A Novel Human Long Noncoding RNA SCDAL Promotes Angiogenesis through SNF5‐Mediated GDF6 Expression

Abstract Angiogenesis is essential for vascular development. The roles of regulatory long noncoding RNAs (lncRNAs) in mediating angiogenesis remain under‐explored. Human embryonic stem cell‐derived mesenchymal stem cells (hES‐MSCs) are shown to exert more potent cardioprotective effects against cardiac ischemia than human bone marrow‐derived MSCs (hBM‐MSCs), associated with enhanced neovascularization. The purpose of this study is to search for angiogenic lncRNAs enriched in hES‐MSCs, and investigate their roles and mechanisms. AC103746.1 is one of the most highly expressed intergenic lncRNAs detected in hES‐MSCs versus hBM‐MSCs, and named as SCDAL (stem cell‐derived angiogenic lncRNA). SCDAL knockdown significantly reduce the angiogenic potential and reparative effects of hES‐MSCs in the infarcted hearts, while overexpression of SCDAL in either hES‐MSCs or hBM‐MSCs exhibits augmented angiogenesis and cardiac function recovery. Mechanistically, SCDAL induces growth differentiation factor 6 (GDF6) expression via direct interaction with SNF5 at GDF6 promoter. Secreted GDF6 promotes endothelial angiogenesis via non‐canonical vascular endothelial growth factor receptor 2 activation. Furthermore, SCDAL‐GDF6 is expressed in human endothelial cells, and directly enhances endothelial angiogenesis in vitro and in vivo. Thus, these findings uncover a previously unknown lncRNA‐dependent regulatory circuit for angiogenesis. Targeted intervention of the SCDAL‐GDF6 pathway has potential as a therapy for ischemic heart diseases.


Culture of hBM-MSCs and HUVECs
hBM-MSCs were isolated from bone marrow aspirates that were obtained from caput femoris upon hip replacement surgery with informed consent according to the guidelines approved by the Ethics Committee of Second Affiliated Hospital, College of Medicine, Zhejiang University (No. 2015-011) as described previously [2] . Briefly, whole bone marrow aspirates were washed with PBS, centrifuged at 1000 rpm for 5 minutes, and plated in 75 cm 2 flasks in 10% FBS-supplemented DMEM. After 2 days, the nonadherent cells were removed and the adherent cells were maintained at 37 o C, 5% CO 2 with medium change every three days until colony confluence, and passaged in a 1:2-1:3 ratio. Characterizations of surface marker expression and multipotency were performed after 3-5 passages of culture as described above. Donor characteristics and passages of hBM-MSCs were listed in Table S1.
Human ECs and HUVECs were maintained in low glucose DMEM (Thermo Fisher Scientific, Invitrogen) containing 10% FBS. HUVECs were transfected with lentivirus encoding green fluorescent protein (GFP) expression (GENECHEM Corp, China) for subsequent tube formation assay.

lncRNA sequencing and data analysis
Total RNA of hES-MSCs from three differentiation experiments of H14 hESCs and hBM-MSCs from three different donors were extracted. First, ribosomal RNA (rRNA) was removed by Ribo-Zero™ rRNA Removal Kit (Epicentre). Sequencing libraries were prepared from the rRNA-depleted RNA by NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina ® (New England Biolabs) following manufacturer's recommendations, and then sequenced on the Illumina Hiseq2000 platform by Novogene (China) and 100 bp paired-end reads were generated. Clean data (clean reads) were obtained through in-house perl scripts by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data, and aligned to the homo sapiens GRCh38.p12 genome using TopHat2 v2.0.9 [3] . The mapped reads of each sample were assembled by both Scripture (beta2) [4] and Cufflinks (v2.1.1) [5] .

In vitro Matrigel tube formation assay
Tube formation was evaluated in HUVECs cocultured with MSCs (Protocol 1) or with the conditioned media (CM) from MSCs (Protocol 2).

Spheroid-based spouting assay
HUVEC spheroids were generated as described previously [10] . Briefly, HUVECs were cultured as hanging drops (400 cells/25 µl per drop) in culture medium containing 0.24% (w/v) methylcellulose for 24 hours to form spheroids. The spheroids were collected and re-suspended in methocel containing 20% FBS. A collagen stock solution was prepared by mixing 8 volume of collagen extract of rat tails (3.83 mg/ml, Corning Incorporated) with 1 volume of 10× M199 (Sigma-Aldrich) on ice, and ~1 volume of 0.2 N NaOH to adjust the pH to 7.4, after which it was mixed with equal volume of spheroid-containing methocel solution. The spheroid-containing collagen was rapidly transferred into 24-well plates (1 ml per well) and allowed to polymerize (4 hours), after which 0.1 ml indicated hES-MSC/hBM-MSC-derived CM or DMEM containing 1% FBS was added on top of each gel. The gels were incubated at 37 o C, 5% CO 2 , and 100% humidity for 24 hours. For quantification of the length of sprouts, six to eight randomly selected spheroids per well were acquired under a fluorescence microscope and analyzed as average spheroid sprout length per well with Image-Pro Plus software 6.0.

