MicroRNA-33a promotes cell proliferation and inhibits apoptosis by targeting PPARα in human hepatocellular carcinoma

Introduction

MicroRNAs (miRNAs) are an endogenous group of small (18–25 nucleotides), non-coding RNA molecules which serve key roles as post-transcriptional regulators by binding to the 3′-untranslated region (3′UTR) of target mRNA (1). Recent results indicate that miRNAs may modulate numerous cellular processes, including differentiation, proliferation, apoptosis and migration (2). Thus, miRNAs are considered to be potential regulators in the development and progression of various human diseases, including cardiovascular disease, diabetes and cancer (3–5).

Hepatocellular carcinoma (HCC) is among the most severe forms of human cancer and is the third-leading cause of cancer-related mortality worldwide (6). Although treatments for HCC have improved, the long-term prognosis for patients with HCC remains poor, with a current 5-year survival rate of ~30% (7,8). A lack of biomarkers for early diagnosis and effective therapeutic targets are primary reasons for the poor disease outcomes. Therefore, studies into the molecular mechanisms of HCC pathogenesis are warranted, in order to identify potential biomarkers and therapeutic targets of HCC (9,10). Recently, the roles of miRNAs in HCC have been investigated, with results indicating that the expression and/or function of miRNAs become aberrant in the pathogenesis of HCC (7).

miR-33a is a member of the highly conserved miR-33 family and is an intronic miRNA located within the genes of sterol regulatory element-binding proteins (11,12), where it principally regulates the metabolism of cholesterol (13) and glucose (14). In addition to its functional roles in metabolism, miR-33a has been implicated in a number of human cancers. In pancreatic ductal adenocarcinoma, it has been observed that miR-33a exerts tumor suppressive effects, by modulating the growth, apoptosis, epithelial-to-mesenchymal transition and chemoresistance of pancreatic cancer cells (15,16). Similarly, a previous study in lung cancer demonstrated that miR-33a had inhibitory effects on the metastasis of cancer cells towards bone tissue (17). It has also been observed in glioma cancer that miR-33a promotes the growth and self-renewal of glioma-initiating cells (18), while previous microarray results have indicated that miR-33a is upregulated in supraglottic carcinoma (19). In osteosarcoma (OS), miR-33a is upregulated in chemoresistant OS and promotes resistance of OS cells to cisplatin through downregulation of the transcription factor, Twist (20). Furthermore, levels of miR-33a and miR-224 expression were elevated in steatotic chronic hepatitis C when compared to control liver tissue (21), and miR-33a in liver tissue has been found to significantly increase in a fibrosis progression-dependent manner (22). Collectively, these data indicate an oncogenic role of miR-33a, though its clinical significance and potential roles in HCC remain unknown.

Therefore, the present study investigated the effects of miR-33a and its underlying mechanisms of action in HCC using BrdU and apoptosis assays as well as dual luciferase reporter assays.

Materials and methods

Human tissues and cell culture

A total of 86 individual primary HCC tissues were collected between January 2010 and January 2012 from patients at The First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China). Patients were monitored for a median time of 31.6 months (range, 2–60 months). Clinical features of the 86 patients are listed in Table I. Age, gender, HBV infection, serum AFP level, tumor size and number of tumor nodules were measured prior to surgery, while other parameters were collected after surgery. Clinical specimens were immediately snap-frozen in liquid nitrogen prior to histological examination. Exclusion criteria included patients who had received chemotherapy or embolization prior to surgical resections. Samples were attained after obtaining informed consent from all patients and all protocols in the present study were approved by the Medicine Ethics Committee of Xi'an Jiaotong University in accordance with the Declaration of Helsinki (as revised in Tokyo 2004) (23).

Table I. Clinical association analysis of miR-33a expression in HCC.

Table I.

Clinical association analysis of miR-33a expression in HCC.

