Effects of Different siRNA Transfection Methods on Expression of ARID4B in HepG2 Cells

Xiaohui WANG, Mingyu GONG

Chengde Medical University, Chengde 067000, China

Abstract [Objectives] This study aimed to optimize the siRNA transfection method for ARID4B gene and lay a foundation for further study on the effect of ARID4B low expression on the biological function of liver cancer cells HepG2. [Methods] HepG2 cells were cultured for 0, 4 and 24 h, respectively and then transfected with ARID4B targeting siRNA. The mRNA expression of ARID4B gene was detected by RT-qPCR, and the protein expression was detected by Western blot. [Results] When HepG2 cells were transfected at 0 h after seeding, the expression of ARID4B was the lowest in both mRNA and protein levels, that is, the inhibition effect was the best. [Conclusions] Transfection at different time points after seeding of HepG2 cells had different inhibition effect on the expression of ARID4B. Transfection at 0 h after seeding showed the best inhibition effect.

Key words siRNA, HepG2 cell, ARID4B, Transfection method

1 Introduction

As one of the most common and most malignant tumors in the world, hepatocellular carcinoma (liver cancer for short) has received widespread attention for its high morbidity and mortality[1]. However, effective control measures are still lacking. Imbalance of oncogene and tumor suppressor gene expression is the basis of tumorigenesis. TheARID4Bgene is a member of the ARID family. It has been recently discovered thatARID4Bis highly expressed in liver cancer tissues compared with adjacent tissues[2]. A precious study found thatARID4Bwas highly expressed in human liver cancer cell line HepG2 than in human hepatic cell line LO2. It is suggested thatARID4Bmay play a role as an oncogene in the occurrence and development of liver cancer. At present, the impact ofARID4Bon liver cancer has not been clearly reported. In this study, liposome LipofectamineTM2000 was used to transfectARID4Btargeting siRNA into HepG2 cells to knock down the expression ofARID4Bgene, in order to investigate the effects of different transfection methods on the inhibition ofARID4Bexpression and lay a foundation for subsequent research.

2 Materials and methods

2.1Materials

2.1.1Reagents. HepG2 cells were purchased from Guangzhou Jennio Biotechnology Co., Ltd. TheARID4Btargeting siRNA was designed and synthesized by Suzhou GnePharma Gene Co., Ltd.ARID4Band GAPDH primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. LipofectamineTM2000 liposome was purchased from Invitrogen Corporation. DMEM medium was purchased from GIBCO Incorporation (USA). Mycoplasma-free fetal bovine serum was purchased from Bio-Excel (Australia) Pty Ltd.ARID4Bantibody was purchased from Novus International Incorporation. β-actin antibody was purchased from ABclonal Biotechnology Co., Ltd. Cell RNA extraction kit was purchased from Changzhou Baidai Biotechnology Co., Ltd.

2.1.2Instruments. Cell carbon dioxide incubator (Thermo, USA), DMIL LED inverted microscope (Leica, Germany), SW-CJ-1F ultra clean bench (Shanghai LNB Instrument Co., Ltd.), Cytation 5 microplate reader (BIO Tek, USA), Tanon 6100 chemiluminescence meter (Shanghai Tanon Science & Technology Co., Ltd.), NanoDrop 2000 instrument (Thermo, USA) and CFX96TM real-time system (BIO RAD, USA).

2.2Methods

2.2.1Cell culture. HepG2 cells were inoculated in DMEM medium containing 10% fetal bovine serum and cultured in a constant temperature incubator (37 ℃, CO2). Passage was performed once every 2-3 d.

