1 Prediction of ZFAS1 over-expression in PAAD by bioinformatic analysis
To investigate the differential expression level of ZFAS1 and other lncRNAs in PAAD, nine GEO datasets were normalized and background adjusted to make differential expression analysis. 44 lncRNAs including ZFAS1 were identified to be up-regulated in PAAD (table 1) (Fig. 1A), ZFAS1 expression level in PAAD were further validated by ONCOMINE, UALCAN and GEPIA databases. Result from Pei 2009 microarray from ONCOMINE supported the GEO results in their 52 samples (16 normal and 36 cancer tissues), AUC (Area Under the Curve) value of ROC (Receiver Operating Characteristic) curve was 0.889 (Fig. 1B-C). UALCAN and GEPIA, which are both online tools based on The Cancer Genome Atlas (TCGA), showed the correlation between ZFAS1 expression level and patients’ gender, tumor grade and drinking habits (Fig. 1D-F), patients also showed survival differences when considering the combined effect of ZFAS1 level and drinking habit (Fig. 1G). However, no significant expression or survival difference of ZFAS1 were found according to TCGA based results alone (Fig. 1H-I). It is noteworthy that once the addition of GTEx data increased the sample size of TCGA-PAAD normal tissues (from 4 to 171), the significance of ZFAS1 expression level difference in PAAD was improved immediately (Fig. 1J).
2 Identification of ZFAS1 expression level and clinical correlation in PAAD by experimental analysis
A microarray with 170 points (71 paired and 28 single PAAD tissues) was obtained from Outdo Biotech (Shanghai, China). The expression level of ZFAS1 in PAAD tissues and its subcellular distribution were investigated by ISH, the expression score were collected by calculating the intensity*area. Result showed that ZFAS1 was significantly higher expressed in cancer than normal tissues and existed both in cytoplasm and nucleus (Fig. 2A-C). According to ZFAS1 expression value, patients were divided into two groups, Furthermore, patients with lower ZFAS1 level have significant longer overall survival time (Fig. 2D) and the AUC of ROC curve was 0.747 (Fig. 2E). However, ZFAS1 expression level showed no correlation with PAAD clinicopathological factors (Table 2). ZFAS1 expression in PAAD cell lines were also detected by qRT-PCR, results showed that ZFAS1 expression level was also significantly up-regulated in PAAD cell lines (bxpc-3, sw1990 and panc1) than the normal cell line hpde6c7 (Fig. 2F). With highest ZFAS1 expression level, sw1990 and bxpc3 cell lines were selected for the following assays.
|
Table1 Up-regulated lncRNAs in the merged GEO dataset
|
Gene
|
LogFC Adj.P.Val Regulated
|
|
ZFAS1
|
1.292114407
|
3.79E-10
|
Up-Regulated
|
|
ZEB1-AS1
|
0.751786689
|
3.16E-08
