Experimental animals and reagents
Yellow catfish (body weight: 22.5 ± 4.4 g) were obtained from a local commercial farm. HEK293T cell lines were purchased from the Cell Resource Center in the Fishery College of Huazhong Agricultural University. Dulbecco’s Modified Eagles Medium (DMEM), 0.25% trypsin-EDTA and fetal bovine serum (FBS) were obtained from Gibco/Invitrogen, USA. Dimethyl sulphoxide (DMSO), penicillin, palmitic acid, oleic acid, streptomycin, trypan blue and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). We ensured that the experiments were performed in accordance with the experimental protocols of Wuhan Polytechnic University (WHPU) and were approved by the ethics committee of WHPU.
Two experiments were carried out. Exp. 1 was conducted to study the transcriptional regulation of pi3kc3 promoter. Exp. 2 was conducted to determine the potential role of PI3KC3 in influencing lipid accumulation in the hepatocytes from yellow catfish under FA incubation.
Exp. 1: Transcriptional regulation assay of pi3kc3 promoter
Promoter cloning and plasmids construction
The genomic DNAs were extracted from the liver of yellow catfish by using a commercial DNA extracted kit (Omega, Norcross, GA, USA). The promoter sequence of pi3kc3 was obtained by RT-PCR according to the genome of yellow catfish (Gong et al. 2018). The primers for pi3kc3 promoter cloning were presented in Table S1. For generating the luciferase reporter construct, we subcloned different plasmids with pi3kc3 promoter into pGl3-Basic vectors (Promega, USA) by using SacI and HindIII restriction sites. On the basic of the distance from its TSS, we named the plasmid as pGl3-1781/+59 of pi3kc3 promoter. Then, we used the template of pGl3-1781/+59 vector to produce the plasmids pGl3-1361/+59, pGl3-848/+59, and pGl3-381/+59 of pi3kc3 vectors. We used ClonExpress II One Step Cloning Kit (Vazyme, Piscataway, NJ, USA) to ligate all of the products. We performed the PCR via the TaKaRa PrimeSTAR® HS DNA Polymerase kit (TaKaRa, Tokyo, Japan). Finally, we sequenced all these plasmids in the Tsingke company (Wuhan, China). The primers for the plasmids construction were presented in Table S2. In addition, The overexpression plasmids of PPARα, PPARγ and STAT3 were obtained from our previous studies (Lv et al. 2021).
We used BLAST network service at the NCBI (http://blast.ncbi.nlm.nih.gov/) to compare the nucleotide sequences with DNA sequences from the GenBank database. Several online softwares, such as the MatInspector database (http://www.genomatix.de/), the JASPAR database (http://jaspar.genereg.net/) and the TFSEARCH database (http://www.cbrc.jp/research/db/TFSEARCH.html), were utilized to analyze the potential transcription factor binding sites (TFBS). The CpG islands were predicted by the online tool MethPrimer (http://www.urogene.org/methprimer/index1.html) with parameters as follows: window 100, shift 1, observed CpG/expected CpG ≥ 0.60 and GC % ≥40.
Plasmid transfections and assays of luciferase activities
HEK293T cells were cultured in DMEM medium with the 10% fetal bovine serum (FBS) (Gibco, C lsbad, CA, USA) in an incubator (5% CO2 and 37°C). Prior to the transfection, HEK293T cells were seeded at a density of 1.2×105 in 24-well plate. They were cultured until the 70-80% confluence. Lipofectamine™2000 (Invitrogen) was utilized to transfect all these plasmids into HEK293T cells, based on the manufacture’s protocol. The 500 ng overexpression plasmids, 400 ng reporter plasmids and 20 ng pRL-TK (the internal control with a Renilla luciferase reporter vector), were co-transfected into HEK293T cells. After 4 h, we replaced the transfection medium by 10% FBS-DMEM or 10% FBS-DMEM + 0.6 mM FA. FA was added as a mixture of palmitic acid and oleic acid at a ratio of 1:1. The form and the concentration of FA were selected according to our pilot trial and the publications of the in vitro studies (Wu et al. 2019; Wu et al. 2020; Chen et al 2020a; Song et al 2020). Then, after 24 h incubation, cells were collected to determine the promoter activity, based on the manufacturer’s instruction of the Dual-luciferase Reporter Assay System (Promega). The relative luciferase activities were obtained by calculating the ratio of Firefly luciferase activity to Renilla luciferase activity. We conducted all these experiments in triplicates.
