Jing Liu,Jinyun Zhu,Xiohong Zhng,Yuzhi Ji,Xuejun Lee,*,Zhiling Go,*

a Department of Infectious Diseases,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou,China

b Department of Pathology,Northwestern University Feinberg School of Medicine,Chicago,IL,USA

Keywords:Liver regeneration Hsa-miR-637 Peroxisome proliferator-activated receptor alpha (PPARA)Thyroid hormone receptor alpha (THRA)Mediator complex subunit 1(Med1)

ABSTRACT Background:Recent studies have shown that mediator complex subunit 1(Med1)can significantly affect hepatocyte proliferation and differentiation.Acting as a tumor suppressor,microRNA-637(hsa-miR-637)can inhibit the growth of hepatocarcinoma cells and further induce cell apoptosis.However,the function of hsa-miR-637 and its target genes during liver regeneration remains to be elucidated.Methods:This study used co-immunoprecipitation (Co-IP) assay,transfection,luciferase reporter assay,functional assay by cell counting kit-8 (CCK-8),Annexin V-FITC/propidium iodide apoptosis assay,and quantitative polymerase chain reaction analysis of chromatin immunoprecipitation (ChIP) for analysis.Results:Hsa-miR-637 has been suggested to suppress the expression of two Med1-interacting nuclear receptors,identified as the peroxisome proliferator-activated receptor alpha (PPARA) and thyroid hormone receptor alpha (THRA) at the transcriptional and translational levels in the human liver HL-7702 cell line.The interaction between Med1 and PPARA/THRA in HL-7702 cells was then confirmed.The transcriptional repression of hsa-miR-637 on PPARA and THRA was also demonstrated.Moreover,hsamiR-637 has been determined to suppress the proliferation of HL-7702 cells.Furthermore,cell cycle arrest of HL-7702 cells was induced by transfection of hsa-miR-637 at the S phase,but its apoptosis failed.Finally,PPARA was indicated to directly bind to the promoter of some transcription factors,like βcatenin,mouse double minute 2 (MDM2),and p53.Conclusions:This study has confirmed that hsa-miR-637 plays an antiproliferative role during liver regeneration,which may contribute in understanding the regenerative process of the liver.

1.Introduction

The liver has been known for its unique ability to regenerate after surgical removal or chemical injury.The process of liver regeneration in mammals is mainly achieved by compensatory growth,which means the damaged liver can regain its original weight and function as the remaining liver tissue grows.1,2This process is mediated by the replication of differentiated liver cells,mainly hepatocytes,through numerous cycles of DNA replication and cell division.3The molecular mechanisms of liver regeneration,however,remain unknown and thus need to be investigated.

Mediator complex subunit 1 (Med1),also called peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP)/PPARBP/thyroid hormone receptor-associated protein 220(TRAP220)/DRIP205/RB18A,is the key subunit of mediator complex that has been identified to play an important role in the development of liver disease and the metabolic pathways essential for normal liver function.4-6Med1 interacts with several nuclear receptors and transcription factors including nuclear receptors like peroxisome proliferator-activated receptor alpha (PPARA),thyroid hormone receptor alpha (THRA),GATA family members,and so on.6,7Early studies have shown that complete deletion of this gene in the mouse results in embryonic lethality,suggesting the important role of Med1 in multi-organ development.4,8To determine the tissue-specific function of Med1,Med1 liver conditional knockout mice (Med1△liv) were generated using Cre-lox technology,which displayed significant loss of liver cell proliferation,PPARA ligand-induced peroxisome proliferation,and the induction of PPARA-regulated genes,suggesting Med1 plays a vital role in liver regeneration and PPARA signaling.9Overexpression of Med1 using adenovirus vector in Med1△liv mouse liver and wild-type control Med1f1/f1liver has been determined to enhance hepatocyte proliferation and the expression of many genes involved in DNA replication and cell cycle progression,suggesting that Med1 alone can induce proliferative response in the liver.10

