Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells

Abstract

Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

Introduction

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths.^1,2 Although chemoembolization and liver transplantation had improved the survival of patients with advanced HCC, the insensitiveness of the chemotherapeutic drugs and recurrence and metastasis still contribute to a poor prognosis.^3,4 Therefore, the identification of targets is important for clarifying the mechanisms underlying HCC progression and testing future therapeutic strategies.

Recent accumulated evidence has revealed that protein arginine N-methyltransferases (PRMTs) are responsible for the transfer of a methyl group from S-adenosylmethionine (SAM) to arginine residues. ⁵ PRMT1, one of the most important types of protein in PRMT family, plays a pivotal role in various cellular processes, including intracellular protein–protein interactions, histone function, transcriptional regulation, and DNA repairing.^6,7 Moreover, PRMT1 was found to be involved in disease progression and prognosis in various solid tumors, and inhibition of PRMT1 significantly suppressed the proliferation potency of various cancer cells.^8,9 Nevertheless, the relevance of PRMT1 in HCC patients has not yet been clarified. In this study, we show that PRMT1 is upregulated in HCC tissues and cells, and this increased expression contributes to HCC cell growth and metastasis. This study identifies a new and important role for PRMT1 in HCC malignant transformation and suggests that PRMT1 may be a novel target for the treatment of HCC.

Materials and methods

Human tissue specimens

All the clinical HCC tissues and their corresponding noncancerous liver tissues used in this study were obtained from Guangdong General Hospital after surgical resection. Histological examination confirmed a diagnosis of HCC in all patients. Informed consents were obtained from each patient to approve the use of their tissues for research purposes. The study protocol was approved by the Institute Research Ethics Committee at Guangdong General Hospital. In total, 28 paired HCC tissues and the corresponding noncancerous liver tissues were collected and immediately snap-frozen and used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis.

Cell culture

Human liver cancer cell lines HepG2, Huh7, and Hep3B, and a normal human liver cell line L02 were obtained from the American Type Tissue Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 100 mg/mL penicillin, and 100 mg/mL streptomycin (Gibco 15140122, Grand Island, NY, USA) at 37°C with 5% CO₂.

qRT-PCR

Total RNA was extracted from tissues and cells using TRIzol Reagent (Invitrogen), and complementary DNA (cDNA) was synthesized using Primescript RT Reagent (TaKaRa). qRT-PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using TaqMan probes for PRMT1 and β-actin. β-actin was used as a reference to obtain the relative fold change for targets using the comparative Ct method. The sequences of the primers (Sangon, Shanghai, China) used in this study were as follows: PRMT1 (forward: 5′-GAGTTCACCCGATGCCACAAG-3′, reverse: 5′-TCCGGTAGTCGGTGGAACAAG-3′) and β-actin (forward: 5′-GAGAGGGAAATCGTGCGTGAC-3′, reverse: 5′-CATCTGCTGGAAGGTGGACA-3′).

Western blot analysis

A total of 30 µg of protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Following blocking with 5% skim milk in tris-buffered saline with 0.1% Tween 20 (pH 7.6 TBST), the membranes were incubated with polyclonal (rabbit) anti-PRMT1, anti E-cadherin, anti-N-cadherin, anti-Vimentin (Santa Cruz Bio-technology, Santa Cruz, CA, USA), and anti-β-actin antibody (Cell Signaling Technology). Goat anti-rabbit IgG (Pierce, Rockford, IL, USA) secondary antibody conjugated to horseradish peroxidase and enhanced chemiluminescence (ECL) detection systems (SuperSignal West Femto, Pierce) were used for detection.

RNA interference of PRMT1 expression

PRMT1-specific small interfering RNA (siRNA) 1 (sense sequence, GUGAGAAGCCCAACGCUGAtt), PRMT1-specific siRNA2 (sense sequence, CCGUCAAGGUGGAAGACCUtt), and a negative control siRNA (si-NC) were purchased from Bonac (Kurume, Japan). Cells were seeded at 2 × 10⁵ cells per well in a volume of 2 mL in six-well flat-bottom plates and incubated in a humidified atmosphere of 5% CO₂ at 37°C. After 24 h of incubation, the siRNAs were mixed with 200 µL of Opti-MEM™ medium (Life Technologies, Carlsbad, CA, USA) and 4 µL of Lipofectamine RNAiMAX (Life Technologies) and incubated for 20 min. Next, the reagents and 800 µL of Opti-MEM were added to each well. After transfected and cultured for 48 h, cells were collected for western blot and qRT-PCR analyses.

