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Supplementary Figure 1: Expression levels of 10 lncRNAs by qRT-PCR in 15 GBC
tissues and paired non-tumor tissues. U6 was used as a loading control.
Supplementary Figure 2: Expression levels of 8 mRNAs by qRT-PCR in 15 GBC
tissues and paired non-tumor tissues. U6 was used as a loading control
Supplementary Figure 3: (A) Schematic representation of the location of GCASPC
within the IGF2R gene is shown. Exons and introns of the IGF2R gene are indicated
by green boxes and green lines, and GCASPC are depicted as the gray box. The location
of the forward (F) and reverse (R) primers used for RT-PCR and nested PCR are shown.
Orientation of arrows indicates the direction of the transcription or amplification
reaction. (B) Linear correlation between the expression of the GCASPC and IGF2R was
not observed. ∆Ct values were used to measure gene expression, which was normalized
by U6 expression levels. (C) GCASPC (mean ± standard deviation) was not
significantly changed in SGC-996 and GBC-SD cells with two different siRNAs against
IGF2R. Statistical differences were analyzed relative to the negative control (NC). *,
p<0.05. (D) IGF2R (mean ± standard deviation) was not significantly changed in GBCSD cells with two different shRNAs against GCASPC. (E) GCASPC is polyadenylated.
The level of GCASPC in purified polyadenylated RNAs and total RNA was detected
using real-time PCR (normalized to GAPDH mRNA). The data represent the average
and standard deviation of three independent experiments. (F) GCASPC gene is
moderately conserved among placental mammals compared with highly conserved
coding genes. Placental mammal conservation of GCASPC by PhastCons, UCSC
(http://genome.ucsc.edu/).
Supplementary Figure 4: Full-length human GCASPC gene cloning. Nucleotide
sequence of the full-length human GCASPC gene.
Supplementary Figure 5: GCASPC has no coding capability. (A) In the translation
assay, a 1958-bp genomic region containing GCASPC was cloned into a pcDNA
vector and expressed using the TnT Quick Coupled Transcription/Translation System
(Promega). The absence of a specific band indicated that GCASPC is a transcript with
no protein-coding capacity. Luciferase in vitro translation served as positive control.
(B,C) GCASPC is a long non-coding RNA. Lack of open reading frame in the 741-bp
GCASPC sense sequence cloned from RACE, as verified by ORF Finder (B) and CPC
(C). The transcript’s noncoding nature was suggested by negative score by PhyloCSF
(-45.0134, meaning that GCASPC is 104.50134 times more likely to be a noncoding
sequence than a coding one). (D) GCASPC was mainly located in the cytoplasm as
shown by qRT-PCR. (E) Prediction of GCASPC structure based on minimum free
energy (MFE) and partition function. Color scale indicates the confidence for the
prediction for each base with shades of red indicating strong confidence.
(http://rna.tbi.univie.ac.at/).
Supplementary Figure 6: (A) The copy number of GCASPC detected in nontumorigenic human intrahepatic biliary epithelial cell line H69 and GBC cell lines,
using RT-PCR and standard curves of known copy numbers of plasmid-derived
reference standard. Error bars show standard deviation. (B) The expression of
GCASPC in GBC-SD cells is comparable to H19, HOTAIR, CCAT1 and MALAT1.
ΔCt values were used to measure gene expression, which was normalized by U6
expression. Error bars show standard deviation. (C,D) SGC-996 Cells were stimulated
with varying concentrations of DNA methylation inhibitor 5-azacytidine (C) and the
histone deacetylase inhibitor trichostatin A (TSA) (D) for 24 hr. The data represent the
average and standard deviation of three independent experiments. *, p<0.05; **,
p<0.01.
Supplementary Figure 7: (A,B) GCASPC expression was detected in NOZ (A) and
SGC-996 (B) cells by qRT-PCR after transfection of lentivirus harboring the fulllength human GCASPC sequence or the empty vector. **, p < 0.01. (C,D) GCASPC
expression was detected in GBC-SD (C) and OCUG-1 (D) cells by qRT-PCR after
transduction of lentiviruses encoding GCASPC shRNA-1 or shRNA-2 or a scrambled
shRNA. **, p < 0.01.
