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Supplementary data for
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A new microRNA signal pathway regulated by long noncoding RNA TGFB2-OT1 in
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autophagy and inflammation of vascular endothelial cells
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ShuYa Huang1, Wei Lu1, Di Ge1, Ning Meng2, Ying Li1, Le Su1, ShangLi Zhang1, Yun Zhang3,
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BaoXiang Zhao2, *, JunYing Miao1,3, *
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Determination of intracellular reactive oxygen species levels
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Intracellular
reactive
oxygen
species
levels
were
measured
by
use
of
2',7'-
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dichlorodihydrofluorescein (DCHF, Sigma-Aldrich, D6883), which is rapidly oxidized into the
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highly fluorescent 2',7'-dichlorofluorescein (DCF) in the presence of intracellular reactive
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oxygen species. As previously described,1 treated cells were washed with basal M199 medium
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for 5 min and incubated with 0.5 ml DCHF at 37°C for 30 min. After washing the cells 3 times
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for 5 min with basal M199 medium, the fluorescence was monitored by confocal laser scanning
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microscopy (Leica, DMIRE2, Wetzlar, Germany) with excitation and emission wavelengths
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488 and 535 nm, respectively. The amount of ROS was quantified as the relative fluorescence
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intensity of DCF per cell in the scan room by Leica Confocal Software (LCS Lite). The
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photographs were representative of 3 independent experiments.
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Cell viability analysis
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Cell viability was determined by sulforhodamine B (SRB) assay as described previously.2 The
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proportion of living cells was calculated by the optical density ratio.
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Table S1. List of primer sequences.
Primer name
Sequence (5’ to 3’)
SQSTM1.F
CTGCCTTCTTCCAGGATCAG
SQSTM1.R
GTGAAAGCCATTAGGCAAGC
CERS1.F
GCTGTTTGGCACCGACTACC
CERS1.R
GGAGGAGACGATGAGGATGAGAG
NAT8L.F
GCTGTGCTTCGCCGTGAG
NAT8L.R
ACCGTGTTGTCCTCCTCGTG
LARP1.F
GGTTCCAGCAAGTTCCTAC
LARP1.R
ATCCAGTGTCCGTCCTTC
IL1B.F
GAGGCACAAGGCACAACAG
IL1B.R
TAGTGGTGGTCGGAGATTCG
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Supplemental results
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Table S2. The top 5 miRNAs can bind TGFB2-OT1 with significantly changed levels on
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miRNA arrays.
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A2 vs. A1 represents the changed ratio induced by 120 μM 3BDO in HUVECs for 24 h. B2 vs.
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B1 represents the changed ratio induced by TGFB2-OT1 overexpression in HUVECs for 24 h.
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The free energy represents the predicted binding ability of miRNA with TGFB2-OT1.
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Figure S1. The miRNA array results and the efficiency of TIA1 knockdown. (A) Differentially
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expressed microRNAs (miRNAs) induced by 120 μM 3BDO in human umbilical vein
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endothelial cells (HUVECs) for 24 h. Depicts miRNAs with >2-fold change in expression:
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upregulation (red) and downregulation (green). The black plots indicated miRNA expression
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changed < 2-fold. (B) Differentially expressed miRNAs induced by TGFB2-OT1
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overexpression in HUVECs for 24 h. Depicts miRNAs with >2-fold change in expression:
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upregulation (red) and downregulation (green). The black plots indicate miRNAs with < 2-fold
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change in expression. (C) Western blot analysis of TIA1 protein level in HUVECs subjected
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to scrambled siRNA (Scr) or TIA1 siRNA (siTIA1, 40 nM) for 24 h, then treated with 1 μg/ml
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LPS for 6 h. *, P < 0.05; **, P < 0.01; n = 3.
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Figure S2. The potential binding sites of MIR3960 and MIR4488 in TGFB2-OT1 and the target
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genes of these 2 miRNAs. (A) Potential sites in Luc-TGFB2-OT1 targeted by MIR3960.
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TGFB2-OT1 fragments and mutant derivatives devoid of binding-site fragments were cloned
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into the luciferase reporter vector, for in 2 constructs, Luc-TGFB2-OT1-WT and Luc-TGFB2-
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OT1-3960Δ. (B) Potential sites in Luc-TGFB2-OT1 targeted by MIR4488. TGFB2-OT1
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fragments and mutant derivatives devoid of MIR4488 binding-site fragments were cloned into
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the luciferase reporter vector, for 2 constructs, Luc-TGFB2-OT1-WT and Luc-TGFB2-OT1-
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4488Δ. (C) Potential sites in the 3’UTR of CERS1, NAT8L and LARP1 targeted by MIR3960,
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MIR4488 and MIR4459, respectively. The 3’UTR of CERS1, NAT8L and LARP1 within the
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binding sites were cloned into the luciferase reporter vector, for 3 constructs, Luc-CERS1-
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3’UTR, Luc-NAT8L-3’UTR and Luc-LARP1-3’UTR.