RNA extraction and qRT-PCR
Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific, Invitrogen). 2 µg of total RNA was reverse-transcribed to cDNA with M-MLV Reverse Transcriptase (Takara). Resultant cDNA samples were subjected to qPCR on 7500 Fast Real-Time PCR System (Applied Biosystems) using SYBR ® Premix Ex Taq TM (Takara) and the primers listed in Table S3. β-actin levels were measured to serve as an internal control, and the relative expression levels were calculated using the 2 -ΔΔCt method.

Western blot
Cells were lysed in RIPA lysis buffer supplemented with Pierce TM protease inhibitors (Thermo Fisher Scientific) and, after centrifugation, the supernatants were collected and quantified using the BCA protein assay. 20-30 μg of protein per sample was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a poly-vinylidene fluoride membrane, and blocked with 5% milk for 1 hour. The membranes were incubated with indicated primary antibodies (Table S2) overnight at 4 o C, and blotted with the corresponding horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 hours. Afterwards, the protein bands could be visualized via enhanced chemiluminescence.

RACE
Total RNA was extracted from hES-MSCs, and converted into first strand cDNA with an M-MuLV First Strand cDNA Synthesis Kit (Sangon Biotech, China). Nested 5' and 3' RACE products were obtained using LA Taq ® polymerase with GC Buffer (Takara) and the primers listed in Table S4. The gel products were extracted with a Gel Extraction kit (Sangon Biotech), cloned into pMD18-T vectors and verified by Sanger sequencing.

Northern blot
Total RNA was extracted from hBM-MSCs and hES-MSCs using standard TRIzol methods. SCDAL probe was generated by RT-PCR using the following primers:

RNA fractionation
Nuclear and cytoplasmic RNA fractions of hES-MSCs were isolated using a PARIS TM Kit (Thermo Fisher Scientific, Invitrogen), according to the manufacturer's instructions. 0.5-1×10 7 freshly cultured cells were collected, lysed thoroughly in ice-cold Cell Fractionation Buffer and centrifuged at 4 o C and 500 g for 5 minutes.
The supernatant was collected as the cytoplasmic fraction, while nuclear pellet was washed and homogenized in ice-cold Cell Disruption Buffer. Both cytoplasmic fraction and nuclear lysate were respectively mixed with an equal volume of 2× Lysis/Binding Solution for further RNA isolation. The SCDAL content in the fractions was determined via standard qRT-PCR protocols. β-actin and glyceraldehyde phosphate dehydrogenase (GAPDH) levels were used as cytoplasmic controls, whereas U6 level was used as a nuclear control.

Lentivirus construction and transfection
Lentiviral vectors expressing either SCDAL shRNA, full-length SCDAL, GDF6 or VEGFR2 were constructed and generated by GENECHEM Corp. For lentiviral infection, cells were plated as 1×10 4 /cm 2 , infected with indicated lentiviruses or lentiviruses containing empty vectors (control) at multiplicity of infection 30-50 with polybrene for 24 hours, and then refreshed with fresh medium. Transfection efficacy was evaluated after 72 hours of infection via qRT-PCR or western blot.

Synthesis and transfection of Ribo TM lncRNA Smart Silencer and siRNAs
Ribo TM lncRNA Smart Silencer for SCDAL was purchased from RiboBio (Table S5).
siRNAs targeting GDF6 and SNF5 were designed and synthesized by GenePharma Table S6). Smart Silencer, indicated siRNAs and their scramble controls were transfected into cells at 50 nM final concentration with Lipofectamine ® RNAiMAX Reagent (Thermo Fisher Scientific).

RNA sequencing in hES-MSCs after SCDAL knockdown
Total RNA was isolated from hES-MSCs transfected with SCDAL shRNA (n=3) or shRNA control lentiviruses (n=3). Sequencing libraries were generated from the rRNA-depleted RNA using NEBNext ® Ultra TM RNA Library Prep Kit for Illumina ® .
Libraries were sequenced on an Illumina platform by Novogene and 125 bp/150 bp paired-end reads were generated. Clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data, and then aligned against the homo sapiens GRCh38.p12 genome using Hisat2 (v2.0.5) [12] . The mapped reads of each sample were assembled by StringTie (v1.3.3b) [13] . FeatureCounts v1.5.0-p3 [14] was used to count the reads numbers mapped to each gene, and then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of two groups was performed using the DESeq2 R package (1.16.1) [15] . The resulting P values were adjusted using the Benjamini-Hochberg method for controlling the false discovery rate. Genes with an adjusted P value <0.05 found by DESeq2 were assigned as differentially expressed. GO as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias was corrected. GO terms and KEGG pathways with corrected P value <0.05 were considered significantly enriched by differential expressed genes.

