MiRNA-200b-3p represses LRH-1 expression and is associated with reduced serum bile acid in human obstructive cholestasis CURRENT POSTED

Background MicroRNAs are closely associated with liver diseases including cholestasis, but the roles of miR-200 family in cholestasis has not been determined. Liver receptor homolog-1 (LRH-1/NR5A2) plays an important role in maintaining bile acid homeostasis by regulating some genes, including CYP7A1 and CYP8B1, but the mechanistic details remain to be elucidated in human cholestasis. Methods The mRNA and or protein expressions of miR-200b-3p, LRH-1, CYP7A1 and CYP8B1 in human liver tissues (11 controls and 24 cholestasis) were analyzed using RT-PCR or Western blot. HepG2 cells was transfected with miR-200b-3p mimic to determine gene expressions. We used luciferase gene reporter assay to identify the potential response element of miR-200b-3p in LRH-1 gene. MiR-200b-3p was increased for about 2 folds in cholestatic liver tissues, which were divided into low and high expression group. Serum TBA was negatively correlated with miR-200b-3p in miR-200b-3p high expression group. Levels of LRH-1, CYP8B1 and CYP7A1 were decreased, LRH-1 and CYP8B1 were negatively correlated with miR-200b-3p in miR-200b-3p high expression group. MiR-200b-3p mimic was transfected into HepG2 cells, the levels of LRH-1, CYP7A1 and CYP8B1 were significantly repressed by 45%-55%. Bioinformatics analysis revealed the potential binding site of LRH-1 and miR-200b-3p and luciferase reporter gene assay showed that miR-200b-3p might directly bind with LRH-1. Our findings indicate that miR-200b-3p represses liver LRH-1, which in turn decreases bile acid synthesis, suggesting that miR-200b-3p may be a potential therapeutic target for cholestasis. (3) Transfection of miR-200b-3p mimic resulted in decreased expression of LRH-1, CYP8B1 and CYP7A1 in HepG2 cells. (4) Luciferase gene reporter assay identified the binding site of miR-200b-3p in LRH-1 gene, and mutation of this binding site abolished miR-200-3p effect in LRH-1 expression. Therefore, we conclude that miR-200b-3p regulates bile acid homeostasis by modulating LRH-1 expression and function in obstructive cholestasis, a novel role of miR-200b-3p in adaptive response to cholestasis. cytochrome P450 7 A 1; CYP8B1: cytochrome P450 8 1; HNF4α: hepatic nuclear factor 4 alpha; FXR: farnesoid X receptor; total ULN: limit of AST: aspartate aminotransferase;

converting cholesterol to bile acid in the liver [1,2], which is tightly regulated in response to bile acid homeostasis. CYP7A1 was found to be reduced not only in patients with cholestasis and alcoholic hepatitis, but also in bile duct ligated (BDL) mice [3,4].
SHP then binds to LRH-1 and represses it transcriptional activity, leading to decreased CYP7A1 expression [8,9]. LRH-1 is encoded by the NR5A2 gene. It is mainly expressed in liver, intestine and pancreas and it regulates the expressions of CYP7A1and CYP8B1, which are critical in maintaining the homeostasis of bile acid and cholesterol [10][11][12][13].
MicroRNAs (miRNAs) are a kind of small non-coding RNA, which contain about 22 nucleotides and play an important role in regulating the expression of genes by RNA silencing. They participate in both physiological and pathological processes. Altered expression of miRNAs has been associated with cholestatic liver diseases [14][15][16][17] both in the serum and liver tissue, including primary biliary cholangitis (PBC) and primary sclerosing cholangitis [16,18].
MiR-200b-3p is a member of miR-200 family, which is initially found associated with cancer metastasis, chemotherapeutic resistance and epithelial-mesenchymal transition (EMT) [19][20][21][22]. Recent studies revealed that miR-200b was also related with cholestatic liver injury. Xiao, et al reported that miR-200b significantly affected the development and progression of liver fibrosis [23]. Upregulation of miR-200b promoted hepatic stellate cell proliferation and migration through PI3K/Akt signaling pathway in cholestatic patients [24]. Furthermore, upregulation of miR-200 and downregulation of miR-124 contributed to cholangiocyte proliferation in the liver of patients with biliary atresia (BA) [25]. However, it is not known whether miR-200b plays any roles in obstructive cholestasis.
In this study, we sought to investigate the functional role of miR-200b-3p in cholestatic liver diseases.
We examined the expression of miR-200b-3p in human obstructive cholestatic patients and the relationship between miR-200b-3p and serum total bile acid (TBA). We found that increased expression of miR-200b-3p is associated with lower expression of LRH-1 and CYP7A1 in obstructive cholestatic patient livers. Further mechanistic studies indicate that miR-200b-3p repressed LRH-1 mRNA expression and resulting in decreased CYP7A1 expression. These findings not only reveal the functional role of miR-200b-3p in cholestatic liver diseases but also provide a therapeutic target for treating cholestatic liver injury.