2.2.2Cell transfection and grouping. Transfection was performed at different time points after cell seeding. In treatment 1, HepG2 cells were seeded in 6-well plates at a density of 6.5×105cells/well and subjected to transfection immediately after seeding. It was ensured that the cell density reached 70% during transfection. In treatment 2, HepG2 cells were seeded in 6-well plates at a density of 6.5×105cells/well and subjected to transfection 4 h after seeding. The cell density was ensured to reach 70% during transfection. In treatment 3, HepG2 cells were seeded in 6-well plates at a density of 5×105cells/well and subjected to transfection 24 h after seeding. It was ensured that the cell density reached 70% within 24 h for seeding. When transfection, appropriate amounts of siRNA and liposome were firstly dissolved in 250 μL of serum-free DMEM, let stand at room temperature for 5 min, let stand at room temperature for 20 min after gently mixing the two to form a carrier complex, and added to the culture plates containing cells, which were then placed in cell incubator to continue culturing. After 6 h, the fresh culture medium was replaced and the culture was continued. In each treatment, the cells were randomly divided into 3 groups: siRNA interference group (transfected withARID4Btargeting siRNA with liposome), negative control group (transfected with non-specific siRNA with liposome) and blank control group (without any treatment).

2.2.3Expression detection ofARID4BmRNA in HepG2 cells by RT-qPCR. Cells of each group were collected 48 h after transfection. RNA was extracted according to the instructions of the Cell RNA Extraction Kit, and the concentration and purity of RNA extracted were examined. Reverse transcription was performed according to the instruction of Takara PrimeScriptTMRT Reagent Kit with gDNA Eraser kit. Primer sequences forARID4Bgene were as follows: upstream, 5′-AACAGCAGGGCCAAAGATCG-3′; downstream, 5′-GCCACAGTCTGCAGTGACTC-3′. Primer sequences for GAPDH gene were as follows: upstream, 5′-GCACCGTCAAGGCTGAGAAC-3′; downstream, 5′-TGGTGAAGACGCCAGTGGA-3′. The reverse transcription products were subjected to PCR according to the following steps: 95 ℃ for 30 s, 95 ℃ for 5 s, 60 ℃ for 30 s, 40 cycles. GAPDH was used as an internal reference. The relative mRNA expression level ofARID4Bgene was quantitatively determined by 2-ΔΔCtmethod. All experiments were repeated in triplicate independently.

2.2.4Expression detection ofARID4Bprotein in HepG2 cells by Western blot. Cells of each group were collected 48 h after transfection for extraction of total protein. The protein extracted was quantified with BCA protein quantification method. Each group was subjected to SDS-PAGE electrophoresis of 8% separation gel with 30 μg of total protein. After electrophoresis, the protein was transferred to PVDF membrane, blocked with 5% skim milk powder at room temperature for 2 h, incubated withARID4Bprimary antibody (1∶4 000) and β-actin (internal reference) primary antibody (1∶100 000) at room temperature for 2 h, washed three times with TBST, incubated with goat anti-rabbit IgG secondary antibody (1∶2 000) for 1 h at room temperature, washed with TBST and subjected to color development with ECL chemiluminescence method.

3 Results and analysis

3.1ExpressionlevelofARID4BmRNAineachgroupofcellsaftertransfectionAfter 48 h of transfection, cells in each group were collected. Total RNA was extracted and detected by RT-qPCR. The relative expression levels ofARID4Bin each group of cells and its inhibition after transfection are shown in Fig.1. The results showed that 48 h after transfection ofARID4Btargeting siRNA, the relative expression levels of mRNA in each group decreased to varying degrees. Compared with the negative control group, the mRNA expression level of the transfection group 0 h after seeding was the lowest (P<0.05); the mRNA expression level 4 h after seeding ranked second (P<0.05); and the expression level in the transfection group 24 h after seeding was higher (P<0.05). That is, whenARID4Btargeting siRNA was transfected 0 h after cell seeding, siRNA showed the best inhibitory effect on the mRNA expression ofARID4B.

Fig.1 Relative expression ofARID4BmRNA in each group

3.2ExpressionlevelofARID4BproteinineachgroupofcellsaftertransfectionCells of each group were cultured for 48 h after transfection, and total proteins were extracted for Western blot detection. ImageJ software was used for grey value analysis of protein bands. The relative expression of the protein and its inhibition are shown in Fig.2 and Fig.3. The results show that 48 h after tansfection ofARID4Btargeting siRNA, compared with the negative control group, the expression level of ARID4B protein was reduced to varying degrees in the siRNA interference groups 0 and 4 h after seeding (P<0.05), and in the transfection group 24 h after seeding, the expression level of ARID4B protein did not change significantly (P>0.05). Among the treatments, the expression level of ARID4B protein in the transfection group 0 h after cell seeding was the lowest, that is, the inhibition effect was the best.

