MiR-200b-3p was upregulated in human cholestatic liver tissues.
To determine whether miRNAs play any roles in human obstructive cholestasis, we initially performed microarray analysis using liver samples from 3 cholestatic patients and 3 control patients. We found that miR-200b-3p expression was increased in the cholestatic patients along with miR-363-5p and miR-3609, while miR-4665-5p was downregulated. To verify these observations, we performed RT-PCR to measure the levels of these four miRNAs using RNA extracted from twenty-four cases of human obstructive cholestatic liver tissues and eleven cases of control liver tissues. We found that miR-200b-3p was significantly upregulated for 2 folds in cholestatic liver tissues when compared with the healthy controls (p<0.05) (Fig. 1A). In contrast, the expressions of miR-363-5p, miR-4665-5p and miR-3609 were not changed (Fig. 1B-D). Of note, previous reports indicated that the expression of these three microRNAs were significantly altered in hepatocellular carcinoma, colorectal cancer, pancreatic cancer, etc[26–29]. Together, these findings indicate that the altered expression of miR-200b-3p is specific to obstructive cholestasis in humans.
Elevated levels of miR-200b-3p is associated with lower serum TBA in cholestatic patients.
To understand the functional role of miR-200b-3p in human obstructive cholestasis, we first performed correlation analysis of miR-200b-3p expression with liver function indexes in all patients. We found that miR-200b-3p was negatively correlated with serum total bile acids (TBA) in some extent, especially in patients with high expression of miR-200b-3p. In order to further determine whether miR-200b-3p was correlated with TBA, cholestatic patients were divided into two groups, i.e. miR-200b-3p low expression group and miR-200b-3p high expression group, where miR-200b-3p high expression group were significantly higher that the low expression group and the health controls (p<0.05) (Fig. 2A). Results showed that serum TBA was significantly lower in miR-200b-3p high expression group (p<0.05) than miR-200b-3p low group (Fig. 2B), whereas there were no significant differences in serum bilirubin between these two groups (Fig. 2C). To explore whether miR-200b-3p was correlated with liver function indexes, correlation analysis was performed. Results showed that serum TBA was negatively correlated with miR-200b-3p levels in miR-200b-3p high expression group (p<0.05) (Fig. 2D), while no significant correlation had been found with TBA in miR-200b-3p low expression group and TBIL in both groups (Fig. 2E-G). These findings indicated that miR-200b-3p was associated with lower serum TBA in cholestatic patients, which indicated that miR-200b-3p might play some roles in regulating bile acid homeostasis in human obstructive cholestatic patients. However, the mechanism remained unknown.
LRH-1 and CYP8B1 was upregulated, while CYP7A1 was downregulated in human cholestatic liver tissues.
To investigate how elevated miR-200b-3p could lead to lower serum TBA in our patients, we asked whether miR-200-3p regulated the expression of genes involved in bile acid synthesis and transport as well as nuclear receptors that regulate the expression of these enzymes and transporters. Computer software analysis (from www.targetscan.org) indicates that LRH-1 may be one of the downstream target genes of miR-200-3p. To determine whether miR-200-3p regulates LRH-1 expression in the liver, we first measured the mRNA expression of LRH-1 and its targets CYP7A1 and CYP8B1 in these cholestatic livers using RT-PCR. As shown in Fig. 3, LRH-1 and CYP8B1 mRNA levels were significantly upregulated in cholestatic liver tissues, while the levels of LRH-1 and CYP8B1 mRNA in miR-200b-3p low expression group were much higher than these in control group and miR-200b-3p high expression group (p<0.05) (Fig. 3A,B). Furthermore, CYP7A1 mRNA levels in miR-200b-3p high expression group were much lower than these in control group (p<0.05) (Fig. 3C). These results indicated that miR-200b-3p might repress the expressions of LRH-1, CYP8B1 and CYP7A1. However, whether these gene expressions were correlated with miR-200b-3p remained unknown.
MiR-200b-3p was negatively correlated with LRH-1 and CYP8B1 in cholestatic patients with miR-200b-3p high expression.