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Figure S3. The transfection efficiency and effects on target genes of the miRNA mimics and
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inhibitors. (A) qPCR analysis of levels of MIR3960 and MIR4488 in HUVECs transfected with
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different concentrations of MIR3960 and MIR4488 mimics or inhibitor, respectively. (B) qPCR
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analysis of mRNA levels of CERS1 and NAT8L in HUVECs transfected with different
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concentrations of MIR3960 and MIR4488 mimics or inhibitor, respectively. *, P < 0.05; **, P
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< 0.01; n = 3.
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Figure S4. The effect of 3BDO on the mRNA levels of CERS1, LARP1 and NAT8L. (A) qPCR
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analysis of mRNA level of CERS1 of HUVECs treated with 3BDO (0, 15, 30, 60 and 120 μM)
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for 12 h. (B) qPCR analysis of mRNA levels of LARP1 and NAT8L in HUVECs treated with
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3BDO (0, 15, 30, 60 and 120 μM) for 24 h. *, P < 0.05; **, P < 0.01; n = 3.
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Figure S5. The effect of TGFB2-OT1 overexpression on ATG3 and ATG7 protein levels.
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Western blot analysis of ATG3 and ATG7 protein levels in HUVECs transfected with 0.01,
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0.02, 0.1, 0.2 μg/ml of pCMV6 or pCMV6-TGFB2-OT1 for 48 h and quantification. **, P <
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0.01; n = 3.
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Relative SQSTM1
mRNA level
Relative SQSTM1
mRNA level
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Figure S6. 3BDO affects SQSTM1 protein level rather than mRNA level. (A) qPCR analysis
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of SQSTM1 mRNA level in HUVECs transfected with 0.1, 0.2, 0.4 μg/ml of pCMV6 or
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pCMV6-TGFB2-OT1 for 48 h. (B) Western blot analysis of SQSTM1 protein level in HUVECs
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transfected with pCMV6 or pCMV6-TGFB2-OT1 for 24 h, then treated with or without 3BDO
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(60 μM) for 6 h. (C) qPCR analysis of SQSTM1 mRNA level in HUVECs exposed to 50 μg/ml
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oxLDL with and without 3BDO (3BDO-L, 60 μM; 3BDO-H, 120 μM) for 12 h. *, P < 0.05; n
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= 3.
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Figure S7. The effect of TGFB2-OT1 overexpression on NFKB RELA nuclear translocation,
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cell viability and reactive oxygen species levels. (A) Percentage of NFKB RELA nuclear
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translocation in HUVECs transfected with 0.4 μg/ml of pCMV6 or pCMV6-TGFB2-OT1
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respectively for 48 h. (B) Percentage of NFKB RELA nuclear translocation in HUVECs
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transfected with pCMV6, pCMV6-TGFB2-OT1, or both siRNA against SQSTM1 and pCMV6-
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TGFB2-OT1 respectively for 48 h. (C) Relative ratio of cells with to without NFKB RELA
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nuclear translocation in HUVECs transfected with pCMV6, pCMV6-TGFB2-OT1, or both
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siRNA against SQSTM1 and pCMV6-TGFB2-OT1 respectively for 48 h. (D) Cell viability was
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detected by SRB in HUVECs transfected with 0.4 μg/ml of pCMV6 or pCMV6-TGFB2-OT1
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for 48 or 72 h, and then exposed with 3BDO (60 μM) for 12 h. (E) Intracellular reactive oxygen
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species levels examined by 2',7'-dichlorodihydroluorescein (DCHF). For reactive oxygen
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species levels in HUVECs, the cells were transfected with 0.1, 0.4 μg/ml of pCMV6-TGFB2-
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OT1 or 0.4 μg/ml pCMV6 for 24 h. Nor, normal HUVECs without any treatment. **P < 0.01,
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n = 3.
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Figure S8. The effect of lncRNA LOC100129973 on SQSTM1 protein level and NFKB RELA
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nuclear translocation. (A) Western blot analysis of SQSTM1 protein level in HUVECs
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transfected with 0.01, 0.05, 0.1 and 0.2 μg/ml of pcDNA or pcDNA-LOC100129973 for 48 h.
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(B) Immunofluorescence staining of SQSTM1 in HUVECs transfected with pcDNA or
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pcDNA-LOC100129973 (0.2 μg/ml) for 48 h. Bar: 16 μm. (C) Western blot analysis of
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SQSTM1 protein level in HUVECs transfected with pcDNA or pcDNA-LOC100129973 (0.2
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μg/ml) for 12 h, and then treated with or without cycloheximide (CHX) for 12 h. *P < 0.05, n
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= 3. (D) Representative photomicrographs of immunofluorescence staining showing NFKB
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RELA nuclear translocation in HUVECs transfected with pcDNA or pcDNA-LOC100129973
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(0.4 μg/ml) respectively for 48 h. DAPI represent the nuclear. Bar: 16 μm.
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