Patients and samples
A total of 527 sporadic nonsyndromic CTD patients were recruited from XinHua Hospital and Shanghai Children’s Medical Center[26] (Table 1). CHD was diagnosed by echocardiography, cardiac catheterization, or surgery. The exclusion criteria included the following: 1) chromosome karyotype confirmed as trisomy 21; 2) family history of CHD; 3) 22q11 microdeletion/duplication with cardiac malformation. Three hundred healthy individuals were also recruited as controls. This study was approved by the Medical Ethics Committee of Shanghai Children's Medical Center and Xinhua Hospital. All parents were informed of the purpose and significance of the experiment and signed an informed consent form. The mean age and sex ratio were matched between CTD patients and the control group.
Peripheral blood was collected, and genomic DNA was extracted using a QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. A UV spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to analyze the DNA purity. All DNA samples were stored at -80°C for future use.
Targeted sequencing
DNA samples were sent to TianHao Biotechnology Co, Ltd, China (a commercial provider) to screen for SRF mutations. Primers were designed by using Primer3 software, and the PCR products were labeled with a barcode for subsequent detection. Base incorporation was performed according to the manufacturer’s standard measurement protocol, and cluster generation was performed on a MiSeq Benchtop sequencer (Illumina, lnc, San Diego, CA, USA)[27].
Mutation validation
The mutations detected by targeted sequencing were validated by Sanger sequencing. The primers (SRF-seq) were designed by using Primer5 for the SRF sequence (NC_000006.12) (Table 3.). PCR amplification was performed, and PCR products were sequenced using an ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA).
Multiple SRF sequence alignments and online function prediction
To evaluate the conservation of mutated sites in the SRF protein, multiple sequences from human(NP_003122.1), macaca mulatta (XP_001093365), chimpanzee (XP_518487), mouse (NP_065239.1), rat (NP_001102772), dog (XP_852302.1), bovine (NP_001192945), chicken (NP_001239070), xenopustropicalis (XP_002942523), and zebrafish (NP_001103996) were aligned with ClustalX. The impact of the mutation on SRF proteins was also predicted by MutationTaster (http://www.mutationtaster.org/), SIFT (http://sift.jcvc.org/www/SIFT_enst_submit.html) and Polyphen-2 (http://genetics.bwh.havard.edu/pph2/).
Plasmids construction
The SRF open reading frame (ORF) clone was purchased from OriGene Technologies (Catalog No: SC118177). Primers for site-directed mutagenesis (Mut-G274D forward/reverse and Mut-G294C: forward/reverse) were designed online (https://www.genomics.agilent.com/primerDesignProgram.jsp) (Table 2), and PCR amplification products were digested using DpnI (NEB, Catalog: R0176S) before being transfected into DH5α and cultured on ampicillin dishes at 37°C for 14h. Selected successful mutant colonies were grown in 120ml LB medium (Beyotime, ST158), and then plasmids were extracted from the bacteria solution.
To construct the SRFluc reporter plasmid, the genomic sequence of SRF was obtained from the NCBI (https://www.ncbi.nlm.nih.gov/). A fragment beginning approximately 1.2kb upstream of the transcription initiation site of SRF was amplified (sense-primer: 5’ –C G G G G T A C C T T T C T G C T G G G C A C G G T G G T - 3’, antisense-primer: 5’ - A T G G C G A G G C C G C T C C T T A T A A G C T T G G G- 3’) from the DNA of a healthy individual and was cloned into the pGL3-basic vector (Promega, USA) between the KpnI and HindIII sites. The atrial natriuretic factor (ANF) promoter plasmid was a kind gift from Professor Mona Nemer[28]. The full-length cDNAs for GATA4 expression constructs in the pcDNA3.1(+) vector were previously generated in our laboratory[29]. All plasmid DNA was confirmed and Sanger sequenced by the Beijing Genomics Institute (BGI) in China.
Cell culture and transfection
The HEK293 and NIH3T3 cell lines were cultured in medium containing Dulbecco’s Modified Eagle’s Media (DMEM), 10% fetal bovine serum (FBS), penicillin (100 unit/ml), and streptomycin (100 µg/ml) before transfection. Cells were transfected with Fugene HD (Promega, E2311) according to the manufacturer’s protocol. Transfected NIH3T3 cells were starved by culturing in DMEM supplemented with 0.5% FBS for 48 h.
Western bloting and real-time PCR
HEK293 cells were transfected with wild-type or mutant SRF plasmid in 12-well plates 24 h after plating, and were harvested after 48 h transfection. Cells were washed with cold DPBS and then lysed on ice using RIPA lysis buffer with PMSF (1/100) for 30 minutes for total protein extraction. For RNA extraction, the transfected cells were lysed with TRIzol for 10 minutes.
For Western blotting, 25 µg of protein per sample was separated using 8% SDS-PAGE (Beyotime, China) and transferred onto PVDF membranes (0.45 μm, Life Technology, ThermoFisher, Scientific). The membranes were blocked for 2 h using 5% skim milk in Tris-buffered saline with 0.2% Tween-20. A rabbit anti-human SRF antibody (1:2000 in 5% BSA, Genview, USA) or mouse anti-human actin antibody (1:2000 in 5% BSA, Genview, USA) was used to incubate the membranes at 4°C overnight. The next day, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody or goat anti-mouse antibody (secondary antibodies) for 2 h. Immobilon ECL (Millipore, USA) and a ChemiDoc XRS+ system (Bio-Rad, USA) were used to visualize the protein bands.
For qPCR, 1000 ng of total RNA was reverse transcribed to synthesize cDNA using a PrimeScriptTMRT reagent kit (Takara, RR037A). Primers (RT-SRF) for the SRF sequence (NM_003131) (forward : 5’-A C T C T C C A C C C C G T T C A G A C-3’/reverse : 5’-T G G T G C A C T T G A A T G G C C T G-3’) and the reference GAPDH sequence (forward : 5’-G A G T C A A C G G A T T T G G T C G T-3’/reverse : 5’-T G A T T T T G G A G G G A T C T C G-3’) were designed using Primer5 (Table 2). Comparative Ct (ΔΔCt) relative quantitation analysis was used to analyze the Ct value in qPCR, and experiments were repeated three times. The results are presented as the mean±SEM.
Luciferase assay
To assess the activation of mutant SRF on the ANF promoter alone, we cotransfected 200 ng of wild-type/mutant SRF constructs, and 66.67 ng of ANF-reporter plasmids; and when in synergy with GATA4, we cotransfected 100 ng of wild-type/mutant SRF constructs, 100 ng of empty vector or 100 ng GATA4 construct, and 66.67 ng of ANF-reporter plasmids per well into starved cells (24 h after plating, the 10% FBS medium was replaced with 0.5% FBS medium before transfection) of 48-well plates. In addition, we cotransfected mutant or wild-type constructs or the pcDNA3.1(+) vector and the SRF-reporter plasmid into NIH3T3 cells using Fugene HD (Promega, USA), and pRL-TK (Promega) was used as an internal control reporter plasmid.
We compared the ANF promoter and SRF promoter expression in the different transfection groups when NIH3T3 cells were cultured in 10% FBS medium or 0.5% FBS medium 48 h after transfection. The luciferase activity of cells was determined by using a Dual-Glo luciferase assay system (Promega, E2920). The results of three replicate experiments are presented as the mean ± SEM.