		No. of patients (n=86)
Clinicopathological features	No. of patients (n=86)	Low miR-33a	High miR-33a	P-value
Age (years)
<50	27	16	11	0.245
≥50	59	27	32
Gender
Male	69	34	35	0.787
Female	17	9	8
HBV
Absent	30	14	16	0.651
Present	56	29	27
Serum AFP level, ng/ml
<20	20	11	9	0.610
≥20	66	32	34
Tumor size, cm
<5	30	20	10	0.024
≥5	56	23	33
No. of tumor nodules
1	66	35	31	0.307
≥2	20	8	12
Cirrhosis
Absent	37	22	15	0.127
Present	49	21	28
Venous infiltration
Absent	42	23	19	0.388
Present	44	20	24
Edmondson-Steiner grading
I+II	49	31	18	0.005
III+IV	37	12	25
TNM tumor stage
I+II	61	35	26	0.033
III+IV	25	8	17

[i] HCC, hepatocellular carcinoma; HBV, hepatitis B virus; AFP, α-fetoprotein; TNM, tumor-node-metastasis.

Human HCC cell lines, HepG2 and Huh7 (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China), were cultured in complete Dulbecco's modified Eagle's medium (Biosera, Inc., Villebon sur Yvette, France) supplemented with 10% fetal bovine serum (Biosera, Inc.), 100 units/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Cell lines were cultured in a humidified atmosphere in a 5% CO2 incubator at 37°C for 2–3 days and collected for further analyses.

RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from frozen tissues or cultured HCC cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Quantification of miR-33a was performed using a TaqMan MicroRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and U6 small nuclear RNA was used as an endogenous control by determining the fold-change in miR-33a expression relative to U6 expression. For PPARα quantification, cDNA was synthesized using Taqman RT reagents (Applied Biosystems; Thermo Fisher Scientific, Inc.). Total RNA (2 µg) was reverse transcribed at 37°C for 15 min, and cDNA was incubated at 85°C for 5 sec to inactivate the reverse transcriptase. cDNA (2 µl) was used for the qPCR, which was performed using a SYBR Premix Ex Taq II Perfect Real Time kit (Takara Bio, Inc., Otsu, Japan) in the ABI PRISM 7300 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reactions were incubated at 95°C for 60 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. Expression of PPARα mRNA was normalized to that of GAPDH and the primer sequences were as follows: For PPARα forward, 5′-ACTGTTGCAAGAGATCTACAGAG-3′ and reverse, 5′-TTGTCTGTCACTGTCTGAATCC-3′ and for GAPDH forward, 5′-AACTTTGGCATTGTGGAAGG-3′ and reverse, 5′-ACACATTGGGGGTAGGAACA-3′. All samples were normalized to internal controls and fold changes were calculated based on relative quantification using the 2−ΔΔCq method (24).

Cell transfection

Overexpression and inhibition of miR-33a in Huh7 and HepG2 cells, respectively, was established using miRNA vectors obtained from GeneCopoeia, Inc. (Rockville, MD, USA). In accordance with the manufacturer's instructions for Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), Huh7 cells at 75% confluence were transfected with an miR-33a expression vector (HmiR0366-MR03) or an miRNA scrambled control vector (CmiR0001-MR03), and HepG2 cells were transfected with an miR-33a inhibitory vector (HmiR-AN0429-AM03B) or a negative non-coding vector (CmiR-AN0001-SN). The efficacy of miR-33a or anti-miR-33a vector transfection was assessed by RT-qPCR as outlined above. The primer sequences used for miR-33a were as follows: forward, 5′-CGCGCGTGCATTGTAGTTG-3′ and reverse, 5′-CACCAGGGTCCGAGGT-3′ and stem loop primer, 5′-TGGATATCCACACCAGGGTCCGAGGTATTCGGTGTGGATATCCATGCAATG-3′.