MicroRNAs (miRNAs) are small noncoding RNA molecules that are usually functional in silencing gene expression at the posttranscriptional level by binding to the 3ʹuntranslated regions(3ʹUTR) of the targeted mRNA.11,12Recently,miRNAs have been proposed to play an important role in liver regeneration.Genomewide miRNA microarray studies have shown that 70%of all miRNAs downregulated 24 h post-partial hepatectomy in rat liver,and this genome-wide downregulation was observed to be required for the recovery of liver cell mass.13Liver regeneration in mice after partial hepatectomy exhibited a delay in cell cycle progression with liverspecific inactivation of DiGeorge syndrome critical region gene 8(DGCR8),an essential regulator of the miRNA processing pathway.14Hsa-miR-637 is a primate-specific miRNA expressed in many tissues including the liver.15,16Functional studies have revealed that hsa-miR-637 can act as a tumor suppressor in liver cancer by inhibiting the growth of hepatocellular carcinoma (HCC) cells and inducing cell apoptosis.16The role of hsa-miR-637 during liver regeneration,however,remains unknown.

In this study,bioinformatics analysis was carried out using TargetScan,and hsa-miR-637 was found to target a number of downstream genes involved in hepatocyte proliferation including PPARA and THRA.As PPARA and THRA have been found to interact with Med1 and Med1 exerts a dominant effect in hepatocyte proliferation,this study sought to determine whether hsa-miR-637 plays a significant role in the regulation of liver regeneration and whether this regulation is mediated through Med1-interacting proteins.

2.Materials and methods

2.1.Cell culture

The human liver cell line HL-7702 was obtained from Shanghai Culture Collection,which was then cultured in RPMI-1640 medium supplemented with 1% (v/v) penicillin-streptomycin and 10% (v/v)heat-inactivated fetal bovine serum(FBS).All cells were maintained in 5% CO2humidified incubator at 37°C.During subculture,cells were detached using trypsin when they reached 80-90% confluency.The well-grown cells were harvested and seeded into 6-well plates at a density of 1-2×105cells per well for experiments.

2.2.RNA extraction and real-time polymerase chain reaction (RTPCR)

The total cellular RNA was isolated using TRIzol Reagent (Invitrogen,Carlsbad,CA,USA) and treated with RNA-free DNase I to remove residue DNA according to the manufacturer’s protocol.This method yielded an average of 20-40 μg of the total RNA from 106cells.cDNA was then synthesized using the TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit (D6210A) and RT Primer Mix(Takara Bio Inc.,Otsu,Japan).The primers were purchased from the Shanghai Biotechnology Inc.,with the following sequences:PPARA forward,5ʹ-TCACCACAGTAGCTTGGAGC-3ʹ,reverse,5ʹ-CATCTGAGCCAGGACAGCTT-3ʹ;THRA forward,5ʹ-CTGCTGATGAAGGGTCCGC-3ʹ,reverse,5ʹ-GGCATGGAGAATTCCGCTTC-3ʹ;GATA binding protein 4 (GATA4) forward,5ʹ-TGTGCAGAGTTTGCCTCACA-3ʹ,reverse,5ʹ-AGAACGAATGCCGAGTCCTG-3ʹ;ADP ribosylation factor 4 (ARF4) forward,5ʹ-ACTTCAGGGGTGCCACATTC-3ʹ,reverse,5ʹ-AGCCCCTCCTCTTTCTTCCT-3ʹ.The relative expression of each product was normalized to an internal control (GAPDH).The quantitative RT-PCR was performed using Bio-Rad CFX96 Real-Time PCR System(Bio-Rad Laboratories,Hercules,CA,USA),and the data were analyzed using Bio-Rad CFX Manager Software 1.6 using 2-ΔΔCt and setting control as 1.