Cell viability assay

Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Measurements were made at 570 nm relative to basal absorbance at 630 nm using Victor3™ multilabel counter (Perkin Elmer, Waltham, MA, USA). Two independent experiments in quintuplicate were performed, and each measurement was expressed as a percentage of maximum absorbance.

Wound healing assay

Wound healing assay was adopted to test the migration ability of HCC cells. In our study, cells were digested after transfection by specific siRNA and control siRNA to PRMT1 for 24 h in 6-well plates, 2 × 10⁵ cells were plated in 24-well plates; when cell confluence reached approximately 100%, the old medium was removed and the monolayer was wounded by scratching with a 10-µL sterile pipette tip lengthwise along the chamber, then cells were washed three times with phosphate-buffered saline (PBS) and cultured with serum-free medium at 37°C. Images of cells migrating into the wound were photographed at 0 and 48 h using an inverted microscope. Wound width (µm) was measured using OpenLab software. Wound healing rate = (0-h scratch width − 48-h scratch width)/0-h scratch width × 100%. The experiments were repeated three times.

Invasion assay

The cell invasiveness through Matrigel (BD Biosciences, Sparks, MD, USA) and cell motility through Transwell (Corning, Inc., Cambridge, MA, USA) were assessed. Cells were cultivated to 80% confluence on the 12-well plates. Then, we observed the procedures of cellular growth at 24 h. Cells invaded to the membrane underside were scored for at least 10 microscope fields (original magnification, ×400). Each experiment was conducted in duplicate inserts, and the mean value was expressed from three independent experiments.

Statistical analysis

All statistical analyses were performed with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Data are expressed as means ± standard deviation (SD). Significant differences between the groups were determined using Student’s t-test. A value of p < 0.05 was considered to indicate a statistically significant difference.

Results

PRMT1 is overexpressed in human HCC patients

To investigate whether PRMT1 is highly expressed in HCC tissues, we detected the expression of PRMT1 in fresh HCC tissues and adjacent normal tissues from 28 cases of HCC patients. The results of qRT-PCR showed that the expression levels of PRMT1 messenger RNA (mRNA) were higher in HCC tumor tissues than in the normal pericarcinomatous tissue, the difference between them was statistically significant (p < 0.01; Figure 1(a)). These results indicated that PRMT1 is commonly overexpressed in HCC. The expression of PRMT1 was also examined by qRT-PCR and western blot in a panel of HCC cell lines and a normal human liver cell line L02. The result showed that PRMT1 expression was higher in HCC cell lines than in L02 (p < 0.05; Figure 1(b) and (c)). Among the HCC cell lines, the expression level of PRMT1 mRNA and protein was highest in the HepG2 cell line, whereas lowest in the Huh7 cell line.

Figure 1.

(a) Relative expression of PRMT1 was higher in HCC tissues as compared to the normal pericarcinomatous tissues, as determined by qRT-PCR. Values are represented as mean ± SEM (**p < 0.01), (b) PRMT1 mRNA expression levels in a panel of human HCC cell lines, and (c) PRMT1 protein expression in a panel of human HCC cell lines.

Knockdown of PRMT1 inhibited the proliferation, migration, and invasion of HCC in vitro

To examine the effect of PRMT1 expression on the development of malignant characteristics in HCC cells, we performed targeted knockdown of PRMT1 expression using RNA interference in HepG2 and Hep3B cells, and the proliferation, invasion, and migration of HCC cells were evaluated. Nonspecific siRNA was used as a negative control. As shown in Figure 2, HepG2 and Hep3B cells showed a significant decreased expression of PRMT1 mRNA and protein after transfection with si-PRMT1 compared to the si-NC group (p < 0.01), suggesting that we successfully downregulated the PRMT1 expression in HCC in vitro. MTT assays showed that the growth of HepG2 and Hep3B cells transfected with si-PRMT1 was impaired compared with control cells (Figure 3(a) and (b)). Knockdown of PRMT1 inhibits the migration and invasion of HCC cells. A wound-healing assay conducted on HCC cells transfected with si-PRMT1 showed that they underwent a slower closing of scratch wounds compared with control cells (Figure 4(a) and (b)). We then used Matrigel to evaluate cancer cell invasion and found that invasion of HepG2 and Hep3B cells was significantly reduced following transfection with si-PRMT1, as shown by Transwell assays (Figure 4(c) and (d)).

Figure 2.