Cell proliferation and apoptosis assays

For the proliferation assay, transfected HCC cells were seeded into 96-well plates (1×103 cells/well) and incubated at 37°C. At 48 h post-transfection, the proliferative ability of HCC cells was assessed using a 5-bromo-2-deoxyuridine (BrdU) Cell Proliferation ELISA kit (Roche Diagnostics, Indianapolis, IN, USA), according to the manufacturer's instructions. For the apoptosis assay, HCC cells were seeded into 6-well plates (6×104 cells/well) and incubated at 37°C. At 48 h post-transfection, an Annexin-V-FLUOS Staining kit (Roche Diagnostics) was used to determine the percentage of apoptotic cells by flow cytometry, according to the manufacturer's instructions. Flow cytometric analysis was conducted using fluorescence-activated cell sorting Calibur (BD Biosciences, San Jose, CA, USA) and Cell Quest Pro v.4.0.2 software (BD Biosciences). Similar results were obtained in three independent experiments performed in duplicate.

Dual luciferase reporter assay

A dual luciferase reporter assay was performed to determine whether PPARα was a downstream target gene of miR-33a in HCC cells. Briefly, the 3′-UTR sequence of PPARα, which was predicted to interact with miR-33a using two publicly available databases (TargetScan 6.2, targetscan.org; miRanDa, microrna.org), or a mutated sequence within the predicted sites were synthesized (GeneChem Co., Ltd., Shanghai, China) and inserted into the XbaI and FseI restriction sites of a pGL3 control vector (luciferase reporter vector; Promega Corporation, Madison, WI, USA) downstream of a luciferase minigene, as previously reported (25). These constructs were named as wild-type (wt) PPARα-3′UTR or mutant (mt) PPARα-3′UTR, respectively. For the reporter assay, HepG2 cells that were seeded into 96-well plates (5×103 cells/well) were cultured in complete Dulbecco's modified Eagle medium (Biosera, Inc.) supplemented with 10% fetal bovine serum (Biosera, Inc.) at 37°C and co-transfected with miRNA mimics or inhibitors as outlined above, the above constructs and Renilla plasmid (Promega Corporation) using FuGENE HD Transfection Reagent (Promega Corporation), according to the manufacturer's protocol. At 48 h post-transfection, cells were harvested and Renilla and firefly luciferase activities were quantified using a Dual Luciferase Assay system (Promega Corporation), according to the manufacturer's protocol. Firefly luciferase activity was normalized to that of Renilla luciferase. Results were obtained from three independent experiments performed in triplicate.

Immunoblotting

HCC cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris pH 7.5, 150 mM sodium chloride, 1% Triton X-100, 5 mM ethylenediaminetetraacetic acid) at 4°C for 1 h, then insoluble material was removed by centrifugation at 12,000 × g for 10 min. A total of 30 µg of the resulting protein samples (per lane) were separated by 4–12% SDS-PAGE and transferred onto a nitrocellulose membrane. Blots were then incubated with primary antibodies against PPARα (sc-398394; 1:1,000; Santa Cruz Technology, Inc., Santa Cruz, CA, USA) and GAPDH (5174; 1:1,500; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C for at least 12 h. After three washes with Tris-buffered saline-Tween-20, blots were incubated with horseradish peroxidase-conjugated goat anti-mouse (sc-2005; 1:5,000; Santa Cruz Technology, Inc.) or anti-rabbit secondary antibodies (1662408; 1:10,000; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at room temperature for 2 h, detected using a Bio-Rad Gel imaging system and quantified using Quantity One v.4.1 software (both from Bio-Rad Laboratories, Inc.).

Statistical analysis

Data are expressed as the mean ± standard error of the mean. Group comparisons were conducted using a two-sample t-test or one-way analysis of variance (multiple comparisons). Categorical variables were compared using χ2 analysis or Fisher's exact test. Kaplan-Meier analysis was used to analyze overall survival and recurrence-free survival. SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) were used for statistical analysis and P<0.05 (two-tailed) was considered to indicate a statistically significant difference.