2.3.Protein extraction and Western blot

The procedure for protein extraction and Western blot has been previously described and modified.Briefly,tissues or cells were homogenized and lysed in an ice-cold RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.5),150 mM NaCl,1 mM ethylene glycol tetraacetic acid(EGTA),1 mM phenylmethanesulfonyl fluoride(PMSF),0.5%NP-40,0.25%sodium dodecyl sulfate(SDS),5 μg/mL leupeptin,and 5 μg/mL aprotinin,and all these were incubated on ice for 30 min.Homogenates were centrifuged at 12,000 g for 10 min at 4°C,and the supernatant was collected.After determining the protein concentration in the supernatant,aliquots of proteins(40 μg) were loaded into the lanes of a SDS-polyacrylamide gel(PAGE).The proteins were separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes.The membranes were blocked with 5% bovine serum albumin (BSA) in 0.01 M tris-buffered saline(TBS)(pH 7.4)and 0.1%Tween-20(TBST)at room temperature for 1 h;then,they were incubated with primary antibodies directed against target proteins overnight at 4°C.The final dilutions for primary antibodies were as follows:anti-PPARA antibody,1:400;anti-THRA antibody,1:2000.After washes with TBST for 3 times,the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP)diluted at 1:5000 in TBST containing 5% BSA for 1 h.The immunocomplexes were detected using the BeyoECL Plus Detection System (Thermo Fisher Scientific,Rockford,IL,USA).GAPDH was then used as a loading control.The density of immunoblotting was quantified using the software Image J.

2.4.Co-immunoprecipitation (Co-IP)

Co-IP was performed using the Co-IP Kit(BersinBio,Guangzhou,China).Briefly,1×107HL-7702 cells were collected and washed 3 times using ice-cold 1 ×phosphate-buffered saline (PBS).Afterward,1 mL of cell lysis buffer was added and incubated on ice for 20 min.The cells were then centrifuged at 12,000 g for 10 min at 4°C,and the supernatant was divided into Input,IP group,and IgG group according to 1:6:3.IP group and IgG group were added with a different 3 μg of target antibodies,that is,anti-Med1 (Abnova,PAB8661,Taipei,Taiwan,China) and IgG antibody (Cell Signaling Technology,4340,Danvers,MA,USA) were incubated at 4°C for 12-18 h;then,200 μL of protein-A/G Magbeads were respectively added to IP and IgG group and incubated for 2 h at 4°C.The immunoprecipitated material were washed using pre-cooled washing buffer for 3 times.Subsequently,the loading buffer was added,and the sample was boiled for 10 min before it was centrifuged to collect the supernatant;the protein sample was subjected to PAGE electrophoresis.Anti-PPARA (Proteintech,15540-1-AP,Rosemont,IL,USA) and anti-THRA (Proteintech,10139-1-AP,Rosemont,IL,USA)were used for Western blot detection of the protein sample.

2.5.Transfection

Hsa-miR-637 mimic or inhibitor was used for the transfection of HL-7702 cells.The hsa-miR-637 negative control(NC)siRNA served as a NC.Transfection was performed using the Hieff Trans™transfection reagent (Xusheng Bio.Inc.China) as per manufacturer’s instructions.Briefly,the day before transfection,the cells were trypsinized and plated into 6-well plates to reach cell confluence of about 70-80% on the second day.For each well of cells,3 μL of transfection reagent was mixed with 1 μg of hsa-miR-637,hsa-miR-637 inhibitor,or hsa-miR-637 NC and 100 mL serumfree media and then incubated for 10 min to allow DNA-liposome complex formation.The cell culture medium was then replaced,and the cells were washed with serum-free RPMI-1640,and 900 μL of serum-free medium was then added.Afterward,100 μL of miRNA/mimic-Hieff mixture was added into each well and mixed gently.The cells were further incubated for 24,48,and 72 h prior to harvesting.

2.6.Luciferase reporter assay

The luciferase assay was conducted using Dual-Luciferase Reporter Assay System (Promega,USA).The following plasmids and miRNAs were used:for the wild-type group,psiCheck-3ʹUTR(WT)was co-transfected with miRNA mimic or miRNA mimic NC,whereas for the mutant group,psiCheck-3ʹUTR (mut) was cotransfected with miRNA mimic or miRNA mimic NC.No plasmid or miRNA was used for the NC group.Growth media were removed and lysed with luciferase lysis buffer,24 h after co-transfection of plasmids and miRNAs.Renilla luciferase reporter was used as a transfection efficiency control.Meanwhile,GloMax 96 Microplate Luminometer (Promega,Madison,WI,USA) was used to measure the luciferase activity of lysate as per manufacturer’s protocol.

