The immortalized human HSC line LX-2 (Institute of Cell and Molecule Biology, Central South University, Changsha, China), a generous gift from Dr. Tan (24), was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), 100 μg/ml streptomycin (Amresco, Solon, OH, USA) and 100 U/ml penicillin (Amresco) and incubated at 37°C with 5% CO₂. Prior to the start of the experiments, cells were tested by PCR for mycoplasma, using commercially available primers, and were shown to be mycoplasma-negative. The cells were activated by administration of transforming growth factor β1 (TGF-β1; Sigma-Aldrich, St. Louis, MO, USA). Human Embryonic Kidney (HEK; Cell Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) 293 cells were maintained in DMEM supplemented with 10% FBS and antibiotics at 37°C in 5% CO₂. The mimics and inhibitors of miR34a and miR-34c, and the respective RNA controls, were obtained from Shanghai JIMA Pharmacy Technology Co., Ltd. (Shanghai, China).

To search for miRNA-34 family target genes, the online miRNA database miRanda (http://www.microrna.org), Targetscan (http://www.targetscan.org) and Pictar (http://pictar.bio.nyu.edu) were used.

Primary HSCs were isolated from normal male Sprague Dawley rats aged between five and six months (weighing 400–500 g; Shanghai Laboratory Animal Center of Chinese Academy of Sciences, Shanghai, China) by in situ perfusion and density-gradient centrifugation, as previously described (25). The rats were maintained at 25°C on a 12/12 h light/dark cycle, with ad libitum access to rodent chow and water. The rats received humane care according to the Guide for the Care and Use of Laboratory Animals of the Chinese Academy of Sciences. The study was approved by the ethics committee of Yiwu Central Hospital. All institutional and national guidelines for the care and use of laboratory animals were followed. Isolated HSCs were suspended in DMEM supplemented with 10% FBS, and penicillin and streptomycin at a cell density of 5×10⁵ cells/ml, seeded in culture flasks and cultured at 37°C in 5% CO₂.

The cells seeded on glass coverslips or 96-well plates were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min at room temperature (RT). After being washed three times with PBS, the cells were incubated in blocking buffer [PBS containing 3% bovine serum albumin (BSA), 0.3% Triton™ X-100 and 10% FBS] for at least 30 min and then in binding buffer (PBS containing 3% BSA and 0.3% Triton X-100) with monoclonal antibodies (mAbs) against α-SMA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100 for 1 h at RT. Following three washes with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (Ig)G (Thermo Fisher Scientific, San Jose, CA, USA) at a 1:100 dilution with binding buffer for 1 h at RT. The cell nuclei were stained with DAPI (Sigma-Aldrich). The stained samples were then examined with a Leica TCS SPII confocal microscope (Leica Microsystems, Wetzlar, Germany).

For general PCR, total RNA was extracted with TRIzol (Invitrogen Life Technologies) from HEK 293 cells and cDNA was synthesized using moloney murine leukemia virus reverse transcriptase (Promega Corp., Madison, WI, USA). For miRNA detection, total RNA was prepared by using the mirVana™ miRNA Isolation kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using the Taqman miRNA RT kit (Applied Biosystems, Foster City, CA, USA). For the detection of the miRNA levels by qPCR, TaqMan^® microRNA assay (Applied Biosystems) was used to quantify the relative expression levels of miR-34a (assay ID: 000426), miR-34c (assay ID: 000428), and U6 (assay ID: 001973) as an internal control, in an Applied Biosystems 7500 Detection system (Applied Biosystems). The relative amount of miRNAs was normalized against U6 small nuclear RNA, and the fold change for each miRNA was calculated using the 2^−ΔΔCt method (26). The relative miRNA expression was calculated from three different experiments.

The 3′-untranslated region (UTR) fragments of the indicated target mRNAs containing putative miR-34a and miR-34c binding sites were amplified by PCR from the cDNA of HEK293 cells. The amplified fragments were cloned into the XbaI sites of pGL3-promoter vector (Promega Corp.) downstream of the luciferase coding region to generate reporter vector pGL-UTRs. The vector pGL-PPARγ-mut with mutated binding sites was mutated with the QuikChange II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. The oligonucleotides used for cloning are demonstrated in Table I. All of the primers were synthesized by Sangong Biotech Co., Ltd. (Shanghai, China). The constructed clones were confirmed by sequencing (Sangong Biotech Co., Ltd.).