Results

Increased miR-33a expression in HCC correlates with adverse clinical features and poor prognosis

To elucidate the expression status and clinical significance of miR-33a in HCC, the levels of miR-33a were measured in HCC tissues and matched adjacent non-tumor tissues from 86 HCC patients. It was observed that levels of miR-33a were significantly higher in HCC tissues relative to adjacent non-tumor tissues (P<0.05; Fig. 1A). The clinical significance of miR-33a expression in HCC patients was subsequently investigated. Expression of miR-33a in HCC patients was determined to be low (n=43) or high (n=43) according to a cutoff value, which was defined as the median level of miR-33a in the patient cohort (0.087). As depicted in Table I, high levels of miR-33a expression were significantly correlated with larger tumor size (P=0.024), higher Edmondson-Steiner grading (poor differentiation; P=0.005) and higher tumor-node-metastasis tumor stage (P=0.033). Furthermore, a Kaplan-Meier analysis demonstrated that patients with high levels of miR-33a exhibited significant decreases in overall survival rate (P=0.015; Fig. 1B) and recurrence-free survival rate (P=0.008; Fig. 1C). These results suggest that miR-33a serves an oncogenic role and may be a prognostic indicator in HCC.

Figure 1. Expression and prognostic value of miR-33a in HCC. (A) Levels of miR-33a expression in HCC tissues relative to matched adjacent NT tissues obtained from 86 HCC patients. (B) Overall survival rate of patients with high vs. low levels of miR-33a expression. (C) Recurrence-free survival rate of patients with high vs. low levels of miR-33a expression. The median level of miR-33a expression in the 86 HCC samples, detected by reverse transcription-quantitative polymerase chain reaction, was used as a cutoff value to define high and low miR-33a expression. *P<0.05, vs. matched adjacent NT tissue, #P=0.015 and &P=0.008 vs. patients with low miR-33a expression. miR-33a, microRNA-33a; HCC, hepatocellular carcinoma; NT, non-tumor; U6, small nuclear RNA.

miR-33a induces proliferation and inhibits apoptosis in HCC cells

As increased proliferation and reduced apoptosis are key hallmarks of cancer cells (26), the oncogenic effects of miR-133a were subsequently determined by evaluating its effects on the proliferation and apoptosis of HCC cells. The HCC cell line Huh7 was transfected with an miR-33a expression vector or an miR control vector. As depicted in Fig. 2A, miR-33a expression was significantly upregulated in Huh7 cells transfected with the miR-33a expression vector, relative to cells transfected with the miR control vector (P<0.05). Subsequently, a BrdU incorporation assay indicated that forced expression of miR-33a in Huh7 cells led to a significant increase in cellular proliferation (P<0.05; Fig. 2B). In addition, an Annexin V/propidium iodide double staining assay demonstrated that the rate of apoptosis was significantly decreased in Huh7 cells overexpressing miR-33a (P<0.05; Fig. 2C). By contrast, transfection of the HCC cell line HepG2 with miR-33a inhibitors resulted in significantly decreased miR-33a expression, relative to cells transfected with a negative control vector (P<0.05; Fig. 3A). In turn, miR-33a downregulation lead to significantly decreased proliferation (P<0.05; Fig. 3B) and increased apoptosis (P<0.05; Fig. 3C) in HepG2 cells. Collectively, these results indicate miR-33a may promote the development and progression of HCC by potentiating proliferation and inhibiting apoptosis of HCC cells.