Note: 1. blank control group; 2. negative control group; 3. siRNA interference group.

Fig.2 Expression level ofARID4Bprotein in different groups

Fig.3 Relative expression ofARID4Bprotein in different groups

4 Discussions

ARID4Bis an important subunit of the histone deacetylation-dependent SIN3A transcriptional co-repressor complex. It plays a role in a variety of cellular processes including proliferation, apoptosis, and tumorigenesis[3]. Studies have found thatARID4Bis highly expressed in breast, lung, colon and ovarian cancers and normal testis, but its expression is restricted in other normal tissues[4]. It shows thatARID4Bgene plays different roles in the occurrence and development of different tumors. Tsaietal.[5]found thatARID4Bis a good biomarker for predicting the behavior of gliomas and meningiomas. Recently, Wangetal.[2]found thatARID4Bis highly expressed in liver cancer tissues compared with adjacent tissues, and the high expression ofARID4Bis closely related to tumor number, vascular invasion, Edmondson-Steiner classification and tumor lymph node metastasis stage.It is suggested thatARID4Bmay play an oncogene role in the occurrence and development of liver cancer, but the specific mechanism of action is unknown. The previous experiments of this research group have confirmed thatARID4Bis highly expressed in HepG2 compared with human normal liver cell line LO2. In order to further investigate the effect ofARID4Bon the biological function of HepG2 cells and further reveal the specific role ofARID4Bin the occurrence and development of liver cancer, down-regulating the expression ofARID4Bgene in HepG2 cells using siRNA interference technology is of great significance.

siRNA cell transfection technology is a technique for introducing exogenous siRNA into eukaryotic cells using physicochemical or biological mediation methods. Cationic liposome transfection is the most commonly used method in nucleic acid transfection for its simple preparation process, fast and easy operation, high safety and easy storage compared to viral vectors[6]. Liposome can form positively charged complex with siRNA. The complex binds to the cell membrane through electrostatic interaction and enters the cell through endocytosis. After small double-stranded RNA (siRNA) is transfected into cells, it can mediate the specific degradation of homologous mRNA sequences[7], thereby inhibiting the expression of targeted genes. Therefore, siRNA interference technology has become an important tool for elucidating the function of genes in organisms.

At present, there are many factors affecting the transfection efficiency of cationic liposomes, such as the ratio of transfection reagent to siRNA, cell growth status and density, medium replacement time and culture time. In this study, it was found that when transfection was performed at different time point after seeding, the transfection effect was quite different. When transfection was performed 0 h after cell seeding, the inhibition effect on the expression ofARID4Bin HepG2 cells was the best in both mRNA and protein levels; the inhibition effect in the transfection group 4 h after cell seeding ranked second; and when transfection was performed 24 h after cell seeding, the inhibition effect on HepG2 cells was the worst. This phenomenon might be related to the phenomenon that HepG2 cells are prone to clustering and stacking[8]. When HepG2 cells are scattered, their contact area with the liposome-siRNA complexes is increased, so that the transfection efficiency is the highest. When the traditional method is used for transfection, the cells adhere to the wall and grow in clumps, making it extremely difficult for liposome-siRNA complexes to enter into cells, so that the transfection efficiency is poor. When transfection was performed 4 h after cell seeding, HepG2 cells just began to attach to the plate and they had not grown into clumps, so the transfection efficiency ranked second. The efficiency of transfection performed before the adherence of the HepG2 cells was significantly higher than that performed at other two time points. In addition, when transfection was performed 0 h after cell seeding, the experiment period was shortened,i.e., time was saved. This study provides a reference for the subsequent research on the effect ofARID4Bon the biological function of HepG2 cells.