The HEK293 cells were plated in 24-well plates the day prior to transfection. The cells were co-transfected with internal control pRL-TK (Promega Corp.), reporter vectors and mimics of miR-34a/c or negative control probes (Shanghai JIMA Pharmacy Technology Co., Ltd.) using Lipofectamine^® 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. A total of 48 h later, the cells were harvested and applied to the luciferase measurement with a Dual-Luciferase Reporter Assay System (Promega Corp.) using a GloMax^® 20/20 detection system (Promega Corp.). Values are represented as the number of relative light units (RLU).

rHSCs or LX-2 cells were seeded into six- or 96-well plates the day prior to transfection. The cells were transfected with inhibitors of miR-34a/c or negative control miRNAs (Shanghai JIMA Pharmacy Technology Co., Ltd.) using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen Life Technologies). Following 6 h of culture with the transfection mix, the cells were cultured in normal culture medium (rHSCs) or medium containing 5 ng/ml TGF-β1 (LX-2 cells). A total of 48 h later, the cells were harvested and subjected to western blot analysis.

The cells were harvested in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland)] were first quantified by the bicinchoninic acid method (Pierce Biotechnology, Inc., Rockford, IL, USA) and then denatured by boiling for 5 min. A total of 30 μg of protein per sample were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with tris-buffered saline (TBS; 20 mM Tris-HCl (pH 7.4), 150 mM NaCl) containing 5% milk for 1 h at RT. Next, the membranes were incubated with monoclonal mouse antibodies against α-SMA (1:100 dilution; sc-53142), against PPARγ (1:100 dilution; sc-7273) or against GAPDH (1:100 dilution; sc-365062) (all Santa Cruz Biotechnology, Inc.) for 1 h at 37°C, followed by three time washes with TBS-T (TBS containing 0.1% (v/v) Tween-20) buffer. Next, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG at a dilution of 1:10,000 in TBS for 1 h at RT, followed by three washes with TBS-T. The proteins were then detected using the Supersignal^® West Pico chemiluminescent substrate (Pierce Biotechnology, Inc.) on an AlphaEase^® FC Imaging System (Alpha Innotech Corporation, San Leandro, CA, USA).

Values are presented as the mean ± standard deviation. The software GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for all statistical analysis and graphical illustrations. The statistical significance was analyzed using Student t-test. P<0.01 was considered to indicate a statistically significant difference.

To identify the potential target mRNAs of miR-34a and miR-34c, which contain the same ‘seed region’, three different prediction tools (miRanda, Targetscan and Pictar) were used. Of all the predicted genes, eight genes were selected for confirmation. The criteria for target selection was not only concerning the binding possibility, but also concerning the potential functions in liver fibrosis. Therefore, the selected genes encoded proteins either correlated with certain important signaling pathways [lymphoid enhancer factor 1 (LEF1), Wnt-2B, PPARγ, amphiregulin (AREG)] (23,27–29) or associated with lipid metabolism [lactate dehydrogenase (LDHA), ACSL-1, fatty acid binding protein 3 (FABP3), X box binding protein 1 (XBP1)] (30,31).

HEK293 cells were co-transfected with reporter vectors pGL-UTRs bearing the predicted binding sites and mimics of miR-34a or miR-34c together with the internal control pRL-TK. The increased expression of miR-34a or miR-34c by mimics transfection was confirmed by qPCR at 48 h following transfection (Fig. 1A), and a concentration of 20 nM mimics was selected for the following experiments. The results demonstrated that the overexpression of miR-34a/34c significantly decreased the relative luciferase activity in HEK293 cells transfected with UTRs of LEF1, PPARγ, AREG, LDHA, ACSL-1 and XBP1. LEF1, LDHA and ACSL-1 have been reported to be targeted by miR-34a/34c previously (15,32). Since PPARγ was reported to be involved in the maintenance of a quiescent HSCs phenotype and the activation of HSCs resulted in the loss of PPARγ (19,33), PPARγ was selected for further confirmation. The reporter vector bearing the mutated UTR of PPARγ was constructed (Fig. 1C). It was identified that the overexpression of miR-34a/34c significantly decreased the relative luciferase activity in HEK293 cells transfected with pGL-PPARγ (Fig. 1D), while it caused no apparent relative luciferase activity changes in HEK293 cells transfected with pGL-PPARγmut (Fig. 1E). These results indicated that PPARγ was a direct target gene of miR-34a and miR-34c.