|
Up-Regulated
|
|
UCA1
|
1.431842139
|
2.46E-08
|
Up-Regulated
|
|
TUG1
|
0.819486239
|
3.85E-12
|
Up-Regulated
|
|
TP53TG1
|
0.635576903
|
9.28E-08
|
Up-Regulated
|
|
TMEM191A
|
0.623671728
|
2.56E-07
|
Up-Regulated
|
|
THAP9-AS1
|
1.613537794
|
7.11E-21
|
Up-Regulated
|
|
SNHG17
|
1.022943348
|
4.65E-12
|
Up-Regulated
|
|
SH3PXD2A-AS1
|
0.740309382
|
1.56E-06
|
Up-Regulated
|
|
RPL32P3
|
0.727465001
|
3.06E-08
|
Up-Regulated
|
|
RNASEH1-AS1
|
0.61011974
|
3.68E-08
|
Up-Regulated
|
|
RARA-AS1
|
0.639837801
|
7.40E-11
|
Up-Regulated
|
|
PP7080
|
0.763093938
|
6.47E-06
|
Up-Regulated
|
|
POTEKP
|
0.642481197
|
4.36E-06
|
Up-Regulated
|
|
PMS2P3
|
0.632825563
|
1.18E-10
|
Up-Regulated
|
|
PMS2P1
|
0.900767335
|
4.30E-13
|
Up-Regulated
|
|
PAX8-AS1
|
0.897048546
|
1.77E-05
|
Up-Regulated
|
|
OTUD6B-AS1
|
0.746209247
|
1.89E-11
|
Up-Regulated
|
|
OR7E14P
|
1.188067838
|
3.01E-11
|
Up-Regulated
|
|
NRAV
|
0.622272261
|
8.29E-13
|
Up-Regulated
|
|
MIR31HG
|
0.691318092
|
5.08E-05
|
Up-Regulated
|
|
MIR210HG
|
0.872969962
|
2.99E-08
|
Up-Regulated
|
|
MIF-AS1
|
0.760101248
|
1.85E-09
|
Up-Regulated
|
|
LINC01503
|
0.713069329
|
2.38E-07
|
Up-Regulated
|
|
LINC01207
|
0.967896702
|
0.001919014
|
Up-Regulated
|
|
LINC01137
|
0.615492033
|
8.09E-09
|
Up-Regulated
|
|
LINC01133
|
0.855940052
|
1.57E-08
|
Up-Regulated
|
|
LINC00857
|
0.852965802
|
3.37E-12
|
Up-Regulated
|
|
LINC00467
|
0.771416443
|
7.23E-10
|
Up-Regulated
|
|
LINC00094
|
0.780486158
|
1.15E-11
|
Up-Regulated
|
|
JHDM1D-AS1
|
0.635281024
|
8.44E-11
|
Up-Regulated
|
|
HOTAIRM1
|
0.617801402
|
0.001358296
|
Up-Regulated
|
|
HMGN2P46
|
0.74496713
|
5.94E-08
|
Up-Regulated
|
|
HLA-J
|
0.959015425
|
5.48E-07
|
Up-Regulated
|
|
HCP5
|
1.086029552
|
1.96E-08
|
Up-Regulated
|
|
GBAP1
|
0.892668634
|
1.67E-12
|
Up-Regulated
|
|
EP300-AS1
|
0.988390142
|
1.40E-09
|
Up-Regulated
|
|
CRNDE
|
1.991179914
|
1.82E-19
|
Up-Regulated
|
|
BLACAT1
|
1.485832133
|
1.33E-14
|
Up-Regulated
|
|
APTR
|
0.752779098
|
2.27E-15
|
Up-Regulated
|
|
ANXA2P3
|
0.711844952
|
3.92E-16
|
Up-Regulated
|
|
ANXA2P2
|
1.753775455
|
5.51E-21
|
Up-Regulated
|
|
ANXA2P1
|
0.667336504
|
1.29E-10
|
Up-Regulated
|
|
AFAP1-AS1
|
1.486798093
|
1.47E-13
|
Up-Regulated
|
|
|
|
|
|
|
|
3 ZFAS1 knockdown inhibits cell metastasis in PAAD
To investigate the function of ZFAS1 in vitro, the synthesized small interfering RNAs (siRNAs) were respectively transfected into sw1990 and bxpc3 for 48 h. Wound healing (Fig. 3A-B) and transwell migration assays (Fig. 3C) showed the metastasis of bxpc3 and sw1990 were inhibited after ZFAS1 knockdown. The transfection efficiency of si-ZFAS1 was detected with qRT-PCR (Fig. 3D). Thus, ZFAS1 knockdown was suggested to inhibit PAAD metastasis in vitro.