Figure 2. Overexpression of miR-33a promotes proliferation and reduces apoptosis in Huh7 cells. (A) Effects of transfection with miR-33a on the level of miR-33a expression in Huh7 cells. (B) BrdU incorporation assay demonstrating the proliferative capacity of Huh7 cells following upregulation of miR-33a. (C) An Annexin V/PI double staining assay demonstrating the effects of miR-33a overexpression in Huh7 cells on the apoptotic rate of cells. Three independent experiments were performed for each assay. Right upper quadrant displays late apoptotic cells and right lower quadrant displays early apoptotic cells. *P<0.05 vs. cells transfected with miR control vector. miR-33a, microRNA-33a; Huh7; hepatocellular carcinoma cell line; BrdU, bromodeoxyuridine; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 3. Down-regulation of miR-33a inhibits proliferation and increases apoptosis in HepG2 cells. (A) Transfection with miR-33a inhibitors significantly decreased the level of miR-33a expression in HepG2 cells. (B) A BrdU incorporation assay demonstrated that the proliferative capacity of HepG2 cells was decreased following downregulation of miR-33a in HepG2 cells. (C) An Annexin V/PI double staining assay demonstrated that inhibition of miR-33a in HepG2 cells significantly increased the apoptotic rate of cells. Right upper quadrant displays late apoptotic cells and right lower quadrant displays early apoptotic cells. Three independent experiments were performed for each assay. *P<0.05 vs. cells transfected with miR control vector. miR-33a, microRNA-33a; HepG2; hepatocellular carcinoma cell line; BrdU, bromodeoxyuridine; PI, propidium iodide; FITC, fluorescein isothiocyanate.

PPARα is a direct downstream target of miR-33a in HCC cells

To identify the underlying mechanisms by which miR-33a exerts its potential oncogenic effects in HCC cells, two publicly available databases, TargetScan 6.2 (www.targetscan.org) and miRanDa (www.microrna.org), were used to predict the target sequences of miR-33a. PPARα, which is considered to be a key regulator of HCC cell proliferation and apoptosis (27), was identified as a miR-33a target. As depicted in Fig. 4A, the 3′-UTR of PPARα mRNA contains a complementary sequence for miR-33a binding, suggesting that PPARα is a direct downstream target of miR-33a. Dual-luciferase reporter gene assays were subsequently performed to confirm whether miR-33a targets the 3′-UTR of PPARα mRNA, using wild type (wt) and mutant (mt) PPARα-3′UTRs (Fig. 4A). As shown in Fig. 4B, overexpression of miR-33a in HepG2 cells significantly inhibited the luciferase activity of PPARα expressing a wt 3′-UTR (P<0.05), while having no effect on that of mt PPARα-3′UTR. Accordingly, downregulation of miR-33a in HepG2 cells lead to significantly increased luciferase activity of wt PPARα-3′UTR (P<0.05), while having no significant effect on that of mt PPARα-3′UTR. These results indicate that PPARα is a direct downstream target of miR-33a.

Figure 4. PPARα is a direct downstream target of miR-33a in HCC cells. (A) A complementary sequence for miR-33a was identified in the 3′-UTR of PPARα. Dual-luciferase reporter gene assays were subsequently performed to confirm whether miR-33a targets the 3′-UTR of PPARα mRNA, using the wt and mt PPARα-3′UTR sequences shown. (B) Effects of overexpression of miR-33a on the luciferase activity of wt PPARα-3′-UTR and mt PPARα-3′-UTR. Three independent experiments were performed. *P<0.05. miR-33a, microRNA-33a; nt, nucleotides; wt, wild type; mt, mutant; PPARα, peroxisome proliferator-activated receptor-α; UTR, untranslated region.

miR-33a regulates the expression of PPARα in HCC cells

RT-qPCR and western blot analysis were subsequently performed to determine whether miR-33a regulates the expression of PPARα in HCC cells. It was observed that overexpression of miR-33a in Huh7 cells significantly decreased the levels of PPARα mRNA (P<0.05; Fig. 5A). Results of western blot analysis also demonstrated that the levels of PPARα protein was significantly reduced following forced expression of miR-33a (P<0.05; Fig. 5B). By contrast, downregulation of miR-33a in HepG2 cells lead to significantly increased PPARα expression at the mRNA (P<0.05; Fig. 5C) and protein (P<0.05, Fig. 5D) levels.

