciferase reporter, and real-time polymerase chain reaction assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells and in serum and bile.

Results: Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile after BDL in control mice. BDL Sct(-/-) mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor (VEGF) A and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA, and those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue.

Conclusions: After liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in up-regulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases.

Keywords: Biliary Epithelium; Gastrointestinal Hormones; Heterogeneity; cAMP.

Figure 1

[A] Large normal cholangiocytes (red arrows) express secretin. The expression of secretin increased in large ducts (red arrows) from BDL WT mice (see Table 1). Orig. magn., x20. [B–C] The expression of secretin increased in large cholangiocytes and S cells from BDL compared to normal mice. Data are mean ± SEM of four real-time PCR reactions and immunoblots. *p<0.05 vs. normal WT mice. [D] In BDL WT mice, there was increased large IBDM compared to control mice (green arrows). Knockout of the Sct gene reduced large IBDM compared to BDL WT mice (green arrows). In Sct^−/− BDL mice there was increased small IBDM compared to BDL WT mice (red arrowheads, see Table 1). Orig. magn., x20. [E] In mice treated in vivo with microRNA 125b or microRNA let7a Vivo-Morpholinos, there was increased IBDM compared to control mice. *p<0.05 vs. mice treated with mismatched Morpholinos.

Figure 2

[A] In large ducts, the expression of VEGF-A/C and NGF increased following BDL and decreased in SEC^−/− BDL compared to controls. Orig. magn., x40. [B] In large cholangiocytes from SEC^−/− BDL mice there was decreased expression for PCNA, VEGF-A/C and NGF compared to BDL cholangiocytes. Data are mean ± SEM of four experiments. *p<0.05 vs. large BDL cholangiocytes. [C] The expression of microRNA 125b and microRNA let7a was lower in BDL compared to normal cholangiocytes. In cholangiocytes from SEC^−/− BDL mice there was enhanced expression of microRNA 125b and microRNA let7a compared to BDL WT cholangiocytes. Data are mean ± SEM of four experiments. *p<0.05 vs. normal cholangiocytes. #p<0.05 vs. large cholangiocytes from WT BDL mice. [D–E] Secretin (both in vivo and in vitro) decreased expression of microRNA 125b and microRNA let7a in cholangiocytes. Data are mean ± SEM of four experiments performed in purified cholangiocytes or cholangiocyte lines. [D] *p<0.05 vs. large cholangiocytes from saline-treated normal mice. [E] *p<0.05 vs. the corresponding basal value.

Figure 3

Secretin increased the proliferation of [A] non-transfected, [B] vector-transfected and secretin shRNA large cholangiocytes and [C] HiBEpiC compared to the cell lines treated with BSA (basal). Data are mean ± SEM of six MTS assays.

Figure 4

[A] In large secretin-shRNA transfected cholangiocytes, there was decreased secretin expression and secretin secretion compared to control cholangiocytes (*p<0.05). Data are mean ± SEM of three real-time PCR reactions, three immunoblots and seven evaluations by ELISA kits. [B–C] In secretin-shRNA transfected cholangiocytes, there was reduced proliferation and expression of PCNA, VEGF-A/C and NGF and [D–E] increased expression of microRNA 125b and microRNA let7a in different transfection time points compared to control vector-transfected cholangiocytes. Data are mean ± SEM of four real-time PCR reactions. *p<0.05 vs. the corresponding value of control vector-transfected cholangiocytes.

Figure 5

Effect of downregulation or overexpression of microRNA 125b and microRNA let7a (Suppl. Figure 7A–B) on biliary proliferation and expression of PCNA, VEGF-A and NGF. [A, B, C] The increase in proliferation occurred in cholangiocytes after incubation with anti-microRNA 125b or anti-microRNA let7a inhibitors. [B, C] Increased expression of VEGF-A occurred in large cholangiocytes after incubation with anti-microRNA 125b inhibitors, whereas enhanced NGF biliary expression was observed when cholangiocytes were treated with anti-microRNA let7a inhibitors compared to control. Following overexpression of microRNA 125b or microRNA let7a (Suppl. Figure 7B), there was reduced biliary proliferation [B, D]. Following overexpression of microRNA 125b, there was reduced VEGF-A expression, whereas reduced NGF expression was observed following overexpression of microRNA let7a in cholangiocytes [B, D). Data are mean ± SEM of four real-time PCR reactions. *p<0.05 vs. controls.