2.7.CCK-8 cell proliferation assay

Cell proliferation has been determined using the cell counting kit-8(CCK-8)(Beyotime,Hangzhou,China).Cells were seeded into 96-well plate at 5 ×104cells/well in 100 μL medium for 24 h and then were transfected with the indicated miRNA or miRNA inhibitors,and the cells were further incubated for 24 h.One hour before the endpoint of incubation,10 μL of CCK-8 reagent was added to each well.The absorbance at 450 nm in each well was measured using a microplate reader.The relative cell activity was then calculated by the following formula:relative cell activity(%)=(mean absorbance of treatment group)/(mean absorbance of control group) ×100.

2.8.Annexin V-FITC/propidium iodide (PI) apoptosis assay

HL-7702 cells were placed in a 6-well culture plate and treated with miRNA or its inhibitor for 24 h with ribo FECT™CP Transfection Reagent (Ruibo Inc.,Guangzhou,China).Annexin V-FITC/PI assay kit(KGA512 Inc.,China)was then used to distinguish normal,apoptotic,and necrotic cells as per manufacturer’s instructions.On the other hand,1×106cells were then washed using ice-cold PBS and resuspended in 250 μL binding buffer.Then,5 μL Annexin VFITC and 10 μL PI solution (20 μg/mL) were added to the samples.After incubation for 15 min at room temperature in the dark,400 μL PBS was added to the reaction tube,and the samples were analyzed using flow cytometry.

2.9.Cell cycle distribution analysis

After being transfected with miRNA mimic or inhibitor,1×105HL-7702 cells were collected and washed three times using ice-cold 1×PBS.The cells were then fixed in 75%ice-cold ethanol overnight at-20°C.The ethanol-fixed cells were then centrifuged and stained with PI in 1 mg/mL RNase A in a 37°C water bath for 30 min before the cell cycle was measured using flow cytometry.

2.10.Chromatin immunoprecipitation (ChIP)

The cell line HL-7702 cells were cultured in RPMI-1640 medium supplemented with 1% (v/v) penicillin-streptomycin and 10% (v/v)FBS,treated by adding formaldehyde directly to the medium to a final concentration of 1%,and incubated for 10 min at room temperature.Approximately 2 ×107cells were used for each immunoprecipitation using the ChIP Kit (BersinBio,Guangzhou,China).Cross-linking reactions were stopped by adding 1/10 volume of 1.375 M PBS-glycine.Cells were then washed twice using ice-cold PBS and centrifuged at 500 g for 5 min.Cells were then resuspended in RIPA buffer (10 mM Tris-HCl,pH 7.6,1 mM ethylenediaminetetraacetic acid(EDTA),0.5 mM EGTA,140 mM NaCl,1%Triton X-100,0.1% Na-deoxycholate,0.1% SDS),sonicated on ice to an average chromatin length of 200-600 bp,and then centrifuged at 13,000 g for 10 min at 4°C.The supernatants were incubated at 4°C overnight with 5 μg of anti-PPARA antibody and 5 μg of antirabbit IgG antibody in 1.5 mL RIPA buffer.Nonimmune IgG was used as NC.The immunoprecipitated material was washed and eluted from the beads by incubation at 37°C for 1 h in 500 μL TE(1 mM EDTA,50 mM Tris,pH 8.0) containing 0.5% SDS and proteinase K(0.1 mg/mL),followed by 12 h at 65°C after adding NaCl to 0.3 M and SDS to 1%.The DNA samples were then extracted using phenol/chloroform and chloroform and ethanol precipitation.