(a and b) HepG2 and Hep3B cells were transfected with si-PRMT1 or si-NC. PRMT1 mRNA expression was analyzed by qRT-PCR, (c and d) HepG2 and Hep3B cells were transfected with si-PRMT1 or si-NC. PRMT1 protein expression was analyzed by western blotting.

Figure 3.

Knockdown of PRMT1 inhibits the proliferation of HepG2 and Hep3B cells. (a and b) Cell proliferation was measured by MTT assay.

Figure 4.

(a and b) Effect of PRMT1 interference on the ability of migration of HepG2 and Hep3B cells according to the scratch-wound assay and (c and d) effect of PRMT1 interference on the ability of invasion of HepG2 and Hep3B cells according to Transwell assays.

Knockdown of PRMT1 reverses epithelial–mesenchymal transition in HCC cell lines

Epithelial–mesenchymal transition (EMT), an early step of tumor cell migration, is important to evaluate cancer aggressiveness and metastatic potential. To understand whether PRMT1 regulates the EMT of HCC cells, we detected the expression level of EMT markers, Vimentin, N-cadherin, and E-cadherin, in HepG2 and Hep3B cells under siRNA treatment. The results of western blotting indicated that E-cadherin expression was significantly increased, while expression of N-cadherin and vimentin was greatly reduced following transfection with si-PRMT1 (Figure 5(a) and (b)).

Figure 5.

(a) Knockdown of PRMT1 reverses EMT in HepG2 cells and (b) knockdown of PRMT1 reverses EMT in Hep3B cells.

Discussion

HCC is currently one of the leading causes of cancer deaths in industrialized countries, and its incidence appears to be increasing.^10,11 Even though outcomes in patients with HCC have been improved by the use of potentially curative resection and adjuvant treatments, such as chemotherapy and biological therapies, early recurrence and metastasis still occur in a substantial proportion of them after these treatments.^12,13 Clarifying the molecular mechanisms involved in carcinogenesis and recurrence that could reflect the metastasis and recurrence of HCC will be necessary.

Accumulated studies revealed PRMT1 is one of the most important types of protein in PRMT family, which play a crucial role in several cellular processes including signal transduction.^14–16 Extensive studies demonstrated that elevated PRMT1 expression is found in a variety of cancers.^9,17,18 In breast cancer cells and clinical samples, the tumor suppressor BRCA1 was methylated by PRMT1, which inhibited its transcriptional function. ¹⁹ However, the role and mechanism of PRMT1 in HCC remain unclear. This study suggests that PRMT1 plays important roles in invasion and metastasis of HCC. Blocking PRMT1 expression was able to suppress growth and metastasis of HCC in vitro. More important, we found PRMT1 is a pivotal modulator of the molecular and functional characteristics of EMT.

HCC clinical samples validate the conclusions that PRMT1 is upregulated in HCC tissues. The qRT-PCR analysis showed that the PRMT1 expression was much higher in HCC tissues compared with adjacent noncancerous tissues. Furthermore, our data demonstrated that PRMT1 protein level was higher in HepG2 and Hep3B cells, which corresponds with its high invasion ability. Inhibition of PRMT1 significantly suppresses growth of various cancer cells. In our study, knockdown of PRMT1 by siRNA could inhibit cell proliferation, invasion, and migration of HCC cells. Of these signatures, PRMT1 is considered as an ideal target for anti-cancer therapy.

The EMT has a crucial role in cancer metastasis. During the EMT process, the expression of epithelial markers (such as E-cadherin and β-catenin) is downregulated, whereas the expression of mesenchymal markers (such as vimentin and fibronectin) is upregulated. Recent studies showed that PRMT1 is a novel regulator of EMT in non-small cell lung cancer. ²⁰ Silenced PRMT1 significantly inhibited lung cancer cell migration by increasing E-cadherin expression and decreasing N-cadherin expression. To explore the role of regulation of the EMT of HCC cells, we detected the expression level in the EMT process and investigated the expression of three EMT-related proteins—E-cadherin, N-cadherin, and Vimentin—by Western blot. As shown in Figure 5, the expression of E-cadherin was increased in si-PRMT1 group compared with NC group, indicating that suppressed expression of PRMT1 might inhibit HCC cell migration and invasion by regulating EMT.

In summary, this study for the first time identified the oncogene role of PRMT1 in HCC development and progression, suggesting that PRMT1 could be an ideal candidate for molecular targeted therapy in HCC. Further studies may contribute to a clearer understanding of the PRMT1-related pathways in HCC.