Introduction

Hepatocellular carcinoma (HCC) [6] is a primary cancer of the liver. It is the fifth most common malignant cancer and the third leading cause of cancer-related death. Worldwide, there were about 626,000 new HCC cases and nearly 600,000 HCC-related deaths each year [6]. Molecular profiling of changes in gene expression has improved our understanding of the HCC mechanism, allowing the identification of biomarkers for HCC diagnosis and HCC patient stratification for prognosis and therapy. Recently, a new group of molecules, microRNAs, has been discovered to be aberrantly expressed in HCC and some of them are functionally involved in HCC carcinogenesis and progression. Further, certain microRNAs are associated with HCC or related to HCC subtypes, implying the potential of microRNAs for HCC patient stratification of diagnosis and prognosis [7].

MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs of approximately 18–25 nucleotides (nt). miRNAs play important roles in post-transcriptional regulation of gene expression by targeting mRNAs [1, 2]. Recently, new human miRNAs have been identified in human genome implicated in development of different cancers (more than 700 distinct miRNAs). They are involved in the control of various biological processes like development, metabolism, signal transduction, cell differentiation, apoptosis, and carcinogenesis [3–5].

Up-regulated gene 4/Upregulator of cell proliferation (URG4/URGCP), a novel gene induced by hepatitis-B-virus-encoded X antigen (HBxAg), was identified by polymerase chain reaction (PCR) select cDNA subtraction, using HBxAg-positive and HBxAg-negative HepG2 cells. URG4/URGCP located on 7p13 and originally identified by Satiroglu-Tufan NL, encodes 922 amino acids. Over-expression of URG4/URGCP in HepG2 cells and GES-1 cells promoted hepatocellular growth and survival in tissue culture and in soft agar [8, 9]. These results showed that over-expression of URG4/URGCP accelerated tumor development in nude mice and also suggest that URG4/URGCP may be associated with initiation of tumorigenesis. Over-expression of URG4/URGCP in osteosarcoma tissues either correlates with tumor recurrence and metastasis, as well as with the proliferative activity of osteosarcoma cells. Patients with high expression of URG4/URGCP had shorter survival time, suggesting that URG4/URGCP may be a valuable prognostic marker in osteosarcoma patients [9–11]. URG4/URGCP is also strongly expressed in hepatocellular carcinoma, gastric cancer and osteosarcoma. Hence, URG4/URGCP may be a putative oncogene that contributes importantly to multistep carcinogenesis.

In this study, we investigated the cellular miRNA expression changes in HepG2 cell line in the presence of URG4/URGCP and in the absence of URG4/URGCP, which was suppressed by RNA interference. To our knowledge, this is the first study analyzing miRNA expression changes in the presence or absence of URG4/URGCP. Results may reveal important roles of miRNAs in the URG4/URGCP gene related disease development through the regulation of different signaling pathways.

Materials and methods

Cell lines and tissue culture conditions

The human hepatoblastoma cell lines, HepG2 (ATCC, Manassas, VA, USA), HepG2-pcDNA3, and HepG2-pcDNA3-URG4/URGCP were cultured at 37 °C with 5 % CO2 on type-1 rat tail collagen (Serva)-coated tissue culture dishes or plates. Cells were grown in Earle’s modified Eagle’s medium (Biological Industries, Beit Haemek, Israel) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) (Biological Industries), 100 mM minimal essential medium non-essential amino acids (Biological Industries), 1 mM sodium pyruvate (Biological Industries), l-glutamine as well as standard concentrations of penicillin (10,000 units/ml) plus streptomycin (10 mg/ml) (Biological Industries).

Plasmids

pcDNA3 (Invitrogen, San Diego, CA, USA) was used to clone full-length URG4/URGCP cDNA under the control of the immediate early cytomegalovirus (CMV) promoter [11] and in the construction of URG4/URGCP-overexpressing HepG2 cells. To study the properties of URG4/URGCP at the cellular level, separate cultures of 1 × 106 HepG2 cells were stably transfected with 10 μg pcDNA3 or pcDNA3-URG4/URGCP using SuperFect transfection reagent (QIAGEN, Valencia, CA, USA) according to manufacturer’s instructions. Cells were selected with G418 (800 mg/ml) for 4 weeks. URG4/URGCP mRNA expression was confirmed by semi-quantitative RT-PCR.

RNAi-mediated URG4/URGCP silencing in HepG2 cells

To study the effects of RNAi-mediated URG4/URGCP silencing, HepG2-pcDNA3 cells were seeded in 6-well plates in duplicate (2.5 × 105 cells/per well) or in 96-well plates in triplicate (1 × 104 cells/per well) and incubated at 37 °C with 5 % CO2 overnight. Separate cultures of HepG2-pcDNA3 cells were then transfected with chemically synthesized siRNA reagents for URG4/URGCP (ON-TARGETplus SMARTpool human URG4/URGCP siRNA, Dharmacon, Lafayette, CO, USA) using Dharmafect I transfection reagent (Dharmacon) according to manufacturer’s instructions. At 48 h after the siRNA transfection, RNA was isolated and the expression level of URG4/URGCP mRNA was determined by semi-quantitative RT-PCR with URG4/URGCP specific primers (Table 1).

