Transcriptional Repression of CYP3A4 by Increased miR-200a-3p and miR-150-5p Promotes Steatosis in vitro

Hepatic cytochrome P450 enzyme activities correlate with non-alcoholic fatty liver disease (NAFLD) and hepatic steatosis. The decreased activity of CYP3A4, an important drug-metabolizing enzyme, is associated with the progression of NAFLD. CYP3A4 is predicted as a target gene of miR-200a-3p and miR-150-5p by MicroInspector and TargetScan algorithms analyses. Here, we found decreased CYP3A4 and increased miR-200a-3p and miR-150-5p in LO2 cells with free fatty acid (FFA)-induced steatosis. Dual-luciferase assay confirmed that both miR-200a-3p and miR-150-5p targeted the 3′-untranslated region (3′-UTR) of CYP3A4 and that such interaction was abolished by miRNA binding site mutations in 3′-UTR of CYP3A4. Using miR-200a-3p and miR-150-5p mimics and inhibitors, we further confirmed that endogenous CYP3A4 was regulated posttranscriptionally by miR-200a-3p or miR-150-5p. Moreover, miR-200a-3p and miR-150-5p inhibitors attenuated FFA-induced steatosis in LO2 cells, and such effect was dependent on CYP3Y4 expression. These results suggest that miR-200a-3p and miR-150-5p, through directly targeting 3′-UTR of CYP3A4, contribute to the development of FFA-induced steatosis.


INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) has been the most common chronic liver disease in the world for the past 30 years (Younossi et al., 2018) with an estimated prevalence of 10-40% (Younossi et al., 2016). NAFLD includes a spectrum of disease from simple liver steatosis to non-alcoholic steatohepatitis (NASH). NASH is considered as a leading indication for liver transplants in the near future, as it increases the risk of hepatocarcinoma (HCC) (Sattar et al., 2014;Diehl and Day, 2017).
Several genetic defects and environmental factors have been implicated in the pathogenesis of NAFLD. Specifically, a few studies have reported that NAFLD is associated with decreased expression and function of CYP3A4, a major member of the hepatic cytochrome P450 superfamily contributing to the metabolism of 45-60% of all drugs used in the clinical setting (Wojnowski and Kamdem, 2006;Anglicheau et al., 2007). Donato et al. (2006) observed a significantly reduced CYP3A4 activity in both human liver tissue with steatosis and fatoverloaded hepatocytes cultured in vitro. Similar results were shown in nutritionally obese mice (Yoshinari et al., 2006;Maximos et al., 2017). Compared with healthy controls, NAFLD patients showed higher plasma concentration of CYP3A4 substrate, indicating impaired CYP3A4 function in NAFLD patients (Woolsey et al., 2015). Decreased protein expression and activity of CYP3A4 were observed with NAFLD development (Fisher et al., 2009). A recent study further confirmed reduced protein level and activity of CYP3A4 in liver tissues of NAFLD patients as compared to those of controls (Jamwal et al., 2018). However, despite all these observations, the underlying mechanism regulating the expression and function of CYP3A4 in NAFLD remains unclear.
Cell Culture Model of Hepatic Steatosis in Vitro LO2 cells were provided by Stem Cell Bank, Chinese Academy of Sciences and cultured in RPMI 1640 medium supplemented with 10% FBS at 37 • C/5% CO 2 . Steatosis was induced as previously described (Feldstein et al., 2004;Ricchi et al., 2009). PA and OA were codissolved in 10% FA-free BSA prepared in H 2 O. In accordance with previous studies (Wang et al., 2014), LO2 cells were exposed to a mixture of 1 mM OA and PA (final ratio 2:1) for 24 h. After 24 h of incubation, lipid droplets were stained by BODIPY according to a previously reported protocol (Qiu and Simon, 2016).

In silico Identification of Putative miRNA Binding Sites
The 3 -UTR sequences of human CYP3A4 (GenBank sequence NM_017460) were searched for the antisense matches to individual miRNAs using MicroInspector (Rusinov et al., 2005) and Target Scan (Lewis et al., 2005).