Supplementary Figure 8: (A) The cell growth rates were determined by performing
CCK-8 proliferation assays. Overexpressing GCASPC in NOZ cells significantly
inhibited cell proliferation, relative to control cells. (B) GCASPC depletion enhanced
the proliferation of OCUG-1 cells. Changes in the proliferation marker, PCNA, were
shown by western blotting analysis and normalized to β-Tublin. (C,D) FACS
determined the relative cell numbers in each cell-cycle phase after propidium iodide
staining of GCASPC-overexpressed NOZ cells (C) or –downregulated OCUG-1 cells
(D). Numbers inside bars represent percentages of cells in each phase. Data are the
mean ± SD. (E) Effects of GCASPC overexpression on tumor growth in vivo.
Representative images of tumors formed in nude mice injected subcutaneously with
NOZ cells overexpressing GCASPC. Tumor weights and tumor growth curves. *, P <
0.05; **, P < 0.01. Right: Representative images of IHC staining of Ki67. (original
magnification ×200) (F) Effects of GCASPC knockdown on tumor growth in vivo.
Representative images of tumors formed in nude mice injected subcutaneously with
the GCASPC-silencing OCUG-1 cells. Tumor weights and tumor growth curves.
Right: Representative images of IHC staining of Ki67. (original magnification ×200)
Supplementary Figure 9: (A) The effect of GCASPC overexpression on the
expression of its neighboring genes in SGC-996 cells. Error bars represent S.D. (n =
3). (B) 3’-end segment of GCASPC (472~741nt) is predicted to have a stable stemloop structure. Prediction of GCASPC structure of a 472~741nt region based on
minimum free energy (MFE) and partition function. Color scale indicates the
confidence for the prediction for each base with shades of red indicating strong
confidence. (http://rna.tbi.univie.ac.at/). (C) PC expression was analyzed by western
blot analysis in GBC samples and adjacent non-tumor liver tissues (cohort 1, n = 20).
PC level was normalized to that of β-Tublin. Horizontal lines in the box plots
represent the medians, the boxes represent the interquartile range, and the whiskers
represent the 2.5th and 97.5th percentiles. The significant differences between
samples were analyzed using the Wilcoxon signed-rank test. **, p < 0.01.
Supplementary Figure 10: The effect of GCASPC on the protein, activity and
mRNA levels of PC. (A,B) The protein levels of PC were detected in GCASPCknockdown OCUG-1 cells (A) and GCASPC-up-regulated NOZ cells (B) by Western
Blot analysis. PC activity were measured in total cell lysate as described in methods.
(C,D) The mRNA levels of PC were detected in GCASPC-knockdown GBC-SD cells
(C) and GCASPC-up-regulated SGC-996 cells (D) by qRT-PCR (n=3). (E) Effects of
the truncated version (472-741 nt) of GCASPC overexpression on NOZ cell growth in
vitro. (F) The truncated version (472-741 nt) of GCASPC was overexpressed in NOZ
cells.
The protein levels of PC were detected by Western Blot analysis. PC activity
were measured in total cell lysate as described in methods.
Supplementary Figure 11: (A) GCASPC stable overexpressing NOZ cells and
control cells were incubated with MG132 (5 μM) for 24 hours. The levels of PC
proteins were detected by western blots. The experiments were performed in
triplicate; the data are expressed as the mean ± SD. *, P<0.05, **, P<0.01. (B) Cell
lysates from NOZ cells stably expressing lncRNA- GCASPC and control vector were
immunoprecipitated with a PC-specific antibody, followed by western blotting with
an antibody to ubiquitin. The bottom panel depicts the input of the cell lysates. MW,
molecular weight. (C) PC specific siRNA 1,2 effectively suppressed the protein level
of PC and the expression of proliferation marker, PCNA in NOZ cells.
Supplementary Figure 12: (A) Enhanced GCASPC expression in SGC-996 and
NOZ cells after the transfection of miR-17-3p inhibitor. **, P < 0.01. (B) miR-17-3p
expression levels in SGC-996 and NOZ cells after GCASPC overexpression. (C) Pulldown of Ago2 by biotin-labeled GCASPC or loc285194 RNA probe, as detected by
western blotting. The loc285194 lane was composed from the same gel with the same
contrast. Empty vector (Beads) was used as a negative control. Loc285194 was used
as a positive control. (E,F) RIP followed by miR qRT-PCR to detect microRNAs
endogenously associated with GCASPC and loc285194. The expression level of miR
was normalized to that in empty vector (Beads). Error bars represent the mean±S.D.
of triplicate experiments. *, P<0.05; **, P<0.01.