Table 1 Primers used for semi-quantitative RT-PCR
Full size table

Real time qPCR reaction for miRNAs

48 h after siRNA transfection, total RNA was isolated with tri-reagent (Sigma, St. Louis, MO, USA) according to manufacturer’s instructions. cDNA synthesis was performed by using poly (A) tailing kit procedure (AM1350, Ambion) and miRNA cDNA synthesis kit (G269, Applied Biological Material, mandatory). Relative quantification of whole genome miRNAs was analyzed by RT-PCR using human whole genome miRNA qPCR profiling kits (ABM catalog # MA003-human). SNORD48, SNORD47, SNORD44, U6 were used as human endogenous controls.

Western blot analysis

48 h after transfection, the cells were washed with PBS and then were lysed in ice-cold. Protein samples (100 μg protein) were separated on 8.5 % polyacrylamide gels. The membranes were incubated with rabbit polyclonal IgG URG4 primary antibodies (1:4,000 dilution) and incubated with the secondary antibody horseradish peroxidase-conjugated secondary antibody (1:12,000). Proteins were detected using SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA), and bands were visualized and recorded using GelQuant image analysis software in a DNR LightBIS Pro image analysis system (DNR Bio-Imaging Systems Ltd. Jerusalem, Israel). Protein bands were quantitated using Scion Image Version Beta 4.0.2 software [25].

Data analysis

Data analysis was evaluated with ∆∆CT method, “LightCycler 480 Quantification Software” program and statistical analysis was evaluated with web based RT2 Profiler PCR array data analysis which is assessed statistically using the “student t test”

Results

Construction of URG4/URGCP overexpressing HepG2 cells

To functionally characterize URG4/URGCP, independent cultures of HepG2 cells were stably transfected with pcDNA3 or pcDNA3-URG4/URGCP, and each of the cell lines were selected in G418. The level of URG4/URGCP mRNA was then determined in each of the cell lines by RT-PCR. The level of URG4/URGCP expression was higher in HepG2-pcDNA3-URG4/URGCP cells (Fig. 1).

Fig. 1
figure 1

Expression of URG4/URGCP mRNA in HepG2 cells stably transfected with pcDNA3, pcDNA3-URG4 cells. One-step multiplex RT-PCR analysis was carried out on RNA isolated from HepG2 (lane 1), HepG2-pcDNA3 (lane 2), and HepG2-pcDNA3-URG4 (lane 3) cells using URG4/URGCP specific primers. M marker

Full size image

RNAi-mediated URG4/URGCP gene silencing in HepG2 cells

In this study, the effects of RNAi-mediated URG4/URGCP gene silencing were determined. To confirm the RNAi-mediated URG4/URGCP silencing in HepG2 cells, mRNA expression of URG4/URGCP was determined in HepG2-pcDNA3, HepG2-pcDNA3 + URG4/URGCP siRNA, HepG2-pcDNA3-URG4/URGCP. As a result of URG4/URGCP siRNA transfection, the expression level of URG4/URGCP mRNA and protein was reduced in HepG2-pcDNA3 + URG4/URGCP siRNA cells compared to that in HepG2-pcDNA3 cells (Figs. 2, 3).

Fig. 2
figure 2

Expression of URG4/URGCP mRNA in RNAi-mediated URG4/URGCP-silenced HepG2 cells. One-step multiplex RT-PCR analysis was carried out on RNA isolated from HepG2-pcDNA3 (lane 1), HepG2-pcDNA3 + URG4 siRNA (lane 2), and HepG2-pcDNA3-URG4 (lane 3) cells using URG4/URGCP specific primers (48 h after siRNA transfection)

Full size image
Fig. 3
figure 3

Expression of URG4/URGCP protein in RNAi-mediated URG4/URGCP-silenced HepG2 cells. Lane 1: HepG2-pcDNA3 + URG4 siRNA, Lane 2: HepG2-pcDNA3, Lane 3: HepG2-pcDNA3-URG4 (48 h after siRNA transfection)

Full size image

miRNA expression profiles in the presence or absence of URG4/URGCP

To investigate potential miRNA expression changes in the presence or absence of URG4/URGCP, miRNA was extracted from HepG2-pcDNA3, HepG2-pcDNA3-URG4/URGCP HepG2-pcDNA3 + URG4/URGCP siRNA cells and miRNA expression changes was analyzed by LightCycler 480 RT-PCR. It is well recognized that appropriate reference genes for normalization is critical for genome-wide quantitative gene expression analyses. Housekeeping small RNAs that were stably expressed across different tissue types are commonly used as endogenous controls in miRNA profiling studies. However, it is still uncertain whether these reference genes are differentially expressed. With this concern, we normalized the miRNA expressions data with a panel of four commonly used endogenous controls, SNORD48, SNORD47, SNORD44 and U6. Among the 1,034 human miRNAs investigated by the arrays, 77 miRNAs were up-regulated (the most up-regulated miRNAs: miR-1274b, miR-4271, miR-4286, miR-3926, miR-4267; p < 0.05) and nine miRNAs were down-regulated (the most down-regulated miRNAs: miR-3664, miR-3663-5p, miR-3134; p < 0.05) in the presence of URG4/URGCP (Tables 2, 3).