Site-mutation analysis of binding sites on the pi3kc3 promoter
To identify the corresponding binding sites on the regions of pi3kc3 promoter, we used QuickChange II Site-Directed Mutagenesis Kit (Vazyme, Piscataway, NJ, USA) to perform site-directed mutagenesis analysis. Several mutations were performed at the sites of -1621/-1611 bp, -1603/-1594 bp, -922/-907 bp, -1083/-1076 bp, and -245/-230 bp of pi3kc3 promoter; The primers used for mutagenesis were shown in Table S3. The DNA sequencing was utilized to confirm these mutations. Then, the Lipofectamine 2000 reagent (Invitrogen) was utilized to co-transfect the plasmids into HEK293T cells. After 4 h transfection, the medium was substituted with 10% FBS-DMEM or 10% FBS-DMEM + 0.6 mM FA. After 24-h incubation, we harvested the cells to determine the luciferase activities, based on the procedures mentioned above.
Electrophoretic mobility-shift assay (EMSA)
The EMSA was conducted to confirm the functional PPARα, PPARγ and STAT3 binding sties on the pi3kc3 promoter according to our and other recent publications (Xu et al. 2017; Zhuo et al. 2018; Chen et al. 2020b). Nuclear and cytoplasmic extracts were extracted according to the method of Read et al. (1993). Protein contents were determined by the BCA method (Smith et al. 1985). The oligonucleotide probes were synthesized in the Tsingke company (Wuhan, China). Nuclear extracts (10 µg) were incubated for 30 min at the room temperature by using the binding buffer (20 mM HEPES, pH7.9, 1 mM MgCl2, 0.5 mM DTT, 4% Ficoll, 110 mM KCl, 0.2 µg Poly(dI-dC)). Then, the biotin-labeled double-stranded oligo nucleotides (Table S4) were added. The reaction continued for 30 min and then the electrophoresis was performed on 6% native polyacrylamide gels. For the competitive binding analysis, a 100-fold excess of unlabeled double-stranded DNA oligo with mutant binding site (Table S4) was added with the corresponding labeled one.
Exp2. FA incubation with hepatocytes of yellow catfish
Hepatocytes were isolated from yellow catfish according to our previous studies and were cultured in M199 medium containing 1 mmol/L L-glutamine, 5% (v/v) FBS, penicillin (100 IU/mL) and streptomycin (100 g/mL) in a humidified atmosphere with 5% CO2 at 28°C (Zhuo et al. 2018). Hepatocytes were counted using a hemocytometer based on the trypan blue exclusion method and only more than 95% cell viability were used for the present experiment. Hepatocytes were plated onto 25 cm2 flasks at the density of 106 cells/mL, and then they were incubated with PBS (control) and 0.6 mM FA. Each treatment was performed in triplicate and three independent experiments were carried out. After 48 h, the hepatocytes were gathered for the following analysis.
TG, NEFA, and lipid drops assay
TG and nonesterified fatty acid (NEFA) concentrations were determined with commercial kits (Nanjing Jian Cheng Bioengineering Institute, China), according to the manufacturer’s instructions. Bodipy 493/503 staining were used to assess the changes of intracellular lipid drops (LDs). Briefly, hepatocytes were cultured in 12-well plates and treated with the corresponding treatments for the required period, and then they were washed twice with PBS. After that they were incubated with 5 mg/ml Bodipy 493/503 (D3922; Thermo Fisher Scientific Waltham, MA, USA) for 30 min, followed by 3 PBS washes. Then the hepatocytes were observed with a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) to visualize the intensity of fluorescence. The green dots were defined as lipid drops, which were quantified with a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA). Data analysis was performed with FlowJo v.10 software (Ashland, OR, USA).