The upregulation of miR-34 family members and downregulation of PPARγ have been previously reported during liver fibrosis (15,23). Since the activation of HSCs is the pivotal process in liver fibrosis, the expression of miR-34a/c and PPARγ was detected during the activation of HSCs. Rat HSCs and human HSCs (LX-2) were used in the studies.

Firstly, primary rat HSCs were isolated, and ~2×10⁸ HSCs were harvested from each rat. The fraction of freshly isolated living HSCs was ≤90%, as defined by trypan blue staining. The morphology and growth characteristics of the freshly isolated cells were observed with an inverted phase contrast microscope. The cells were small and round with the quiescent phenotype when freshly isolated; however, they demonstrated a weak adhesive growth pattern following culture for two days and presented a wall-adhesive growth pattern following culture for ten days (Fig. 2Aa–c). IFA and western blot analysis of α-SMA were applied for demonstrating the activated phenotype of HSCs. The IFA results demonstrated that the cells expressed no or little α-SMA on day 2 (Fig. 2Ad) and expressed abundant α-SMA on day 10 (Fig. 2Ad). Western blot analysis also demonstrated that the expression of α-SMA was increased progressively with the time in culture, and reached the highest expression at day 10 (Fig. 2B). These results indicated that following culture for ten days, HSCs had been highly activated.

Next, the expression of miR-34a and miR-34c in the rat HSCs was detected by qPCR. As demonstrated in Fig. 2C, the expression of miR-34a and miR-34c in rat HSCs revealed a significant ~15-fold and 5-fold increase, respectively, following culture for seven days, and peaked with the highest expression at day 10, with an increase of ~20-fold and 10-fold, respectively. The expression of miR-34a was always marginally higher than that of miR-34c.

Next, the expression of PPARγ was detected by western blot analysis. As demonstrated in Fig. 2B, PPARγ decreased progressively with the time in culture and demonstrated a negative correlation with the expression of miR-34a/c and α-SMA.

The expression of miR-34a/c and PPARγ was also analyzed during the activation of human HSC LX-2 cells. TGF-β1 is one of the critical factors for the activation of HSC during chronic inflammation (34). LX-2 cells were treated with TGF-β1 for the indicated time (from 0 to 48 h) in 5 ng/ml or in the indicated concentration (from 0 to 5 ng/ml) for 48 h. The upregulated expression of α-SMA in both conditions indicated the activation of LX-2 by TGF-β1 stimulation (Fig. 2D). In addition, the expression of PPARγ, miR-34a and miR-34c demonstrated changes that were consistent with the results in the rat HSCs during the activation of LX-2 (Fig. 2D and E). In conclusion, during activation of HSCs, miR-34a and miR-34c were upregulated and PPARγ was downregulated, and the expression of PPARγ was negatively correlated with the expression of miR-34a and miR-34c and the degree of HSC activation.

To further investigate the association between the miR-34 family and PPARγ and their effect on the activation of HSCs, activated HSCs were transfected with miR-34a and miR-34c inhibitors alone or together, and the expression of PPARγ and α-SMA was detected. As demonstrated in Fig. 3A, PPARγ was upregulated following transfection with miR34a and miR34c alone or together, compared with the negative control miRNA in TGF-β1-stimulated LX-2 cells, while α-SMA was downregulated in HSCs transfected with miR-34a/c compared with negative control microRNA, which suggested that activation of HSCs was reversed. Furthermore, application of the miR34a inhibitor resulted in higher expressional regulation than the miR34c inhibitor did, and the two inhibitors in combination induced the highest expressional regulation. However, in the activated rat HSCs, no expression change was observed (Fig. 3B). These results indicated that the miR-34a family accelerated the activation of human HSCs by modulating PPARγ, and the decreased expression of PPARγ may have resulted from differential regulatory mechanisms in rat and human HSCs.