Figure 5. miR-33a regulates the expression of PPARα in HCC cells. (A) RT-qPCR and (B) western blot analysis of PPARα expression in Huh7 cells transfected with miR-33a mimic or miR control vectors. (C) RT-qPCR and (D) western blot analysis of PPARα expression in HepG2 cells transfected with miR-33a inhibitor or negative control vectors. PPARα expression levels are relative to GAPDH for both the RT-qPCR and western blot data. Three independent experiments were performed. *P<0.05 vs. respective control transfections. miR-33a, microRNA-33a; PPARα, peroxisome proliferator-activated receptor-α; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Discussion

miRNAs regulate gene expression at the post-transcriptional level and have been demonstrated to participate in numerous biological processes, including embryogenesis, differentiation, morphogenesis and tumorigenesis (1,3,4,28). In the last two decades, studies have investigated the potential roles of miRNAs in cancer. It has been indicated that miRNAs may have oncogenic or tumor-suppressive effects in human malignancies and may also serve as promising biomarkers and therapeutic targets in the diagnosis and treatment of cancer (3,29). miR-33a is an established regulator of glucose and cholesterol metabolism (14,30) and has been demonstrated to be an active regulator in the pathogenesis of various human cancers, including pancreatic cancer (15,16), lung cancer (17), glioma (18), osteosarcoma (31) and colon cancer (32). In the present study, significant overexpression of miR-33a was confirmed in HCC tissues. In turn, increased expression of miR-33a was associated with adverse clinical features and a poor prognosis for HCC patients. These results indicate that miR-33a may serve an oncogenic role in HCC and as a potential biomarker for the diagnosis and prognostic prediction of HCC.

Functionally, miR-33a is a key regulator of cholesterol metabolism through its manipulation of adenosine triphosphate-binding cassette transporter A1 levels (13). In addition, it regulates glucose metabolism through targeting of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (14). However, miR-33a is also implicated in numerous aspects of cancer biology. In pancreatic cancer cells, miR-33a has been demonstrated to enhance gemcitabine sensitivity (16), while in lung cancer, the miRNA acts a bone metastasis suppressor through its targeting of parathyroid hormone related protein (17). It has also been suggested that miR-33a may promote the self-renewal of glioma-initiating cells (18). In the present study, gain- and loss-of-function experiments demonstrated that miR-33a may promote the growth of HCC by potentiating proliferation and inhibiting apoptosis of HCC cells. Specifically, overexpression of miR-33a promoted proliferation and inhibited apoptosis of Huh7 cells, while inhibition of miR-33a decreased proliferation and increased apoptosis of HepG2 cells. Therefore, these results indicate that miR-33a may promote the development and progression of HCC, at least in part through modulation of cell proliferation and apoptosis.

PPARα is a member of the nuclear hormone receptor superfamily and participates in the metabolism of glucose and lipids (33). In addition, it has been identified as a tumor suppressor in colorectal carcinoma (34) and ovarian cancer (35). Regarding HCC, a previous study has demonstrated that PPARα exerted anti-tumorigenic effects in HCC cells through modulation of nuclear factor-κB signaling (27). It has also been observed in HCC cells that ectopic expression of PPARα significantly suppressed proliferation while inducing apoptosis (27). In the present study, a complementary sequence for miR-33a was identified in the 3′-UTR of PPARα. In turn, alterations in miR-33a expression led to significant changes in luciferase activity of wt PPARα-3′-UTR, while having no influence on that of mt PPARα-3′-UTR. Furthermore, ectopic expression of miR-33a significantly reduced the expression of PPARα, while downregulation of miR-33a led to a significant increase in PPARα expression. Collectively, these data indicate that PPARα is a direct downstream target of miR-33a in HCC cells. Thus, the regulatory effects of miR-33a on HCC cell proliferation and apoptosis may be due to its targeting of PPARα.

In conclusion, the present study demonstrated that miR-33a is overexpressed in HCC tissues, with elevated expression of miR-33a correlated with adverse clinical features and poor prognosis in HCC. Functional experiments also demonstrated that miR-33a may promote cell growth by modulating the proliferation and apoptosis of HCC cells. Furthermore, PPARα was verified to be a direct downstream target of miR-33a. Collectively, these data suggest that miR-33a may be a novel clinical biomarker for the diagnosis and prognosis of HCC, as well as a potential therapeutic target for the treatment of HCC.

References