mRNA level determination by real-time Q-PCR
Total RNA was isolated using Trizol reagent (TaKaRa, Dalian, China) according to the manufacturer's instruction. cDNA was then reverse-transcribed from normalized RNA using oligo (dT) primers and M-MLV reverse transcriptase (TaKaRa, Dalian, China). The mRNA levels of (pparα, pparγ, stat3, dnmt1, dnmt3a, dnmt3b, and pi3kc3) were examined by quantitative PCR (Q-PCR). Q-PCR assays were performed in a quantitative thermal cycler (MyiQ™ 2 TwoColor Quantitative PCR Detection System, BIO-RAD, USA) with a 20 µL reaction volume containing 10 µL SYBR Premix Ex Taq™ II (TaKaRa, Japan), 1 µL of diluted cDNA (10-fold), 10 mM each of forward and reverse primers (0.4 µL), and 8.2 µL H2O. Primers are given in Table S5. The Q-PCR parameters consisted of initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, 57°C for 30 s and 72°C for 30 s. All reactions were performed in duplicates and each reaction was verified to contain a single product of the correct size by agarose gel electrophoresis. The melting curve was generated for every PCR product to confirm the specificity of the assays. A set of seven common housekeeping genes (β-actin, 18s-rrna, gapdh, rpl7, hprt, ubce, and tuba) were selected from the literature (Vandesompele et al. 2002) in order to test their transcription stability. Two most stable control genes (gapdh and 18srrna, M=0.35) were selected by using geNorm software. The relative expression levels were calculated with the “delta–delta Ct” method (Pfaffl 2001), and normalized in terms of the geometric mean of two genes by geNorm.
Analysis of protein expression by western blot
Western blotting was performed according to the previous study (Wu et al. 2020). Hepatocytes were lysed in RIPA buffer (Sigma, USA). Equal amounts of protein were separated on 12% SDS-PAGE, transferred onto PVDF membranes, and then blocked with 8% (w/v) dry milk. After that, the membranes were incubated with primary antibodies as follows: rabbit anti-PPARα (15540-1-AP, Proteintech, USA), rabbit anti-PPARγ (16643-1-AP, Proteintech, USA), rabbit anti-STAT3 (10253-2-AP, Proteintech, USA), rabbit anti-PI3KC3 (AbClone, A12295, USA), and anti-GAPDH (10494-1-AP; Proteintech, USA) overnight at 4°C. Then, HRP-conjugated anti-rabbit secondary antibody (CST, USA) was used to probe with. Finally, the protein bands were visualized with enhanced chemiluminescent (ECL) and quantified by Image J software.
Methylation analysis of CpG islands of pi3kc3 promoter
Genomic DNA from hepatocyte was extracted using AxyPrep DNA Kit (Axygen Biotechnology, Hangzhou, China) according to the manufacturer's instructions. The genomic DNA extracted above was modified according to the manufacturer's protocol using the DNA Methylation Gold Kit (Zymo research, Orange, CA). Two CpG islands on the pi3kc3 promoter were predicted. The bisulfite modified DNA was amplified by nest polymerase chain reaction (PCR) with two BSP (bisulfite sequencing PCR) specific primer pairs (list in Table S6), under the following conditions: 95°C denaturation for 3 min; 30 cycles of 95°C for 30 s, 53°C for 30 s and 72°C for 40 s; and 72°C extension for 5 min. The PCR products were gel purified and were subjected to cloning into a PMD 19-T Vector (TaKaRa, Dalian, China). After the cloning, total of 20 clones from each treatment were randomly selected for DNA sequencing. The methylation level was analysis by the online website (http://quma.cdb.riken.jp/).
We used SPSS 19.0 software for all these statistical analysis. All of these data were expressed as means ± SEM (standard errors of means). Before statistical analysis, we evaluated all data for normality using the Kolmogorov-Smirnov test. In order to test the homogeneity of variances, we performed Bartlett’s test. We analyzed data with Duncan’s multiple or Student’s t-test where appropriate. Difference was considered significant at p < 0.05.