Table 2 Up-regulated miRNAs with in the presence of URG4/URGCP
Full size table
Table 3 Down-regulated miRNAs with in the presence of URG4/URGCP
Full size table

Discussion

Hepatocarcinogenesis is a multistep process that is consisted by accumulation of molecular alterations from background liver disease. The involvement of miRNA deregulation in human carcinogenesis has been potentially identified. Previous array analyses have clearly demonstrated that the miRNA expression profile is essentially altered in cancer samples [12, 13]. miRNA deregulation is an early event in human hepatocarcinogenesis and almost 30 % of protein-coding genes are predicted to be regulated by miRNAs [14]. In addition, the levels of some miRNAs have arisen as biomarkers for various pathological situations. In this study we evaluated the expression profiles of cellular miRNA expression changes in HepG2 cell line in the presence or absence of URG4/URGCP gene expression. The expression levels of the 77 miRNAs were up-regulated and nine miRNAs were down-regulated in URG4/URGCP over-expressed HCC cells compared with URG4/URGCP suppressed cells by RNA interference. With the development of miRNA detection technologies, these results may serve as indicators of disease progression and prognosis in URG4/URGCP gene related disease development. Research data show that miRNA expression emerged to be deregulated in cancer via normal tissue. For example, miRNA deregulation has been shown in B cell lymphoma, chronic lymphocytic leukemia, hepatocellular carcinoma, and breast cancer [15–17]. Several recent studies have performed miRNA profiling in human HCC. The miRNAs are aberrantly expressed in HCC and explanation of their contribution to the pathophysiology of HCC which is likely to be useful in establishing a role for miRNA. These profiling studies’ findings which are associated with cancer or pre-cancerous changes can be identified.

miRNA expression has been studied in HCC tissues in comparison with non-neoplastic liver tissues. Among the miRNAs that are aberrantly expressed are several that are increased in expression, including miR-21, miR-181, and miR-221/-222, whereas others are significantly decreased in expression, such as miR-29, miR-101, miR-122, miR-148a, miR-200, and mir-199a [18–22]. Thus, expression profiling may be useful for the diagnosis of HCC or prognosis.

Some miRNAs are involved in HCC carcinogenesis by controlling cell proliferation and apoptosis; others are associated with HCC progression by controlling cell migration and invasion. Tan et al. [22] suggested that their study provides mechanistic insight of the consequence of reduced expression of miR-198 in HCC cells and its connection with HCC invasion. miR-198, as a conditional metastasis suppressor in HCC cells. These findings will facilitate a better understanding of the molecular pathogenesis of HCC and suggest that reduction of miR-198 expression may be utilized as novel strategy for HCC therapy. Furuta and his colleagues’ results showed that miR-124 and miR-203 are novel tumor suppressive miRNAs for HCC epigenetically silenced and activating multiple targets during hepatocarcinogenesis [23]. Remarkable progress has been made in the techniques used to identify the differences in gene expression between cells. Previously, Satiroglu-Tufan et al. [8], have analyzed the differentially expressed genes in the presence of HBxAg in HepG2 cells, and showed that HBx up-regulates the expression of a novel gene. URG4/URGCP is differentially expressed in tumor compared to non-tumor liver specimens from HBV-infected HCC patients. Over-expression of URG4/URGCP in HepG2 and GES-1 cells promoted cell growth and survival in tissue culture and soft agar, and accelerated tumor development in nude mice, suggesting that URG4/URGCP may be associated with the onset of tumorigenesis [8, 9, 24]. Over-expression of URG4/URGCP in osteosarcoma tissues is well correlated with tumor recurrence and metastasis, as well as with the proliferative activity of osteosarcoma cells [10].

In conclusion, our results showed that URG4/URGCP overexpression led to altered expression of some cellular miRNAs. Some of these miRNAs may play roles in the URG4/URGCP gene related disease development through the regulation of different signaling pathways. This knowledge may be very helpful in understanding of the mechanism of URG4/URGCP gene function and its interaction within the cell. Thus, our data suggests that expression changes in miRNAs of miR-1274b, miR-4286, miR-3926, miR-4271, miR-320d, miR-3134, miR-3663-5p, miR-3664 in the presence or absence of URG4/URGCP gene expression may be important in URG4/URGCP gene related disease development need to be investigated with further studies. In addition, miRNAs would provide easily accessible, sensitive, reliable data resources to shed light on the research of early stage HCC development and progression. Combined with other technologies would identify novel miRNA biomarkers that could be used for more accurate early detection, prognosis and targeted therapy. Importantly, scientific approaches on the functional investigation of miRNAs and their targets will elucidate the intrinsic molecular mechanisms of HCC development and progression.