The human liver cancer cell line HepG2 was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose Dulbecco's modified Eagle medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C, 5% CO₂ (v/v). A total of ~1×10⁵ HepG2 cells were treated with DGKθ inhibitor R59949 at 10 µM, or DGKθ agonist GW4064 (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 1 µM for 24 h at 37°C.

The targeting regions for four pairs of single-guide RNA (sgRNA) located in exon 6, exon 7 or exon 8 of human DGKθ were selected using the CRISPR Design website (http://crispr.mit.edu). The sgRNAs were synthesized at the Beijing Genomics Institute (Beijing, China). The sequences of the oligonucleotides are shown in Table I. According to a previously described method (12), the human U6 promoter and sgRNA backbone were sequentially cloned into pUC19 (Clontech Laboratories, Inc., Mountain view, CA, USA), the obtained plasmid was called the pUC19/U6-BsaI-sgRNA backbone vector. The synthesized oligos were annealed, and ligated into the BsaI sites of the pUC19/U6-BsaI-sgRNA backbone vector under the control of the U6 promoter. The resultant plasmids were referred to as pUC19/U6-DGKθ sgRNA1, pUC19/U6-DGKθ sgRNA2, pUC19/U6-DGKθ sgRNA3 and pUC19/U6-DGKθ sgRNA4.

To construct the donor vector, an up homologous arm, 909 bp in length and located upstream of the targeting sites, was amplified through nest polymerase chain reaction (PCR) using two pairs of primers (Table II) based on a template of human genomic DNA. PCR was conducted in a 50 µl reaction volume, consisting of 5 µl 10X PrimeSTAR buffer, 100 ng genomic DNA template, 0.2 µM each primer, 10 mM dNTPS and 1 unit PrimeSTAR HS DNA Polymerase (all from Clontech Laboratories, Inc.) according to the following conditions: 29 cycles of 94°C for 30 sec; 98°C for 10 sec, 58°C for 15 sec and 72°C for 1 min and a 10 min extension step at 72°C. Similarly, a down homologous arm 975 bp in length was obtained using two pairs of primers (Table II). Subsequently, the donor vector pAd5/DGKθ-up/down-arm was constructed by sequentially inserting the up and down homologous arms into the backbone vector according to the previously described method (12). This vector also contained an eGFP-T2A-Neomycin expression cassette between the up and down homologous arms for positive selection, and a PGK-TK-T2A-mCherry expression cassette located at the 3′-terminal of the down homologous arm for negative selection (Fig. 1D).

Genomic DNA was extracted using the TIANamp Blood DNA kit (Tiangen Biotech, Inc., Beijing, China). The target site was amplified by nest PCR, the product of which was then purified using an AxyPrep DNA Gel Extraction kit (Axygen Biotechnology, Hangzhou, China). The purified product was then denatured and re-annealed, and digested with T7E1 (New England Biolabs, Ipswich, MA, USA). The digested product was then separated by 1.2% agarose gel electrophoresis. The gel was stained in running buffer containing 0.5 µg/ml ethidium bromide at room temperature for 15–30 min, then the images were captured under FR-98A Gel Imaging System (Shanghai Furi Science & Technology Co., Ltd., Shanghai, China).

To construct the human DGKθ gene-knockout Liver cancer cell line, pUC19/CMV-Cas9-U6-sgRNAX (X represents the sgRNA with the highest activity) was generated according to a previously described method (12). A total of ~1.5×10⁵ HepG2 cells were then co-transfected with 4 µg of pUC19/CMV-Cas9-U6-sgRNAX and 8 µg of pAd5/DGKθ-up/down-arm at 37°C for 48 h at an efficiency of ~20% using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) followed by screening in DMEM containing G418 (1 mg/ml) and GCV (1 mg/ml). A single cell clone was obtained through limited dilution following positive and negative selection, which was then confirmed by PCR that was performed in a 50 µl reaction volume, consisting of 5 µl 10X PrimeSTAR, 100 ng genomic DNA template, 0.2 µM each primer, 10 mM dNTPs, and 1 unit PrimeSTAR HS DNA Polymerase (all from Clontech Laboratories, Inc.) according to the following conditions: 30 cycles of 94°C for 30 sec; 98°C for 10 sec, 58°C for 15 sec and 72°C for 90 min, followed by a 10 min extension step at 72°C, then the PCR products were sent to Bejing Genomics Institute Genomics Co., Ltd. (Shenzhen, China) for sequencing.

