Expression analysis of a panel of long non-coding RNAs (lncRNAs) revealed their potential as diagnostic biomarkers in bladder cancer

Genomics xxx (xxxx) xxx–xxx

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Genomics journal homepage: www.elsevier.com/locate/ygeno

Expression analysis of a panel of long non-coding RNAs (lncRNAs) revealed their potential as diagnostic biomarkers in bladder cancer Feraydoon Abdolmalekia, Soudeh Ghafoui-Farda, Mohammad Taherib, Alireza Mordadic, Mandana Afsharpadd, Sajad Varmazyare, Bashir Nazparvarf, Vahid Kholghi Oskooeia, ⁎ Mir Davood Omranig, a

Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran c Department of Microbiology, Hamadan University of medical science, Hamadan, Iran d Cancer Control Research Center, Cancer Control Foundation, Iran University of Medical Sciences, Tehran, Iran e Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran f Department of Anatomy, Legal Medicine Research Center, Tehran, Iran g Urology and Nephrology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran b

ARTICLE INFO

ABSTRACT

Keywords: Bladder cancer lncRNA HOTAIR NEAT1 TUG1 FAS-AS1 GHET1 HOTAIRM1 DLEU2 THRIL DSCAM-AS1

Introduction: Long non-coding RNAs (lncRNAs) have fundamental roles in cell migration, proliferation, invasion and metastasis. Methods: In the current study, we evaluated expression of a panel of lncRNAs in bladder cancer tissues, adjacent non-cancerous tissues (ANCTs) and normal bladder tissues to evaluate their diagnostic power. Results: PV1 was down-regulated in tumor tissues compared with both ANCTs and normal controls (Expression ratios of 0.48 and 0.14; P values of 0.4 and < 0.001 respectively). HOTAIR, NEAT1, TUG1 and FAS-AS1 were significantly down-regulated in tumor tissues compared with normal controls (Expression ratios of 0.4, 0.68, 0.54 and 0.11; P values of 0.04, 0.02, 0.02 and < 0.001 respectively). Conclusion: Combination of transcript levels of seven lncRNAs improved both sensitivity and specificity values to 100%. The current study shows dysregulation of lncRNAs in bladder cancer and implies their role as diagnostic markers in this malignancy.

1. Introduction Long non-coding RNAs (lncRNAs) comprise the major part of the human transcriptome and are involved in the regulation of fundamental aspects of cell life. Their diverse roles in the regulation of cell migration, proliferation, invasion and metastasis have endowed them the potential to be used as diagnostic markers or therapeutic targets in malignancies. Several studies have reported dys-regulation of lncRNAs in bladder cancer [1]. Bladder cancer is the ninth most common human malignancy for both sexes combined. The worldwide mortality from this malignancy is estimated to be 165,000 in 2012 [2]. Currently, the bladder cancer diagnosis is based on the concomitant use of cystoscopy and urine cytology with the first method being an invasive method associated with risk of trauma and infection [3]. There are numerous ongoing researches for identification of dys-regulated transcripts in bladder tissues or urine whose expression levels might be used as novel biomarkers in this type of cancer [4,5]. Based on the

⁎

recent highlighting of the significance of lncRNAs in bladder cancer [6], we designed the current study to evaluate expression levels of a panel of lncRNAs in three kinds of bladder tissues including normal, adjacent noncancerous tissues (ANCTs) and tumoral tissues. The selected lncRNAs were involved in the regulation of fundamental cancer-related processes such as apoptosis and proliferation (TUG1 [7], DLEU2 [8], PVT1 [9], HOTAIRM1 [10], GHET1 [11], DSCAM-AS1 [12] and HOTAIR [13]), regulation of genes expression (NEAT1 [14]), regulation of cell cycle (FAS-AS1 [15], DSCAM-AS1 [12] and HOTAIR [13]) or immune response (THRIL [16]). 2. Material and methods 2.1. Study participants A total of 50 patients with definite diagnosis of bladder cancer were recruited for the present study. Both tumoral and ANCTs were excised

Corresponding author. E-mail address: [email protected] (M.D. Omrani).

https://doi.org/10.1016/j.ygeno.2019.04.020 Received 8 January 2019; Received in revised form 22 April 2019; Accepted 30 April 2019 0888-7543/ © 2019 Published by Elsevier Inc.