Transfection
All transfections were performed by Lipofectamine TM 2000 according to the manufacturer's instructions.
Twenty-four hours after transfection, cells were treated with a mixture of 1 mM OA and PA (final ratio 2:1) for 24 h. To confirm the effect of miR-150-5p or miR-200a-3p inhibition or induction on CYP3A4 mRNA, total RNAs were extracted from LO2 cells with TRIzol (Life Technologies, CA, United States). Total mRNAs were reverse transcribed into cDNAs by the PrimeScript RT Reagent kit. Real-time PCR was performed by the SYBR Green PCR Master Mix using the following conditions: 95 • C for 10 min followed by 40 cycles of amplification at 95 • C for 10 s and 59 • C for 30 s. GAPDH (forward primer: AGAAGGCTGGGGCTCATTTG; reverse primer: AGGGGCCATCCACAGTCTTC) was used as an internal control to normalize CYP3A4 expression (forward primer: CCCGTTGTTCTAAAGGTTGA; reverse primer: TCTGGTGTTCTCAGGCACAG). qPCR was quantified using the formula 2 − CT and plotted as x-fold to the control.

Western Blot
Cells in six-well plates were harvested posttransfection and treatment, and whole-cell lysates were prepared with RIPA lysis buffer (Beyotime, Beijing, China) supplemented with complete protease inhibitor and phenylmethanesulfonyl fluoride (Beyotime, Beijing, China). Protein concentrations were determined with the BCA Protein Assay Kit (Beyotime, Beijing, China). Whole-cell protein (20 µg) was separated on SDS-PAGE and electrophoretically transferred onto PVDF membrane (Millipore, CA, United States). The membrane was incubated with a selective rabbit antihuman CYP3A4 polyclonal antibody (Millipore, CA, United States) or mouse anti-human GAPDH antibodies (Zhongshan Inc., Guangzhou, China), and subsequently with the secondary antibody of HRP goat anti-rabbit IgG (Zhongshan Inc., Guangzhou, China) or rabbit anti-mouse IgG (Zhongshan Inc., Guangzhou, China). Images were acquired with GE Healthcare ImageQuant 350, and band densities were quantified with GeneTools (SynGene, Cambridge, United Kingdom).

Construction of Reporter Plasmids
The 3 -UTR of CYP3A4 gene corresponding to 1,620-2,792 nt (1,173 bp; accession no. NM_001202855) was cloned into pmiR-RB-REPORT TM vector via XhoI and NotI restriction sites. The primers used for construction of wild-type CYP3A4 3 -UTR reporter plasmid were as follows: h-CYP3A4-F, 5 -CTTGACTCGAGATTTTCCTAAGGACTTCTGC-3 ; h-CYP 3A4-R, 5 -ATTGCGGCCGCAGGCTTATTGCTCAATC-3 . The sequence of the recombinant clones was confirmed by DNA sequencing and named as pmiR-RB-REPORT TM CYP3A4 3 -UTR WT. miRNA-200a binding site mutant (AGTGTTA changed to TGTGCCA) and miRNA-150 binding site double mutant (TTCCCAG changed to ATCCGAT and TGGGAGA changed to AGGCATA) were constructed using Q5 R Site-Directed Mutagenesis Kit (NEB, MA, United States) and confirmed by DNA sequencing. The firefly luciferase gene used 3 -UTR of CYP3A4 as the report luciferase, with Renilla luciferase gene as an internal control.

Dual-Luciferase Assay
The LO2 cells were seeded into 24-well plates. Firefly luciferase (0.1 µg) containing 3 -UTR of CYP3A4 in pmiR-RB-REPORT TM vector, along with miR-200a-3p or miR-150-5p mimic, was transfected into LO2 cells with Lipofectamine 2000. After 24 h of incubation, luciferase activities were measured with a luminometer (Tecan Infinite 200 Pro, Switzerland) using the Dual-Luciferase Reporter Assay System (Promega, Valencia, CA, United States). Firefly luciferase activity was normalized by Renilla luciferase activity and compared between different treatments.

Statistical Analysis
All values were expressed as the mean ± SEM. Comparisons of variables between groups were performed with an unpaired two-tailed Student's t-test. P < 0.05 was considered statistically significant. . LO2 cells were cotransfected with a firefly luciferase reporter vector containing the indicated CYP3A4 3 -UTR constructs, a Renilla luciferase reporter as internal control, and treated with miR-200a-3p (B) or miR-150-5p (C) mimics. Values were mean ± SEM for three independent experiments. * p < 0.05, * * p < 0.01, versus control; ns, not significant.

CYP3A4 Expression Is Decreased in FFA-Induced Steatosis Cells
CYP3A4 activity is reduced in human NAFLD as well as in mouse and in vitro cell models of the disease (Kolwankar et al., 2007;Woolsey et al., 2015). Here, we investigated CYP3A4 expression in FFA-induced steatosis cells in vitro. As shown in Figures 1A,B, CYP3A4 protein level was decreased in FFA-induced steatosis cells compared to control. Similarly, a significant decrease of CYP3A4 mRNA was observed in FFA-induced steatosis cells compared to control (Figure 1C).