2.11.qPCR analysis of ChIP samples

The iCycler iQ Real-Time PCR Detection System (Bio-Rad) was used to quantify the DNA sequences in the ChIP fraction.The primer sequences were as follows:bone morphogenetic protein (BMP)forward,5′-ACACGCTTGCCATGGTTTTC-3′,reverse,5′-TCGATCTCCTGACCTCGTGA-3′.CTNNB1 forward,5′-GCGCCATTTTAAGCCTCTCG-3′,reverse,5′-TAAGGAAAGGAGCGCCCAAG-3′.Mouse double minute 2 (MDM2) forward,5′-TGCACCGGTGTATAAGCCTG-3′,reverse,5′-ATTAAGCCCACTCCACCAGC-3′.Retinoblastoma transcriptional corepressor 1 (RB1) forward,5′-TGCCTCACGTTACAATGGCT-3′,reverse,5′-AGTCTGCTGGGAGCCTGATA-3′.Tumor protein p53 (P53) forward,5′-CCTGCCCCGTTGTTATCCTT-3′,reverse,5′-AGCGCTTGGAACTACAGCTT-3′.Wnt1 forward,5′-GTGGTGGGGCATGATAACCA-3′,reverse,5′-AGTCCCAGCTACTCAGGAGG-3′.PCR amplification was performed in triplicate in 50 μL TaKaRa SYBR Premix Ex Taq II(Perfect Real-Time)for each ChIP fraction.Enrichment of each DNA sequence was normalized to GAPDH and mock immunoprecipitation values.One sample t-test was performed in order to identify sequences that were enriched in ChIP fractions above background;Student’s t-test was used to determine the significance of differences between the two samples.

2.12.Statistical analyses

All data were expressed as the mean±standard deviation(SD).Statistical analysis was performed using SPSS version 18.0 (SPSS Inc.,Chicago,IL,USA).Comparison between different groups was determined using LSD-t-test (two groups) or one-way analysis of variance (more than two groups).A P-value of less than 0.05 was considered to be statistically significant.

3.Results

3.1.Hsa-miR-637 decreases the viability of liver cells

To determine the function of hsa-miR-637 in cell proliferation,HL-7702 was transiently transfected with hsa-miR-637 mimic or inhibitor,and the cell growth was later measured.Compared with the hsa-miR-637 mimic NC,the transfection of hsa-miR-637 mimic has been determined to suppress cell activity of HL-7702 by 26%(P <0.05) (Fig.1A).In contrast,the transfection of hsa-miR-637 inhibitor enhanced cell activity by 23% (P <0.05) (Fig.1A).These results indicated that hsa-miR-637 had a suppressive effect on liver cell viability.

3.2.Hsa-miR-637 has no effect on the liver cell apoptosis

The effect of hsa-miR-637 on cell activity of HL-7702 may be a result of hsa-miR-637-mediated apoptosis in liver cells.To address this possibility,flow cytometry (FCM) analysis for apoptosis was performed after HL-7702 cells were treated with Annexin V-FITC and PI staining.In the untreated control sample,no significant difference was noted in the apoptotic population labeled by Annexin V+PI-(2.79%) and that of samples transfected with hsamiR-637 mimic (4.74%) or its inhibitor (3.77%) (Fig.1B).The viable and non-apoptotic cells of untreated control sample,labeled by Annexin V-PI-,were identified to be the major cells(97.2%)and not significantly different from those of samples transfected either with hsa-miR-637 mimic (95.2%) or its inhibitor (96.2%) (Fig.1B).The above results suggested that hsa-miR-637 was not involved in the liver cell apoptosis.

Fig.1.Hsa-miR-637 suppresses liver cell proliferation but not apoptosis.

3.3.Hsa-miR-637 induces cell cycle arrest at the S phase

The suppressive effect of hsa-miR-637 on cell activity may result from hsa-miR-637-mediated regulation on the cell cycle of liver cells.FCM analysis for cell cycle was performed after PI staining.As per the results,the percentage of HL-7702 cells at S phase after being transfected with hsa-miR-637 mimic (42.95%) has been determined to be significantly higher than those in the mimic NC group (38.09%) (Fig.2).In contrast,the treatment hsa-miR-637 inhibitor had a reverse effect on the cell cycle.The percentage of S phase population in HL-7702 cells after being transfected with hsa-miR-637 inhibitor (41.49%) was significantly lower than those from the inhibitor NC group (45.61%) (Fig.2).These results suggested that hsa-miR-637 could induce cell arrest at the S phase.

Fig.2.Hsa-miR-637 induces cell cycle arrest at the S phase.