To examine the relationship between miR-200b-3p and these bile acid-synthetic genes, we performed correlation analysis between the levels of miR-200b-3p and LRH-1, CYP8B1 and CYP7A1 in cholestatic patients. As shown in Fig. 4B,D, LRH-1 and CYP8B1 were negatively correlated with miR-200b-3p in patients with miR-200b-3p high expression (p<0.05) whereas the mRNA expression of CYP7A1 was not correlated with miR-200b-3p level (Fig. 4A,C), despite their levels were lower in these patients. These results indicated that MiR-200b-3p was negatively correlated with LRH-1 and CYP8B1 in cholestatic patients with miR-200b-3p high expression, suggesting that miR-200b-3p may directly regulates LRH-1 expression.
LRH-1, CYP7A1 and CYP8B1 were significantly repressed after miR-200b-3p mimic transfection in HepG2 cells.
To determine whether miR-200b-3p directly control the expression of LRH-1 and its targets of bile acid-synthetic enzyme, we transfected miR-200b-3p mimic into HepG2 cells. As shown in Fig. 5A, the expression of MiR-200b-3p was significantly increased compared to the un-transfected cells and negative control (p<0.05). The mRNA expressions of CYP7A1 and CYP8B1 were significantly repressed after miR-200b-3p mimic transfection (p<0.05), while no significant difference had been found in CYP7B1, CYP27A1 and CYP3A4, compared to mock and miR-NC (Fig. 5B). Furthermore, we found that LRH-1 was also significantly repressed(p<0.05), while there were no significant differences in other nuclear receptors, such as FXR, HNF4α, HNF1α, SHP (Fig. 5C). To further explore the functions of miR-200b-3p, detoxification enzymes, such as GSTM1-4, GSTA1-4, CYP3A4, UGT2B4, UGT2B7 and SULT2A1 were also detected using RT-PCR. However, no significant differences had been found in these enzymes (Fig. 5D-F). Membrane transport protein MRP3 was significantly repressed (p<0.05), while no significant differences had been found in MRP2, OSTα and OSTβ (Fig. 5G). Western blot results showed the expressions of LRH-1, CYP7A1, CYP8B1 and MRP3 were significantly repressed after miR-200b-3p mimics transfection (p<0.05) (Fig. 5H,I). These results indicated that miR-200b-3p repressed the expressions of LRH-1, CYP7A1, CYP8B1 and MRP3. However, the detail mechanism remained unclear. Further studies showed that LRH-1 promoted MRP3 expression[30] and facilitated the expressions of CYP7A1 and CYP8B1[10–13]. Therefore, we assumed that miR-200b-3p repressed expressions of CYP7A1, CYP8B1 and MRP3 through repressing LRH-1.
MiR-200b-3p repressed CYP7A1 and CYP8B1 levels through binding with LRH-1.
LRH-1 plays an important role in regulating bile acid homeostasis through promoting CYP7A1 and CYP8B1 expression[10–13], to further study the mechanism, luciferase reporter gene assay was carried out to examine whether LRH-1 was the direct target gene of miR-200b-3p. The potential binding sequences of miR-200b-3p and LRH-1 was predicted from www.targetscan.org (Fig. 6A). And pmirGLO-basic vectors containing the sequences in 3'UTR region of LRH-1 and its truncated mutants were generated, which were named as pmirGLO-LRH-1-wt and pmirGLO-LRH-1-mut. After post-transfection with pmirGLO vectors for 24 h and miR-200b-3p mimic transfection for 24 h, relative luciferase activities were analyzed. Results showed that cotransfection of miR-200b-3p mimic and pmirGLO-LRH-1-wt luciferase construct into HepG2 cells led to a marked inhibition of luciferase activity by 40–45% compared with controls, which was reversed after the mutation of pmirGLO-LRH-1-wt sequence in pmirGLO-LRH-1-mut (p<0.01) (Fig. 6B). Together, these results indicated that miR-200b-3p could directly bind with LRH-1, therefore, repressing the expressions of CYP7A1 and CYP8B1.