Non-Coding RNA 2016, 2, 7; doi: 10.3390/ncrna2020007
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Supplementary Materials: Promoter-Associated RNAs
(paRNAs) Regulate HSPC152 Gene Expression in
Malignant Melanoma
Hamutal Bonen Nitzan Kol, Noam Shomron, Raya Leibowitz-Amit, Luca Quagliata,
Thomas Lorber, Yechezkel Sidi and Dror Avni
1S paHSPC152 sequence
>hg18_refGene_NM_001286082 range=chr11:63841575-63842632
GGGACTGTCCCGGGAGGTGAGGCCCCCACGATTGGCGAGAAAGGTGGGCA
GCCACGAGGGCCTTCTGAAGGGATGAGCCGGGCGGGCGGAGGGGAGGGGT
CTGGCAGGCAGGAAATAAAGCGATACTCTCAGACTAGCGGGACGGGGATT
GCCATGGGGGTAGTGGGGGATGTCAGGCCCGGCCGGGCCAGCGGTCACCT
TGATTGGGGCCATGGCTGCAGCGGCTCTGCTGAAACTGCGGACCCCGCCA
GACGCCCACTCTCCTTCACTGTACCGTCTTGCTGCCGCTGCCGCAGACTG
ACCGCCGGCCCCACCGACGAGTATATAGCCCGCTGAGCGTCTCAGGGCGC
ACACGCCAGCTAGTCCCATACCCGCCCCACGGCCACTTCCACTCCGCCTC
CTGGCTGGTGCCACCGCAGCAGGCGGGGCACCGCTAGCGGCACAGCGGCG
ACACCCTGCGGAAGGCGGGCCTGGGCCGCCTCCACTGCGCAGGCGCGAGC
GGCCACCCGCAGAACAGAGCTTCCGGGACCCACGCCTCGTTTGCACTGGG
TGCTGGACAGCCGACGCAACTACAAATGGGGCGGAGCTTTCGGCACTGGA
GCAGCTAATTTGCATATAGGAATGAGGTGCGGCTCGGCTTCCATGGGCCT
AATTTACAGATAGGGCGGTATTTCTGCCCCTTAACCGAAAGTGGGATACA
GAGGACGACGGTGTTAGGCGCCTGTGTAGGAGTAAAATGTGTTTATTTTG
CATTCAACGAGAGCTCCTGCATTGCAGCTATTTTGCATATGATTTGCATC
TTACGAAGAATTTGTGGCAAAAAAAAGCTGGGCGTGCGCCGTAGGAACCT
CCTGCTGAGACGCTTCCGGTAGCGGCGCGTGACCCGACAGGTCTTTCACC
TACCTACCTCAGCTCCCACAAACACGAGAAGTTCCAGCAAGTTCGCCACT
TCCGGTTCTCCTGGCTATCCAATAGCATCGAGAGGAGCATCCCCGGAAGT
GAGGCAGCGGAGGACGACCTTTTTCCGGTTCCGGCCTGGCGAGAGTTTGT
GCGGCGAC
Figure S1. The promoter region of HSPC152. - The paHSPC152 sequence that does not overlap with
the mRNA is marked in red. Marked in blue is the part of the paHSPC152 sequence that overlaps
with the mRNA. The yellow denotes the sequence targeted by the siRNA.
Non-Coding RNA 2016, 2, 7; doi: 10.3390/ncrna2020007
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Table S1. Identified paRNA. The positions of the transcriptional start sites (TSS) and the genes are numbered according to human Genome assembly: GRCh37. The TSS were taken from the TSS database available at DataBase of Transcriptional Start Sites (DBTSS, [3]). The p value and q value were calculated by the CUFFDIFF differential
expression method [1]. The 11 genes that withstood all our criteria are marked in yellow.