3.4.Hsa-miR-637 suppresses the expression of PPARA,THRA,GATA4,and ARF4

Hsa-miR-637 has been identified as a tumor suppressor in liver cancer,which induces apoptosis of HCC cells.20To explore the function of hsa-miR-637 in liver regeneration,this study first sought to identify the potential downstream targets of hsa-miR-637.As miRNAs have been known to exert functions by suppressing target genes at the posttranscriptional level,RNA was extracted from nontumorigenic HL-7702 cells,and quantitative RT-PCR assay was performed to search for the targeted genes.The RT-PCR results indicated that nuclear receptors PPARA and THRA,as well as transcription factor GATA4 and tumor suppressor ARF4 were downregulated in hsa-miR-637 mimic-transfected cells,compared with those in the mimic NC group(Fig.3A);this only suggests that these genes were potential downstream targets regulated by hsamiR-637.Due to their involvement in the regulation of cell cycle in hepatocytes,the expression of PPARA and THRA at the protein level was examined further.The immunoblotting analysis indicated that hsa-miR-637 significantly decreased the protein levels of PPARA and THRA (Fig.3B).

Fig.3.Hsa-miR-637 decreases the expression of PPARA,THRA,GATA4,and ARF4.

3.5.PPARA and THRA are targets of hsa-miR-637

To further verify whether PPARA and THRA were the targets of hsa-miR-637,luciferase assay was performed to assess the interaction between hsa-miR-637 and 3ʹUTRs of PPARA and THRA.According to our bioinformatics analysis using TargetScan,the 3ʹUTR of PPARA was predicted to have one binding sequence which matches the hsa-miR-637 seed,whereas the 3ʹUTR of THRA was predicted to have two binding sequences that matched with the hsa-miR-637 seed(Fig.3A).Therefore,the target sequence of their 3ʹUTRs (WT) or the one with site-directed mutation (Mut) was cloned into the luciferase reporter vector,respectively.The WT or Mut vector was then co-transfected with hsa-miR-637 or scramble control miRNA into HL-7702 cells,and the luciferase activity was measured.The results indicated that luciferase activity in HL-7702 cells co-transfected with hsa-miR-637 mimic,and PPARA WT or THRA WT was significantly lower than those in cells treated with hsa-miR-637 mimic associated with PPARA Mut or THRA Mut(Fig.4).Moreover,the luciferase activity in HL-7702 cells cotransfected with scramble control,and WT or Mut remained unaltered for either PPARA or THRA (Fig.4).

Fig.4.Hsa-miR-637 suppresses PPARA and THRA translation activities.

3.6.Med1 interacts with PPARA and THRA

Previous study has indicated that Med1 binds to PPARA and THRA,thus,this study sought to determine whether Med1 was associated with PPARA and THRA.Anti-Med1 antibody was first utilized in this study to immunoprecipitate native Med1-containing protein complexes from HL-7702 cell nuclear extracts.Immunoblotting was then applied using anti-PPARA antibody against Med1-containing protein complex to assess the binding of Med1 and PPARA.The results shown in Fig.5 have indicated the presence of PPARA,suggesting that PPARA interacted with Med1 in HL-7702 cells.Similarly,immunoblotting using anti-THRA antibody against Med1-containing immunoprecipitated complex demonstrated the interaction between THRA and Med1 (Fig.5).The reciprocal immunoprecipitation assays with anti-PPARA or anti-THRA antibody followed by immunoblotting using anti-Med1 antibody have confirmed the interaction between Med1 and PPARA or the interaction between Med1 and THRA.These results showed that Med1 interacted with PPARA and THRA under physiological conditions.

Fig.5.Med1/PPARA/THRA protein complex binds to promoters β-catenin,MDM2,and p53.