GDF6 ELISA
GDF6 levels in the cell lysates and conditioned supernatants of culture cells were quantitatively determined by a Human GDF6 ELISA Kit (mlbio, China) following the manufacturer's instructions.

Structural modeling
The

Adhesion frequency assay
The adhesion frequency assay was conducted as described previously [16] . Before assay, HUVECs infected with lentiviruses expressing VEGFR2 or lentiviruses containing empty vectors were characterized by FACS with VEGFR2-PE antibody (Table S2).
Briefly, human red blood cells (RBCs) were biotinylated with a biotinylation ester (Jenkem technology) and coated with recombinant human GDF6 protein (abcam, Cat# ab245811) via biotin-streptavidin coupling, and the RBCs and HUVECs were suspended in DMEM with 1% BSA. Single indicated HUVEC and RBC were respectively aspirated with micropipettes and driven in and out of contact with controlled duration (2 seconds) by a computer-programmed piezoelectric actuator.
Adhesion was observed from stretching of the RBC on HUVEC retraction. Four to six pairs of cells were tested, and the contact-retraction cycle was repeated 50 times for each pair of cells.

RNA pull-down and mass spectrometry
Biotinylated SCDAL sense and antisense were transcribed in vitro using the T7 RNA  (Table S2) or corresponding IgG were respectively   incubated with magnetic beads at room temperature for 30 minutes for each immunoprecipitation, and then the supernatants were incubated with indicated antibody-beads complexes overnight at 4 o C with rotating. Co-immunoprecipitated RNAs were extracted, reverse-transcribed to cDNA, and subjected to qPCR examination of SCDAL enrichment using the primers listed in Table S4.

ChIP-qPCR
ChIP assays were performed using the SimpleChIP ® Plus Sonication Chromatin IP Kit (Cell Signaling Technology) and followed the manufacturer's instructions.

Luciferase reporter assay
The indicated region of promoter was cloned into GV238 luciferase reporter plasmid to Renilla activity and presented as relative luciferase activity.

Acute MI model, cell transplantation and lentivirus delivery
Male adult C57BL/6J mice were purchased from Shanghai Slac Laboratory Animal Technology Corporation (China), fed a standard laboratory diet, and maintained with a 12:12-hour light/dark cycle. Acute MI was surgically induced as described previously [2a] , and all surgical procedures were performed by experienced technicians in a blinded manner. Briefly, mice (8-10

Cardiac function assessments by echocardiography and hemodynamics
All echocardiography and hemodynamics were performed by skilled technicians using identical settings and analyzed blinded to the experimental status of the animals.
Echocardiography was performed on conscious, gently restrained mice with a Vevo 2100 system before MI (baseline) as well as 3, 7, 14 and 28 days after MI.
Two-dimensional M-mode images of the left ventricle were captured at the level of the papillary muscles. Left ventricular internal diameters were measured at end-diastole (LVIDd) and end-systole (LVIDs), and then EF and FS were calculated as described previously [17] . Cardiac hemodynamics was measured after the final echocardiographic examination. Briefly, mice were anesthetized with pentobarbital sodium (60 mg/kg). The right common carotid artery was dissected under a dissecting microscope (SZX7, Olympus) and cannulated with a 1.4-Fr micro-tipped Millar (Millar Instruments). The transducer was connected to a computerized data acquisition system (PowerLab, AD Instruments) to record heart rate, blood pressure, ventricular pressures and ±dp/dt. Baseline zero reference was obtained by placing the sensor in normal saline before insertion.

Triphenyltetrazolium chloride (TTC) staining
Four weeks after MI, the entire hearts were snap-frozen, sectioned into six 1-mm-thick transverse slices from the apex to the base, incubated in a 1% solution of phosphate-buffered TTC (Sigma-Aldrich) at 37°C in the dark for 10 minutes. The infarct area (white) and viable myocardium (brick red) were delineated and calculated using Image-Pro Plus software 6.0. The ratio of the infarct area to the LV area was calculated and presented as a percentage.

Fibrotic area determination and immunofluorescence staining
Heart tissues were respectively harvested 3 days after MI to measure the survival of Endothelial proliferation was quantified as the number of CD31 + /Ki67 + cells per HRF, and inflammation was quantified as the number of CD68 + and CD3 + cells per HRF.
For assessments of apoptosis, frozen and fixed sections were detected by TUNEL staining (In Situ Cell Death Detection Kit, TMR red; Roche Applied Science).
Cardiac apoptosis was quantified as the percentage of TUNEL + nuclei per HRF in the border area of hearts, whereas apoptotic transplanted hES-MSCs/hBM-MSCs were quantified as the percentage of TUNEL + /GFP + cells per HRF.

Data availability
The RNA sequencing data used in the study are available in a public repository from NCBI. The accession numbers are SRP192798 for the lncRNA sequencing data, and SRP192797 for the mRNA sequencing data.