Culturing HEECs
HEECs[31] were kindly provided by Daniel G. Cyr (INRS-Institut Armand Frappier, University of Quebec, Laval, Quebec, Canada). HEECs were cultured in DMEM/HAM F12 media with penicillin (50 U/ml ), streptomycin (50 lg/ml), L-glutamine (2 mM), insulin (10 lg/ml), transferrin (10 lg/ml of), hydrocortisone (80 ng/ml), testosterone (5 nM), epidermal growth factor (10 ng/ml) , cAMP (10 ng/ml), sodium selenium (2 ng/ml), tocopherol (200 ng/ml), retinol (200 ng/ml), and 10% fetal bovine serum [FBS] [Sigma-Aldrich]. Cells were seeded in culture plates coated with collagen IV (BD Biosciences, Mississauga, Canada) and incubated in a humidified chamber at 32°C with 5% CO2.
Construction of the Prdx6 shRNA expression plasmid
Short hairpin RNA (shRNA) sequences targeting the mRNA of the human Prdx6 gene (Gens ID: 9588) were designed using the online tool (http://www.genesil.com/siRNA design.asp)[32] (Table 1). The Prdx6 shRNA sequences were then inserted into the endonuclease loci of BamHⅠ and Hind III in the pGenesil-1 vector (Wuhan GeneSil Biotechnology, Wuhan, China). The shRNA sequences were specific and did not target mRNA sequences of other known human genes (Table 5).
Plasmid Transfection
HEECs at 80% confluency were transfected with the Prdx6 shRNA expressing construct using the FuGENE HD Transfection Reagent (Roche, Mannheim, Germany). In addition, HEECs were transfected with non-targeting shRNA constructs to be used as the control. Transfections were performed in triplicate.
Protein extractions and Western blot analysis
Total proteins were extracted from 48hr post-transfected HEECs using RIPA lysis buffer (P0013B, Beyotime Biotechnology, China) supplemented with PMSF (ST506, Beyotime Biotechnology) and protease phosphatase inhibitors (P1050, Beyotime Biotechnology). Total proteins were then separated on a 10% (w/v) SDS-PAGE for JAK1 and STAT1 detection and 12% (w/v) SDS-PAGE for Prdx6, SOCS3 and GAPDH detection. After electrophoresis, transferred and blocked membrane, the membrane was were incubated with primary antibodies overnight at 4°C. Anti-JAK1 antibody (ab133666, Abcam, UK) was used at 1:800; anti-JAK1 (phospho Y1022 + Y1023) (ab138005, Abcam) at 1:500; anti-STAT1 (ab47425, Abcam) at 1:500; anti-STAT1 (phospho Y701) (ab29045, Abcam) at 1:500; anti-Prdx6 (ab92322, Abcam) at 1:1000; anti-SOCS3 (ab16030, Abcam) at 1:1000; and anti-GAPDH (ab181602, Abcam) at 1:2000. After incubation, the membranes were washed with PBS-Tween (1000:1) and then incubated with the relevant HRP-labeled secondary antibody for 2h at room temperature. X-ray film (Bio-Rad, Hercules, CA) was used for chemiluminescent detection of target proteins. Experiments were performed in biological triplicates.
The MDA levels and total antioxidant capacity in HEECs of control and Prdx6-RNAi were detected with Commercial kits (S0131S, Beyotime Biotechnology; A015-2-1, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s protocol.
Sample preparation and RNA isolation
Total RNA was extracted from 48 hr post-transfected cells using TRIZOL (Invitrogen, Carlsbad, CA, USA). RNA quality was measured using ultraviolet spectrophotometry and denaturing agarose gel electrophoresis.
DGE library preparation and sequencing
Library preparation and sequencing were performed by BGI using the Illumina Gene Expression Sample Prep Kit and Solexa Sequencing Chip (flowcell) on the Illumina Cluster Station and Illumina HiSeq™ 2000 System.
Data transformation and gene annotation
Raw solexa sequences were transformed using the following steps: ① removal of the 3' adaptor sequence, empty reads, and low-quality tags; ② selection of 21nt read length tags; ③ removal of single copy tags; ④ generation of Clean Tags. After quality assessment, the clean tags were used to generate alignment statistics between the P6-RNAi and control cells.
All clean tags were mapped to the reference sequence and only 1bp mismatch was considered during alignment. The number of unambiguous clean tags for each gene was calculated and then normalized to TPM (number of transcripts per million clean tags)[33-34].
Detection of differential gen expression
Differentially expressed genes between the two groups of transfected HEECs were determined using the Audic-Claverie method (1997)[35]. The threshold used to determine significant differences in gene expression was based on False Discovery Rate (FDR) ≤0.001 and the absolute value of log2Ratio≥1, as described in Benjamini, Yekutieli (2001)[36].
Gene ontology functional enrichment analysis for DEGs
Gene Ontology (GO) functional enrichment and Genomes (KEGG) pathway enrichment analysis were performed to uncover biological function and metabolic pathways of the differentially expressed genes, respectively. GO analysis was performed based on the methods used by Ye et al. (2006)[37]. Significantly enriched metabolic and signal transduction pathways were identified using the KEGG public database[38].
Pathway enrichment analysis for DEGs
Pathway enrichment analysis was performed similarly to that of GO analysis.
Real-time PCR
Total RNA was extracted using TRIZOL (Invitrogen, Carlsbad, CA, USA) based on the manufacturer’s instructions. The first strand of cDNA was generated with ReverTra Ace (MMLV Reverse Transcriptase RNase H–) (TRT-101, Toyobo Co., Ltd., Osaka, Japan). Real-time PCR was performed with EvaGreen 2× qPCR Master Mix (Applied Biological Materials, Inc., Vancouver, Canada) with Rotor-Gene Q (QIAGEN, Hilden, Germany). The results were expressed as the real-time quantity of the target gene / GAPDH. Gene-specific primer sequences and PCR conditions are shown in Table 6.