4 ZFAS1 functions as a sponge of miR-3924
Bioinformatic tools DIANA lncbase, Starbase and RNAhybrid were used to explore the target miRNAs of ZFAS1 (Fig. 4A). According to the intersection of Starbase and Diana tool, miR-3924 was chosen and the binding site was predicted by RNAhybrid (Fig. 4B-C), dual luciferase report results showed that miR-3924 mimic could inhibit the luciferase activity of ZFAS1-wt but not of ZFAS1-mut (Fig. 4D). To further demonstrate whether ZFAS1 played its role through miR-3924, we established bxpc3 and sw1990 cell lines stable ZFAS1 silenced by using shRNA. By inhibiting miR-3924 expression level in ZFAS1 stable silence bxpc3 and sw1990 cell lines, wound healing (Fig. 4E-F) and transwell migration assays (Fig. 4G) showed the reversed results of both two cell lines, indicating the antagonistic effect of ZFAS1 and miR-3924. Besides, in view of few miR-3924 related studies has been reported, we also investigated the function of miR-3924 in PAAD cells, results showed that the over-expression of miR-3924 could inhibit the metastasis of bxpc3 and sw1990 (Fig. 4H-J). The sh-ZFAS1 transfection efficiency was detected with qRT-PCR (Fig. 4K). Taken together, ZFAS1 functions as a sponge of miR-3924.
5 ROCK2 functions as the target of ZFAS1/miR-3924
GSEA was made according to the merged GEO data, the results showed that high ZFAS1 expression level was most correlated with focal adhesion (Table 3)(Fig. 5A), by the intersection of GSEA results and miR-3924 target mRNAs prediction, ROCK2 was selected as the mRNA target (Fig. 5B-C). Dual luciferase report showed that the luciferase activity of ROCK2-wt/miR-3924 mimic group was significantly lower than ROCK2-Wt group, furthermore, no difference between ROCK2-Mut group and ROCK2-Mut/miR-3924 mimic group was found (Fig. 5D). Western blot results showed the expression level of FAK, RHOA and ROCK2 were decreased after ZFAS1 knockdown or miR-3924 over-expression (Fig. 5E-H). Taken together, ROCK2 was suggested to function as the target of ZFAS1/miR-3924.
Table2 Correlation between ZFAS1 expression and PAAD clinicopathological factors
|
|
|
ZFAS1 level
|
|
Characteristics
|
N
|
Low high
|
|
Total cases
|
83
|
63
|
20
|
|
|
Gender
|
83
|
|
|
|
|
Male
|
|
37
|
13
|
|
p=0.794
|
Female
|
|
26
|
7
|
|
|
Age(years)
|
82
|
|
|
|
|
<60
|
|
30
|
11
|
|
p=0.798
|
≧60
|
|
32
|
9
|
|
|
TNM stage
|
82
|
|
|
|
|
Ⅰ-Ⅱ
|
|
61
|
19
|
|
P=0.431
|
Ⅲ-Ⅳ
|
|
1
|
1
|
|
|
Tumor grade
|
18
|
|
|
|
|
Ⅰ
|
|
8 1
|
P=0.576
|
Ⅲ
|
|
6 3
|
|
Tumor size(cm)
|
82
|
|
|
|
|
<5
|
|
41
|
16
|
|
p=0.279
|
≧5
|
|
21
|
4
|
|
|
Perineural invasion
|
36
|
|
|
|
|
No
|
|
3 1
|
p=1
|
Yes
|
|
23 9
|
|
Lymph nodes
|
76
|
|
|
|
|
metastasis
|
|
|
|
|
|
No
|
|
29 11
|
p=0.285
|
Yes
|
|
30
|
6
|
|
|
Table3 ZFAS1 overexpression related pathways by GSEA
|
Name
|
Size
|
Es
|
Nes
|
Nom p-val
|
|
KEGG_FOCAL_ADHESION
|
194
|
0.53620225
|
1.7095456
|
0.004081633
|
|
KEGG_ECM_RECEPTOR_INTERACTION
|
81
|
0.5935364
|
1.644135
|
0.043737575
|
|
KEGG_MTOR_SIGNALING_PATHWAY
|
50
|
0.5283579
|
1.5339531
|
0.035196688
|
|
6 ZFAS1 silence inhibited tumor metastasis in vivo
To investigate the biological function of ZFAS1 in vivo, stable sh-ZFAS1 and sh-NC transfected sw1990 cells were selected by puromycin and tumor metastasis models were established by intravenous injection. Results showed that comparing with NC group, ZFAS1 silence could significantly inhibit liver but not lung metastasis in vivo (Fig. 6).