3.7.Med1/PPARA/THRA complex directly binds to promoters of βcatenin,MDM2,and p53

To determine whether the effect of Med1/PPARA/THRA protein complex on its downstream genes was mediated through direct physical association of the proteins with the targeted gene promoter,ChIP was used on cross-linked chromatin from HL-7702 cells.Cross-linked chromatin was prepared from HL-7702 cells and further immunoprecipitated with antibody against PPARA.Primers were designed to screen from ～2 kb of upstream region to 500 bp of downstream region of the target gene transcription initiation site.Quantitative RT-PCR was then used to measure the enrichment of each sequence in ChIP fraction.Six candidate genes have been identified,including β-catenin,Wnt,proto-oncogene MDM2,tumor suppressor p53,Rb1,and BMP.Those genes were selected because of their involvement in hepatocyte proliferation or differentiation and shown to be downregulated in Med1△liv liver compared with those in wild-type Med1f1/f1liver from our preliminary studies (data not shown).As summarized in Fig.6,significant enrichment of sequences in the promoters of β-catenin,MDM2,and p53 was observed in the PPARA fraction but not in the IgG ChIP fraction from HL-7702 cells.No enrichment of promoter sequences was observed for Wnt,Rb1,and BMP.Using an anti-Med1 or anti-THRA antibody to probe chromatin from HL-7702 followed by PCR using promoter-specific primers,similar results were obtained (data not shown).Those results confirmed that Med1/PPARA/THRA complex regulated the expression of β-catenin,MDM2,and p53 by binding directly to their promoters.

4.Discussion

The liver has been known for its remarkable ability to regenerate after loss of hepatic tissue.As an example,the rat liver was reported to regain its original size even after the removal of two-thirds of its liver mass.17This regenerative process of the liver can be completed within 5-7 days after the surgical removal.The extraordinary ability of the liver to regenerate in response to tissue injury is crucial to liver homeostasis as it is the main site for metabolism and detoxification of drugs.18Various studies using models including partial hepatectomy and chemical-induced liver injury have shown that liver regeneration is mainly achieved by expanding the hepatocytes of the remaining tissue and sometimes involving the stem/progenitor cells.1,19

Med1 has been determined to play a pivotal role in liver regeneration and liver tumor development.An early study from our group has demonstrated that Med1 alone can induce liver cell proliferation as demonstrated by overexpression of Med1 by adenoviral vector,which then induced a wide range of genes involved in DNA replication and cell cycle progression and hepatocyte proliferation.10Med1 conditional knockout mice in the liver(Med1△liv) displayed a severe impairment in liver regeneration after partial hepatectomy.20Gene expression analysis indicated that the Med1△liv hepatocytes displayed no DNA synthesis and failed to exit the G0/G1 phase.Med1 then interacts with several nuclear receptors,factors,and cofactors including PPARA,GATA family members,and tumor suppressor p53,which may be an indication of its role in nuclear receptor-mediated hepatic metabolism.20,21In this study,Med1 was shown to interact with PPARA and THRA in HL-7702 cells,and the interactions among Med1 and PPARA and THRA were deemed essential in the regulation of hepatic proliferation.

PPAR is a nuclear hormone receptor that functions as a liganddependent transcription factor,regulating the expression of genes involved in cell differentiation,proliferation,metabolism,and tumorigenesis in many tissues such as the liver,kidney,heart,and skeletal muscle.22-24In the liver,PPARA is activated by PPARA ligands known as the peroxisome proliferators and can regulate lipid metabolism,hepatocyte proliferation,and inflammation.22,25,26Chronic exposure to peroxisome proliferators has been observed to cause sustained activation of PPARA and transcription of PPARA downstream target genes that may contribute to defects in energy metabolism,oxidative DNA damage,and excessive liver cell proliferation,which in turn led to liver cancer.27,28The PPARA ligandinduced hepatic response,however,is completely abolished in Med1 null hepatocytes,suggesting an essential role of Med1 for the function of PPARA.29,30Interestingly,chronic exposure to PPARA ligands triggered a proliferative response and clonal expansion from residual Med1-positive hepatocytes but not the Med1-negative hepatocytes in Med1△liv,indicating a dominant role of Med1 in PPARA ligand-induced liver cell proliferation and hepatocarcinogenesis.20,31

Similar to the PPARA,THRA has been identified as another nuclear hormone receptor that regulates gene expression and plays a role in metabolism,growth,and development.7THRA is ubiquitously expressed that can be activated by its ligand,the active form of thyroid hormone,3,3ʹ,5-triiodo-L-thyronine(T3).32,33In the liver,THRA is expressed mainly in the pericentral hepatocytes that have been known to specifically express glutamine synthetase,a marker for hepatocyte progenitor cells during liver regeneration.34,35A recent study has shown that T3-induced hepatocyte proliferation was achieved through protein kinase A(PKA)-dependent β-catenin activation,suggesting the involvement of Wnt/β-catenin pathway in THRA-mediated liver regeneration.36