Please cite this article as: Feraydoon Abdolmaleki, et al., Genomics, https://doi.org/10.1016/j.ygeno.2019.04.020

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during surgery from all patients. Patients did not receive and chemo/ radiotherapy before tissue excision. All specimens were examined by clinical pathologists to assess the presence of tumor cells. An additional 30 normal bladder samples were obtained from dead bodied and used as controls. Control subjects had no history of urogenital diseases or malignancies. The study protocol was approved by the ethical committee of Shahid Beheshti University of Medical Sciences. All patients have signed written informed consent forms.

the receiver operating characteristic (ROC) curve. The area under curve (AUC) values were used for judgment about appropriateness of putative biomarkers (Fig. 1). 3. Results 3.1. Clinical characteristics of study participants Table 2 shows general data of study participants.

2.2. Expression study

3.2. Relative expression of lncRNAs in tumor tissues, ANCTs and normal tissues

A literature-based method was used for selection of lncRNAs. Ten lncRNAs were selected based on their roles in the fundamental aspects of carcinogenesis process. Total RNA was extracted from all tissues using TRIzol™ Reagent (Invitrogen, Carlsbad, CA, USA) according to instructions provided by the company. Next, cDNA was produced from approximately 50–100 ng of RNA samples using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Transcript levels of lncRNAs were compared between tumor tissues, ANCTs and control samples in the Rotor Gene 6000 Real-Time PCR Machine (Corbett, Australia) using TaqMan® Universal PCR Master Mix (Applied Biosystems, USA). Expression levels were normalized to HPRT1 transcripts. The sequences of primers and probes are shown in Table 1.

PV1 was down-regulated in tumor tissues compared with both ANCTs and normal controls (Expression ratios of 0.48 and 0.14; P values of 0.4 and < 0.001 respectively). HOTAIR, NEAT1, TUG1 and FAS-AS1 were significantly down-regulated in tumor tissues compared with normal controls (Expression ratios of 0.4, 0.68, 0.54 and 0.11; P values of 0.04, 0.02, 0.02 and < 0.001 respectively). Finally, GHET1 and HOTAIRM1 were significantly up-regulated in tumor tissues compared with normal controls (Expression ratios of 9.61 and 8.62; P values of < 0.001 and 0.004 respectively). Expression levels of DLEU2, THRIL and DSCAM-AS1 were not significantly different between three sets of samples. Table 3 shows relative expression levels of lncRNAs in tumor tissues, ANCTs and normal tissues.

2.3. Statistical analysis

3.3. Associations between relative expression of lncRNAs and patients' data

REST 2009 software was used for assessment of relative expression levels of genes in tumor tissues vs. ANCTs/controls. The significance of difference in expression of lncRNAs between tumor tissues and ANCTs/ controls was appraised using t-test. The association between tumor features and relative expression of lncRNAs was evaluated using Chisquare test. P values < .05 were considered as significant. The diagnostic power of lncRNAs in bladder cancer was appraised by plotting

Significant associations were detected between relative expression levels of DLEU2 and patients' age (P = .04), GHET1 and hematuria (P = .04) and FAS-AS1 and tumor recurrence (P = .02). Table 4 shows the results of association analysis between relative expression of lncRNAs and patients' data.