A total of 1×10³ wild-type (WT) HepG2 cells or DGKθ gene-knockout HepG2 cells were cultured in 96-well plates. MTT solution (20 µl; American Type Culture Collection) was added to each well at 37°C at 24, 48, 72 and 96 h, respectively. Following incubation with MTT for 4 h, 200 µl DMSO was added to each well for 35 min at 37°C. The absorbance in each well was then measured at 570 nm on a microplate reader (Thermo Fisher Scientific, Inc.). Each group contained six replicates and the experiment was repeated three times.

The WT HepG2 cells and DGKθ-knockout HepG2 cells grown in 24-well plates were harvested. The cells were stained with Oil Red O (Sigma-Aldrich; Merck KGaA), and quantification of Oil Red O-based steatosis was performed, as previously described (13). The cell nuclei were stained with hematoxylin for 15 sec and washed with saturated Li₂CO₃ solution. Images were captured using a Leica DFC 420 C microscope (Leica Microsystems GmbH). The experiments were performed in triplicate.

Total RNA (1 µg), purified with an RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) was used to synthesize cDNA, followed by amplification of the target gene that was carried out in a 25 µl reaction volume, consisting of 150 ng cDNA, 0.2 µM each primer, 12.5 µl 2X SYBR buffer (Takara Biotechnology Co., Ltd., Dalian, China) containing 10 mM dNTPs and 1 unit DNA Taq polymerase according to the following conditions: 39 cycles of 95°C for 30 sec; 95°C for 5 sec and 60°C for 30 sec. The sequences of the primers used are listed in Table II. All tests were performed in triplicate and the data were normalized to GAPDH and quantified using the 2^−ΔΔCq method (14). ΔCq was calculated by subtracting the Cq value of GAPDH from the Cq value of the target gene. The fold change was generated using the formula ^2-ΔΔCq.

The proteins were extracted from the cells using extraction buffer as previously described (13) and quantified using Pierce bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.), which were then applied (80 µg/lane) to a gel for 10% SDS-PAGE and subsequently electrotransferred onto methanol-pretreated polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with PBS buffer containing 3% bovine serum albumin (Sigma-Aldrich; Merck KGaA) and 0.5% v/v Tween-20 for 1 h at room temperature. The membranes were then incubated with primary antibodies targeting FAS (1:500; cat no. ab82419), CPT1a (1:300; cat no. ab128568), PPARγ (1:500; cat no. ab66343), mTOR (1:500; cat no. ab25880), phosphorylated (p-)mTOR (1:300; cat no. ab109268), PKCε (1:500; cat no. ab63638), p-PKCε (S729; 1:500; cat no. ab63387), IRS1 (1:500; cat no. ab52167) and p-IRS1 (Y632; 1:300; cat no. ab109543) from Abcam (Cambridge, UK), and primary antibodies targeting DGKθ (1:500; cat no. 17885-1-AP), SREBP-1c (1:500; cat no. 14088-1-AP), HADHα (1:500; cat no. 10758-1-AP), AKT (1:500; cat no. 10176-2-AP) and p-AKT (1:300; cat no. 66,444-1-Ig) from ProteinTech Group, Inc. (Chicago, IL, USA) at 37°C for 1 h. Finally, the membranes were incubated with secondary antibodies, horse radish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) polyclonal antibody (1:10,000; cat no. ZB-2301; Beijing Zhongshan Jinqiao Biological Technology Ltd., Beijing, China) or HRP-conjugated goat anti-mouse IgG polyclonal antibody (1:10,000; cat no. ZB-2305; Beijing Zhongshan Jinqiao Biological Technology Ltd., Beijing, China) at 37°C for 1 h and visualized on a Tanon 5500 Chemiluminescence Imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China), and the protein levels were visualized using a Supersignal West Pico chemiluminescent detection system (Tanon Science and Technology Co., Ltd.), according to the manufacturer's protocol. Protein levels were determined using ImageCal software (version 4.0; Tanon Science and Technology Co., Ltd.).

The WT HepG2 cells and DGKθ-knockout HepG2 cells were grown on 60 mm plates for 48 h and lysed with RIPA buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS and 1 mM phenylmethanesulfonyl fluoride. The total lipids in the lysates were harvested by centrifugation at 10,000 × g for 10 min at 4°C. The PA content was quantified using a Total PA kit (HZbscience, Shanghai, China) according to the manufacturer's protocol. The quantity of DAG in each sample was determined using a Human DAG ELISA kit (Cusabio Biotech Co., Ltd., Barksdale, DE, USA).

