Cell culture and transfection
Hela cells were purchased from American Tissue Culture Collection (ATCC; Rockville, USA) and cultured in DMEM medium supplemented with 10% FBS in an incubator with 5% CO2 at 37 °C. The pEGFP-C1/enJSRV-Env vector, used for transient transfections, was previously generated by our laboratory[10], and was transfected by using PEI transfection reagent (Sigma-Aldrich, Saint louis, USA). Cells were harvested 48 h after transfection for the following experiments. The empty plasmid was employed as negative control.
Cell-cell fusion assays
The determination of the fusion activity of the transfected cells was performed 48 h after transfection. Giemsa solutions (Solarbio, Beijing, China) were added to visualize the nuclei of the cells in accordance with the manufacturer’s recommendations.
Cell proliferation assay
Cell proliferation was determined by MTT method (Solarbio, Beijing, China). In details, cells were plated in 96-well plates at a density of 5×103 per well and incubated at 37 ℃ in the presence of 5% CO2. After 24, 48 and 72 h incubation, cells were processed by adding 20µL /well (5mg/mL) MTT and incubated for 4 h at 37℃. Then, 150 µL DMSO was added to each well, and the absorbance was examined at a wave length of 490 nm by a microplate reader.
RNA Extraction and cDNA Library Construction
The total RNA was extracted from the cells transfected with pEGFP-C1/enJSRV-Env (Env group) or pEGFP-C1 (NC group) by Trizol, and the mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. First strand cDNA was synthesized using random oligonucleotides and SuperScript II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). The sequencing library was then sequenced on a HiSeq platform (Illumina).
Transcriptome Sequencing and Bioinformatics Analysis
Firstly, the HTSeq (0.9.1) was launched to compare the Read Count values on each gene as the original expression of the gene, and then the FPKM was employed to standardize the expression. Then, the DESeq (1.30.0) was used to analyze the DEGs with screened conditions as follows: expression difference multiple |log2FoldChange| > 1, significant P-value < 0.05. Both annotations of GO and KEGG were carried out to identify functional genes.
qRT-PCR analysis
The total RNA from cells was extracted with Trizol reagent (XYGEN, CA, USA) and subsequently reverse transcribed into cDNA by utilizing PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Real-time PCR was carried out with SYBR Premix EX Taq™ II kit (TaKaRa, Dalian, China) on the PCR detection instrument (Opticon CFD-3200). GAPDH were used as reference gene. The quantification of the relative expression of targeted genes was carried out using the 2-ΔΔCt method[11]. The primer sequences are list in table 1.
Table 1. The primer sequences for qRT-PCR
Name of primers
|
Primers sequences (5′–3′)
|
GAPDH
|
FORWARD
|
TGACTTCAACAGCGACACCCA
|
REVERSE
|
CACCCTGTTGCTGTAGCCAAA
|
BHLHE4
|
FORWARD
|
CTCAGCTGAAAGATTTACTGCC
|
REVERSE
|
AGGCGGTTAAAGCTTTTAAGTG
|
CCN1
|
FORWARD
|
CTTGTGAAAGAAACCCGGATTT
|
REVERSE
|
ACTCAAACATCCAGCGTAAGTA
|
DLX2
|
FORWARD
|
TTGAGCCTGAAATTCGGATAGT
|
REVERSE
|
CAGCTGGAAACTGGAGTAGATG
|
DUSP6
|
FORWARD
|
ATGATAGATACGCTCAGACCCG
|
REVERSE
|
GATGTGCGACGACTCGTATAG
|
SH2D5
|
FORWARD
|
AGGAGCTGCCAGAGTCGGAAG
|
REVERSE
|
CGGATCACCTTGCTGCGAATGG
|
HIST1H4K
|
FORWARD
|
GCGGGAAGGGTCTTGGCAAAG
|
REVERSE
|
TCGTAGATGAGGCCGGAGATGC
|
NKD1
|
FORWARD
|
ACCATTGCGTAGATGAGAACAT
|
REVERSE
|
CCAAATTGGGACGTGTAGTTTT
|
Western blotting
Cells were lysed with RIPA Buffer (Thermo Fisher Scientific, USA), and the protein concentration was determined with the BCA Kit (Beyotime, Beijing, China). The equivalent amounts of proteins were separated on 12% SDS-PAGE and subsequently transferred to PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk dissolved in BSA solution, the membrane was incubated with the primary antibody at 4℃ overnight, followed by incubation with secondary antibody at room temperature for 1 h. Finally, the bands were detected with enhanced chemiluminescence reagent ECL detection kit (Thermo Fisher Scientifc, Inc.) on ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA). Antibodies for Erk1/2(cat: 4695, Dilution: 1:1000), Phospho-Erk1/2(cat:4370, Dilution: 1:2000), p38(cat: 9212S, Dilution: 1:1000) and Phospho-p38(cat: 4511P, Dilution: 1:1000) were purchased from Cell Signaling Technology; antibodies for JNK(cat: ab179461, Dilution: 1:1000), Phospho-JNK(cat: ab124956, Dilution: 1:1000), GAPDH(cat: ab8245, Dilution: 1:2000) and DUSP6(cat: ab76310, Dilution: 1:1000) were purchased from Abcam.
Statistical analysis
All data were shown as means ± SD, and at least three independent experiments were performed for each condition. Statistical analysis was conducted by employment of SPSS 21.0 software. Student’s t test and one-way ANOVA were applied for comparison of the differences in two groups or more respectively. Differences were defined as statistically significant when P < 0.05.