TSS Id
Gene Start
99186112
85132880
122110877
7146068
2735181
TSS
Length
498
400
402
491
400
127281
357320
51638
826084
1452833
39466245
39466676
431
17419957
88910743
24737713
150131593
64084913
24126329
154320510
24117374
113344441
67275946
17990997
31430662
34633726
64781406
57335657
97355963
55972965
30595733
8093350
20937265
88878217
62446333
48963729
25701904
17420409
88911143
24738248
150131993
64085351
24126827
154320910
24117811
113344920
67276366
17991404
31431254
34634126
64781806
57336093
97356466
55973523
30596310
8093750
20937665
88878646
62446845
48964129
25702304
452
400
535
400
438
498
400
437
479
420
407
592
400
400
436
503
558
577
400
400
429
512
400
400
chr
TSS Start
TSS end
chr10
chr2
chr5
chr17
chrY
99185614
85132480
122110475
7145577
2734781
chr19
chr19
chr11
chr7
chr1
chr11
chr22
chr5
chr1
chr12
chr11
chr17
chr6
chr15
chr11
chr11
chr9
chr19
chrX
chr17
chr14
chr16
chr11
chr12
chr6
99186026
85132762
122110749
7143737
2709622
Gene
Name
PGAM1
TMSB10
SNX2
GABARAP
RPS4Y1
Overlap with
mRNA
86
118
128
176
216
# Reads
Control
23
192
40
60
27
# Reads
Melanome
1
45
3
6
0
265536
39432041
FBXO17
135
49
266332
613993
1098749
926386
591544
485419
12961
228650
903811
1291151
833316
790921
717480
583952
590943
665101
285142
729659
1473435
68394
638932
601713
866749
1013282
17420336
88911039
24737973
150122169
64084165
24115035
154320632
24117645
113344738
67273967
17991282
31430957
34633916
64781584
57319127
97321002
55964345
30576940
8091650
20937537
88875876
62443971
48961466
25652428
DDA1
TYR
DFNA5
PLEKHO1
HSPC152
MMP11
MRPL22
LYPLA2
OAS1
CDK2AP2
DRG2
HCP5
NOP10
ARL2
UBE2L6
FBP2
ISOC2
CXorf21
C17orf59
NP
APRT
UBXN1
LALBA
SCGN
73
104
275
232
120
174
278
166
182
156
122
297
210
222
146
112
84
300
214
128
125
194
100
104
11
0
9
8
1
8
7
6
0
5
16
4
6
2
2
0
1
1
1
1
1
0
1
1
p Value
q-Value
0
0
0
0
0
0
0
0
0
0
2
0
0
0
9
0
0
9
0
0
0
6
0
2
0
8
4
0
2
0
0
0
2
2
1
0
0
0.001
0.002
0.003
0.005
0.005
0.006
0.009
0.014
0.014
0.025
0.03
0.046
0.094
0.132
0.133
0.157
0.225
0.249
0.25
0.286
0.286
0.293
0.317
0.317
0
0
0
0.001
0.001
0.001
0.001
0.003
0.003
0.005
0.006
0.008
0.011
0.024
0.016
0.027
0.026
0.028
0.028
0.049
0.049
0.033
0.051
0.051
Non-Coding RNA 2016, 2, 7; doi: 10.3390/ncrna2020007
chr12
chr10
chr10
chr11
chr19
chr19
chr22
chr20
chr4
chr20
chr2
chr7
chr12
chr1
chr22
chrX
chr5
chr15
120765482
99446796
99078703
125773890
49954941
1095150
46663581
44098264
147559833
3026376
118571967
27196040
10456736
22979406
29196423
9754178
174905263
77712737
120765962
99447282
99079204
125774421
49955388
1095550
46664034
44098664
147560254
3026806
118572399
27196476
10457175
22979853
29196885
9754652
174905696
77713344
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480
486
501
531
447
400
453
400
421
430
432
436
439
447
462
474
433
607
887799
99759
117173
597581
263512
288852
490592
1080343
1428019
823244
353507
1164998
892404
257218
486904
737226
26298
717017
120759913
99437180
99079021
125774271
49949549
1086577
46663860
44098393
147560044
3024267
118572254
27193337
10457049
22979681
29190547
9754495
174905513
77713242
PLA2G1B
AVPI1
FRAT1
DDX25
PIH1D1
POLR2E
TTC38
WFDC2
POU4F2
GNRH2
DDX18
HOXA7
KLRD1
C1QB
XBP1
SHROOM2
SFXN1
HMG20A
110
219
183
150
150
241
174
271
210
15
145
256
126
172
137
157
183
102
1
1
0
1
19
4
4
0
1
4
87
2
5
4
6
10
33
18
0
0
1
0
12
1
1
1
0
0
31
0
0
0
0
6
31
3
0.317