Data are presented as the mean ± standard error of the mean. Differences between the means of each group were analyzed using one-way analysis of variance with Dunnett's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference. The statistical analyses were performed using Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS Statistics 20 software (IBM SPSS, Armonk, NY, USA).

In the present study, the DGKθ gene-knockout liver cancer cell line HepG2 was established using the Cas9/sgRNA technique, as follows. Firstly, the pUC19/CMV-Cas9-U6-sgRNA4 vector was constructed according to the previously described method (12). The vector carried sgRNA4, which had the highest cleavage activity among the four pairs of human DGKθ-targeting sgRNAs (Fig. 1A-C). Secondly, the donor vector pAd5/DGKθ-up/down-arm, which contained up- and down-homologous arms for homologous recombination, a neomycin-T2A-eGFP expression cassette for positive selection and a TK expression cassette for negative selection (Fig. 1D), was generated according to the previously described method (12). The Liver cancer cell line was then transfected with pUC19/CMV-Cas9-U6-sgRNA4 and pAd5/DGKθ-up/down-arm donor vector followed by screening with G418. As the donor vector contained an eGFP expression cassette, the cells with homologous recombination exhibited green fluorescence (Fig. 1E). Finally, the DGKθ gene-knockout liver cancer cell line HepG2 carrying a targeted integration in one allele (Fig. 1F) and a 26 bp deletion in the other allele was confirmed by sequencing (Fig. 1G).

RT-qPCR and western blot analyses were performed to confirm the knockout of the DGKθ gene in the liver cancer cell line HepG2 (Fig. 2A and B). The effect of knockout of the DGKθ gene on the growth of liver cancer cells was then investigated using an MTT assay. The knockout of the DGKθ gene promoted the growth of the liver cancer cells (Fig. 2C). Oil Red O staining showed that the DGKθ gene-knockout HepG2 cells had 32% higher intracellular lipid content (Fig. 2D), compared with the WT HepG2 cells (Fig. 2E). As expected, the content of intracellular PA was significantly decreased (Fig. 2F), whereas the content of DAG was increased (Fig. 2G) in the DGKθ gene-knockout HepG2 cells.

Subsequently, the present study examined the expression levels of genes associated with lipid synthesis (FAS, PPARγ and SREBP-1c) and genes associated with lipolysis (CPT1a and HADHα) in the DGKθ-knockout HepG2 cells at the mRNA and protein levels. The results indicated that DGKθ gene-knockout increased the expression levels of FAS, PPARγ and SREBP-1c, and suppressed the expression of CPT1a (Fig. 3A-C), compared, with the levels in the WT liver cancer cell line HepG2. These changes were observed at the mRNA and protein levels. Similar results were found in WT HepG2 cells treated with the DGKθ inhibitor, R59949. However, the DGKθ agonist, GW4064, had an opposite effect at the mRNA level for FAS only, compared with the knockout of DGKθ and treatment with DGKθ inhibitor. No effects on HADHα were observed in any of the treatment groups (Fig. 3A-C).

The expression levels of the signaling proteins involved in the glucose metabolism pathway, mTOR and Akt, were also analyzed. The results showed that DGKθ-knockout affected neither the mRNA nor the protein levels of mTOR and Akt (Fig. 3D-F). The effect of DGKθ-knockout on the levels of protein phosphorylation were then determined. No significant changes were observed in the total protein levels of mTOR and Akt, however, the phosphorylation levels of these proteins were significantly decreased in the DGKθ-knockout group (Fig. 3E and F). Treatment with the DGKθ inhibitor R59949 decreased the level of p-Akt, whereas the DGKθ agonist GW4064 significantly increased the level of p-Akt. Neither R59949 nor GW4064 treatment affected the level of p-mTOR.

Finally, the present study examined whether the DGKθ-knockout affected the expression levels of insulin resistance mediators, PKCε and IRS-1. The results showed no significant change in the expression levels of PKCε and IRS-1 by DGKθ knockout at the mRNA or protein levels. However, the level of p-PKCε (serine 729) was significantly increased, and the level of p-IRS-1 at tyrosine 632 (a stimulatory site for insulin signaling) was significantly decreased in the DGKθ-knockout group (Fig. 3G-I). In addition, the DGKθ inhibitor R59949 decreased the level of p-IRS-1, and the DGKθ agonist GW4064 significantly decreased the level of p-PKCε.