Methods
Patients and liver samples collection 24 cholestatic liver samples were obtained from patients with pancreatic carcinoma, intrahepatic bile duct calculi and gallbladder cancer, and 11 control liver samples were obtained from patients suffering the resection of liver metastasis that without cholestasis. All patients had the typical symptoms of human obstructive cholestasis, including itchiness, jaundice, dark urine and so on; the level of ALP was 1.5 times higher than the upper limit of normal (ULN), and the level of GGT was 3 times higher than ULN. All patients provided written informed consent. Individuals were excluded if they were suffering with liver diseases, including autoimmune liver disease, virus hepatitis, hepatic cancers, heart dysfunction, renal dysfunction, etc. Liver samples were obtained and were immediately cut into small pieces and then stored in liquid nitrogen until usage. The general clinical characteristics and features of the control group and the patient group were shown in Table 1 and Table 2. No significant differences were found in age and sex between the two groups, however, significant differences were found in AST, ALT, ALP, GGT, TBIL, DBIL, IBIL and TBA between the two groups (p<0.05). This research was approved by the Faculty of Medicine's Ethics Committee of Southwest Hospital, in agreement with the Helsinki declaration.  and sequences were listed in Table 3. SYBR® premix Ex Taq™ II kit (Takara Biotech, Tokyo, Japan) was used to detect the mRNA levels of miR-200b and U6, and a LightCycler® 480 Probes Master (Roche, IN, USA) was used to detect the mRNA levels of CYP7A1 and GAPDH. The mRNA expressions were normalized to U6 or GAPDH and 2-ΔΔCT method was used to detect the relative gene expression. Table 3 Sequences of primers for RT-PCR GSTM2  ACCTTCCTTCCTGTTAGTG  TCAATGCTGCTCCTTCAT  GSTM3  GCTCACCTTTGTGGATTTTCTC  TGTAAGTAGGCAGCGATTTTCTC  GSTM4  ACTTTCCCAATCTGCCCTACTT  TAGCCTGGTTCTCCAAAATGTC  GSTA1  AATGGTTGAGATTGATGGGATG  GGGCTCTCTCCTTTATGTCTTTC  GSTA2  CCTTCTTCTGCCCTTTAGTCAA  GTAGTCTTGTCCGTGGCTCTTT  GSTA3  TGAAAACCAGAATCAGCAACCT  ACAACAGGCACAATCAACACTT  GSTA4  GAGTCCGTGAGATGGGTTTTAG  TTGGAACAGCAGGTGGTTAC  UGT2B4  CCTATGTGCCTGTTGTTATGTCA  AACATTTGGTAAGAGTGGGTGAG  UGT2B7  CTCTGGGGTCAATGGTCAGTA  CCACTTGTAGAGCCGAGTATTG  SULT2A1  AACAGGACACAGGAAGAACCAT  CAGTCCCCAGATACACCTTTTC  MRP2  CATTCTGCATGGCAGGACCTA  GGTGAGGAAGGCCCAAAACT  MRP3 AAAAGCAGACGGCACGACA Shanghai, China) and exposed to an X-ray film using an ECL detection system (Thermo Fisher Scientific, Shanghai, China).

Luciferase gene reporter assays
The potential binding sequences of miR-200b-3p and LRH-1 was predicted using software from www.targetscan.org, which was shown in Fig. 4A. The pmirGLO-basic vectors containing the 3'UTR region of LRH-1 and its truncated mutants were produced by TSINGKE Biological Technology (TSINGKE Biological Technology, Beijing, China), which were named as pmirGLO-LRH-1-wt and pmirGLO-LRH-1mut. pmirGLO vectors or miR-200b-3p mimic were transfected into HepG2 cells. Twenty-four hours post-transfection, luciferase activities were assayed using dual-luciferase reporter assay kit from Promega (Madison, WI, USA).

Statistical analysis
All data were analyzed by SPSS version 21.0 (SPSS Inc., Chicago IL, USA). Data were expressed as means ± SD. The methods of independent-sample t-test and one-way ANOVA were used to analyze these data, and multiple comparison between these groups was performed by using S-N-K method.
The value of p<0.05 was statistically significant.
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][27][28][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.

Discussion
In this study, we assessed the expressions and functions of miR-200b-3p in human obstructive cholestasis due to blockage of bile ducts. We had obtained the following findings.