MicroRNAs are a relatively new class of regulators that control hepatocyte proliferation in the liver.21,37Hepatic-specific inactivation of DGCR8,a key component of the miRNA processing pathway,resulted in a delay in cell cycle progression from G1 to S phase in mice after partial hepatectomy,which only suggests that miRNA is essential in liver regeneration.14Hsa-miR-637 is a primate-specific miRNA expressed in multiple tissues including the liver according to miRBase.Hsa-miR-637 can regulate inflammatory process by modulating the expression of the inflammation marker C-reactive protein through directly binding to its 3ʹUTR.38In smooth muscle cells from pulmonary hypertension patients,hsa-miR-637 has been shown to suppress the cell cycle progression by targeting cyclindependent kinase 6 (CDK6),thus increasing the risk of hypoxic pulmonary hypertension.39Enforced overexpression of hsa-miR-637 suppressed cell growth of HCC and triggered apoptosis of HCC partially by disrupting the phosphorylation of signal transducer and activator of transcription 3 (STAT3),suggesting a tumor suppressor role of hsa-miR-637 in the liver.16

This study has further showed that hsa-miR-637 could regulate the expression of PPARA and THRA through luciferase assay using their 3ʹUTR as the reporter constructs;hsa-miR-637 were also shown to suppress the proliferation of HL-7702 liver cells and induce cell cycle arrest at the S phase.Thus,hsa-miR-637 was verified to play a role in Med1-directed hepatocyte proliferation by regulating the expression of PPARA and THRA,the two Med1-interacting proteins.ChIP allowed us to further identify the molecular targets regulated by Med1/PPARA/THRA complex.Our preliminary study suggested six candidate genes,β-catenin,Wnt,MDM2,p53,Rb1,and BMP,because they were downregulated at the mRNA level and protein level in Med1△liv liver compared with those in the wild-type Med1f1/f1liver.The Wnt/β-catenin canonical pathway,activated by a Wnt-protein ligand via the transcription co-activator β-catenin,has also been determined to be critical for liver development,liver regeneration,metabolism,and liver diseases.40,41The MDM2-p53 pathway,a feedback loop involving tumor suppressor p53 and its downstream target and negative regulator MDM2,has been shown to play a vital role in the development of HCC.41,42Meanwhile,BMPs are a group of growth factors that have been shown to carry out distinct functions in hepatocyte proliferation in response to partial hepatectomy.43,44The RB tumor suppressor is known to inhibit cell cycle progress in cancer and has been shown to mediate checkpoint responses in the development of liver cancer.45,46The results of ChIP assay,however,indicated that β-catenin,Mdm2,and p53 were the direct targets of Med1/PPARA/THRA complex,thus suggesting the involvement of their signaling pathway in Med1/PPARA/THRA complex-mediated hepatocyte proliferation.

According to the above experimental results,hsa-miR-637 was found to suppress the proliferation of HL-7702 liver cells,thus inducing cell cycle arrest at the S phase,and the mechanism of hsamiR-637 was determined to be related to targeting for Med1/PPARA/THRA complex which directly binds to promoters of β-catenin,Mdm2,and p53.The process of the above mechanism was presented in a diagram(Fig.6).

Fig.6.Graph summary of hsa-miR-637 mechanism in the experiment.

5.Conclusions

In summary,this study was able to determine the molecular mechanism underlying the cooperative actions of Med1 and hsamiR-637 in regulating hepatocyte proliferation in human liver HL-7702 cell line.This may help in understanding the process of liver regeneration,thus contributing to the treatment of liver diseases.

Authors’ contributions

J.Liu and J.Zhu contributed equally to this work.Study concept and design:J.Liu and Z.Gao;Database management and experiments:J.Liu and J.Zhu;Data interpretation:X.Zhang and Y.Jia;Drafting and revising the manuscript:X.Lee and Z.Gao.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study was supported by the National Science and Technology Major Project of China(2018ZX10302204-002).The authors would like to thank BersinBio (Guangzhou,China) for their technical assistance.