Table 1 Nucleotide sequences of primers and probes. Gene name

Primer and probe sequence

Primer and probe length

Product length

HPRT1

F: AGCCTAAGATGAGAGTTC R: CACAGAACTAGAACATTGATA FAM -CATCTGGAGTCCTATTGACATCGC- TAMRA F: CCAGTGTGAGTCCTAGCATTGC R: CCTGGAAACAGAACATTGGAGAAC FAM- ACCCTGGAGGAGAGAGCCCGCC - TAMRA F: ACCGGAGGAGCCATCTTGTC R: GAAAGAGCCGCCAACCGATC FAM - ACCGCACGCCCGTTCCTTCGC -TAMRA F: GAAAAGGTGCCGTTCTTCCG R: CTGGCAGTTCTCAGACGTAGG FAM - CGGCTTAACCACTGCTTCGGTGCT -TAMRA F: CTTCTTTGATTGAATACTTACATA R: TATTGTGGTCTTCATTCTATC FAM- GCATTGGAACATGACATGAGATTAAGG- TAMRA F: CCCATTACGATTTCATCTC R: GTTCGTACTCATCTTATTCAA FAM- AGCAAGCACCTGTTACCTGTC - TAMRA F: GAAGAGCAAAAGCTGCGTTCTG R: CTCTCGCCAGTTCATCTTTCATTG FAM-CCCGACTCCGCTGCCCGCCC-TAMRA F: GAGTGCAGTGGCGTGATCTC R: AAAATTAGTCAGGCATGGTGGTG FAM- CTCACCGCAACCTCCACCTCCCAG- TAMRA F:AGTCAGCTCCCTACAGAGGTG R:TCCTTAGGTGGTGGTTTCTGTTC FAM-TCCCACTGCCCAAGATCCCTGCCT-TAMRA F:GATGAATTAAAATGGAGTGGAAC R:AAAACGTGCTGAGAATTCTAG FAM-GTTATCCACTCACTGACTTAGGTGC-TAMRA F:GGAATGGAACGGATTTAGAAG R:CTGGTCTTGTAAACATCAGAC FAM-GAGAGAGGGAGCCCAGAGTTACAG-TAMRA

18 21 24 20 22 23 24 24 24 20 20 23 24 21 27 20 21 20 22 24 20 20 20 23 21 23 24 23 21 25 21 21 24

88

NEAT1 TUG1 FAS-AS1 DLEU2 PVT1 HOTAIRM1 THRIL GHET1 DSCAM-AS1 HOTAIR

2

78 149 81 83 131 135 121 94 104 141

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Fig. 1. Relative expression of lncRNAs in bladder cancer tissues, ANCTs and normal tissues (Horizontal lines inside the bar show median values. Maximum and minimum values are also shown in each bar). Table 3 Relative expression of lncRNAs in tumor tissues, ANCTs and normal tissues.

Table 2 Clinical characteristics of study participants. Study groups

Total Numbers

Variables

Values

Patients

50

Age (mean ± SD) Gender

61.78 ± 18.29 (9–88) Male 47 (94%) Female 3 (6%) Negative 14 (28%) Positive 36 (72%) Negative 25 (50%) Positive 25 (50%) Negative 32 (64%) Positive 18 (36%) Microscopic 39 (78%) Macroscopic 11 (22%) Inconclusive 18 (36%) Positive 32 (64%) High Grade 32 (64%) Low Grade 18 (36%) 71.33 ± 6.97 (59–84) Male 28 (93.3%) Female 2 (6.7%)

Smoking Opium Recurrence Hematuria Cytology Grade Normal Individuals

30

Age (mean ± SD) Gender

LncRNAs

Parameters

HOTAIR

Expression P-value Expression P-value Expression P-value Expression P-value Expression P-value Expression P-value Expression P-value Expression P-value Expression P-value Expression P-value

DLEU2 NEAT1 TUG1 THRIL FAS-AS1 DSCAM-AS1 PVT1 GHET1 HOTAIRM1

ratio ratio ratio ratio ratio ratio ratio ratio ratio ratio

Tumor Tissue vs. ANCT

Tumor Tissue vs. Normal control

1.6 0.1 1.03 0.92 0.91 0.76 1.19 0.65 0.53 0.09 0.65 0.23 0.54 0.08 0.48 0.04 1.12 0.75 1.001 0.99

0.4 0.04 0.77 0.17 0.68 0.02 0.54 0.02 1.82 0.39 0.11 < 0.001 0.78 0.15 0.14 < 0.001 9.61 < 0.001 8.62 0.004

3.6. Assessment of diagnostic power of lncRNAs in tumor tissues vs. normal controls

3.4. Correlations between expression levels of lncRNAs

As seven lncRNAs had differential expression in tumor tissues vs. normal controls, we assessed diagnostic power of these lncRNAs (Fig. 3). Other lncRNAs had similar expression tumor tissues and control tissues, so they were not included in ROC curve analysis. Based on the AUC values, FAS-AS1 had the highest diagnostic power. Combination of transcript levels of seven lncRNAs improved both sensitivity and specificity values to 100% (Table 7).