0.317
0.317
0.317
0.319
0.593
0.593
0.317
0.317
0.046
0.379
0.157
0.025
0.046
0.014
0.532
0
0.086
0.051
0.051
0.051
0.051
0.051
0.093
0.093
0.051
0.051
0.008
0.06
0.027
0.005
0.008
0.003
0.084
0
0.015
Non-Coding RNA 2016, 2, 7; doi: 10.3390/ncrna2020007
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Figure S2. Effect of paHSPC152 sense or antisense overexpression on putative HSPC152 promoter
methylation in 014mel cells by bisulfite sequencing in a stable line. The 014mel cells were stably
transfected with a paHSPC152-expressing plasmid. Represented is a sequencing chromatogram of
DNA extracted from 014mel cells. Each DNA sample was treated with bisulfite to convert
unmethylated residues of cytosine into thymine, PCR amplified and then sequenced. PCR products
were sequenced and analyzed by the BioEdit Sequence Alignment Editor software[2].
Figure S3. The effect of paHSPC152 overexpression on soft agar colony formation. Representative
photomicrographs of colony-formation assays are shown. The assays were conducted using (A)
014mel; (B) 014mel overexpressing empty vector; (C) 014mel overexpressing paHSPC152. 6 × 103
cells were suspended in media containing 0.6% SELECT Agar (BD Difco™ Agar, Franklin Lakes, NJ,
USA) and plated on a bottom layer of media containing 1% SELECT Agar in a 96-well plate. Plates
were incubated at 37 °C for 16 days before imaging. The colonies were photographed at 5×
Non-Coding RNA 2016, 2, 7; doi: 10.3390/ncrna2020007
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magnification.
Figure S4. The effect of paHSPC152 overexpression on the cell cycle. MelST cell lines transfected
with sense-oriented paHSPC152 were subjected to fluorescence-activated cell sorting (FACS) cell
cycle analysis. paHSPC152 effect on cell cycle was tested 48 h (a) or 72 h (b) from transfection. Cells
were harvested and kept in 70% ethanol in phosphate-buffered saline (PBS) solution at −20 °C until
analyzed. Next, 15 min prior to FACS analysis, propidium iodide was added to the cells. The 5000
014mel or 8000 melST cells were read from each sample and analyzed using the “FlowJo” software
(FlowJo, LLC, data analysis software , Ashland, OR, USA), using “24 h mock" as the baseline. G1—
pre-synthesis phase, S—synthesis, G2M─ post-synthesis and mitosis phase. Graphs show the
average results of at least five repeats. * = p value < 0.0149 as calculated by t-test.
Non-Coding RNA 2016, 2, 7; doi: 10.3390/ncrna2020007
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Figure S5. The effect of paHSCP152 on cell proliferation in (A) 014mel cells or (B) MelST cells. Cells
were transfected using plasmid sense paHSPC. Proliferation was examined using the
bromodeoxyuridine (BrdU) incorporation assay. ΔOD was measured for all time points, starting at
time 0 (4 h from transfection) and up to 96 h. The graph represents the average of four different
experiments. Graphs show the average results of at least three repeats. * = p value < 0.05 as
calculated by t-test.
References
1. Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, et al. (2013) Differential analysis of gene
regulation at transcript resolution with RNA-seq. Nat Biotechnol 31: 46-53.
2. Hall AT (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for
Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98.
3. http://dbtss.hgc.jp