Increased Mature miR-200a-3p and miR-150-5p in FFA-Induced Steatosis Cells
To investigate whether decreased CYP3A4 in FFA-induced steatosis cells was due to miRNAs regulation, we used MicroInspector and TargetScan algorithms to screen antisense matches of CYP3A4 3 -UTR against human miRNAs. CYP3A4 was predicted to be a target gene of miR-200a-3p and miR-150-5p (Figure 2A). Next, we determined the expression levels of mature miR-200a-3p and miR-150-5p in FFA-induced steatosis cells and found that both mature miR-200a-3p and miR-150-5p were higher in FFA-induced steatosis cells than in control cells ( Figure 1C).

DISCUSSION
In recent years, NAFLD has emerged as a major public health concern characterized by elevated serum FFAs and hepatocyte lipoapoptosis (Feldstein et al., 2003). Several reports have indicated the importance of both quantitative and qualitative (e.g., saturated versus unsaturated FAs) changes in dietary FAs as underlying causative mechanisms for NAFLD in both rodent After 24 h, cells were exposed to 1 mM FFA or vehicle. The BODIPY 493/503 staining was performed to assess cellular steatosis. Green fluorescence indicated lipid droplets. The cell nucleus was stained by DAPI (Blue). * p < 0.05, * * p < 0.01, bar = 10 µm. models and humans (Nehra et al., 2001;Musso et al., 2003;Wang et al., 2006). Increased supply of FAs or decreased lipid clearance in hepatocytes can set off esterification of FAs and formation of lipid droplets (Anderson and Borlak, 2008). In terms of mechanism, enzymes, important regulators of plasma and tissue FA composition, signaling pathways, and transcriptional factors controlling FA synthesis and gluconeogenesis have all been implicated in NAFLD (Malhi and Gores, 2008;Mota et al., 2016). Here, we demonstrated that miR-200a-3p and miR-150-5p, through directly targeting 3 -UTR of CYP3A4, contributed to the development of FFA-induced steatosis in vitro.
Previous studies have evaluated the relationship between NAFLD and hepatic CYP3A activity. Fisher et al. (2009) explored the relationship between hepatic CYP3A4 and non-alcoholic hepatic steatosis in human liver samples. It was found that CYP3A4 protein expression and activity decreased with the progression of NAFLD. Other studies (Kolwankar et al., 2007;Jamwal et al., 2018) showed that liver samples with steatosis had significantly lower hepatic CYP3A4 activity than those without steatosis, which was consistent with in vitro findings in longchain FFA-induced steatosis. CYP3A4 mRNA and protein also decreased significantly in fatty mice induced by a high-fat diet (Yoshinari et al., 2006).
Although most of the CYPs are found within endoplasmic reticulum, CYP3A4 localizes in mitochondria and synthesizes arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promotes electron transport chain/respiration and mitochondrial function (Guo et al., 2017). It is well known that CYP enzyme activities are affected by steatosis, which poses major impact on drug metabolism and drug-induced hepatotoxicity (Gomez-Lechon et al., 2009). However, our study suggests that increased CYP3A4 expression prevents LO2 cells from FFA-induced steatosis. Our results demonstrate that CYP3A4 activity impairment is not only induced by hepatic steatosis but also a factor further promoting hepatic steatosis. This could explain the phenotype of cyp3a-null male mice, including increases in weight, liver triglycerides, and total lipids (Kumar et al., 2018).
It was previously shown that CYP3A4 could be regulated by miRNAs via direct and indirect targeting (Takagi et al., 2008;Pan et al., 2009;Swathy et al., 2017;Yan et al., 2017). Hepatic miRNA changes in NAFLD have been confirmed in several studies of dietary NASH in mice and rats (Pogribny et al., 2010;Alisi et al., 2011), as well as serum and liver tissue of NAFLD patients (Leti et al., 2015;Liu et al., 2018). In this study, we reported two new CYP3A4 regulators, including miR-200a-3p and miR-150-5p, in FFA-induced steatosis. Our results showed that expressions of miR-200a-3p and miR-150-5p increased significantly in hepatocytes with steatosis as compared to normal hepatocytes, which is consistent with previous studies reporting an increase of miR-200a-3p in NAFLD (Cheung et al., 2008;Alisi et al., 2011). More importantly, miR-200a-3p or miR-150-5p inhibitor can alleviate FFA-induced steatosis. Thus, our findings provide a potential therapeutic target for the use of miR-200a-3p and miR-150-5p inhibitors to protect against steatosis development.