Significant pairwise correlations were detected between expression levels of lncRNAs in certain tissue types. Such correlations tended to be more prominent in tumor tissues rather than normal controls (Table 5). 3.5. Assessment of diagnostic power of PVT1 in tumor tissues vs. ANCTs As PVT1 was the only lncRNA with differential expression in tumor tissues vs. ANCTs, we only assessed diagnostic power of this lncRNA in these two sets of samples (Fig. 2). ROC curve analysis showed that this lncRNA has 86% sensitivity and 40% specificity in this regard (Table 6).

4. Discussion In the current study, we evaluated the diagnostic power of some lncRNAs in bladder cancer and introduced a panel of seven genes which 3

4

Age < 60 years 6 (42.9%) ≥60 years 17 (47.2%) Smoking Yes 14 (38.9%) No 9 (64.3%) Opium addiction Yes 9 (36%) No 14 (56%) Recurrence Positive 12 (66.7%) Negative 11 (34.4%) Hematuria Macroscopic 7 (63.6%) Microscopic 16 (41%) Cytology Positive 13 (40.6%) Inconclusive 10 (55.6%) Grade High grade 9 (50%) Low grade 14 (43.8%)

FAS-AS1 upregulation

Age < 60 years 8 (57.1%) ≥60 years 25 (69.4%) Smoking Yes 20 55.6%) No 13 (92.9%) Opium addiction Yes 15 (60%) No 18 (72%) Recurrence Positive 13 (72.2%) Negative 20 (62.5%) Hematuria Macroscopic 6 (54.5%) Microscopic 27 (69.2%) Cytology Positive 23 (71.9%) Inconclusive 10 (55.6%) Grade High grade 12 (66.7%) Low grade 21 (65.6%)

HOTAIR upregulation

9 (50%) 18 (56.3%)

19 (59.4%) 8 (44.4%)

4 (36.4%) 23 (59%)

6 (33.3%) 21 (65.5%)

16 (64%) 11 (44%)

22 (61.1%) 5 (35.7%)

8 (57.1%) 19 (52.8%)

FAS-AS1 downregulation

6 (33.3%) 11 (34.4%)

9 (28.1%) 8 (44.4%)

5 (45.5%) 12 (30.8%)

5 (27.8%) 12 (37.5%)

10 (40%) 7 (28%)

16 (44.4%) 1 (7.1%)

6 (42.9%) 11 (30.6%)

HOTAIR downregulation

0.67

0.3

0.18

0.02

0.15

0.1

0.78

P value

0.94

0.24

0.36

0.148

0.37

0.01

0.41

P value

7 (38.9%) 15 (46.9%)

13 (40.6%) 9 (50%)

7 (63.6%) 15 (38.5%)

9 (50%) 13 (40.6%)

10 (40%) 12 (48%)

13 (36.1%) 9 (64.3%)

6 (42.9%) 16 (44.4%)

DSCAM-AS1 up-regulation

10 (55.6%) 19 (59.4%)

21 (71.9%) 8 (44.4%)

6 (54.5%) 23 (59%)

11 (61.1%) 18 (56.2%)

12 (60%) 17 (72%)

19 (52.8%) 10 (71.4%)

5 (35.7%) 24 (66.7%)

DLEU2 upregulation

11 (61.1%) 17 (53.1%)

19 (59.4%) 9 (50%)

4 (36.4%) 24 (61.5%)

9 (50%) 19 (59.4%)

15(60%) 13(52%)

23 (63.9%) 5 (35.7%)

8 (57.1%) 20 (55.6%)

DSCAM-AS1 downregulation

8 (44.4%) 13 (40.6%)

11 (28.1%) 10 (55.6%)

5 (45.5%) 16 (41%)

7 (38.9%) 14 (43.8%)

13(40%) 8 (28%)

17 (47.2%) 4 (28.6%)

9 (64.3%) 12 (33.3%)

DLEU2 downregulation

0.58

0.52

0.17

0.52

0.56

0.07

0.91

P value

0.79

0.41

0.79

0.78

0.15

0.34

0.04

P value

8 (44.4%) 12 (37.5%)

12 (37.5%) 8 (44.4%)

7 (63.6%) 13 (33.3%)

11 (61.1%) 13 (40.6%)

8 (32%) 12 (48%)

13 (36.1%) 7 (50%)

3 (21.4%) 17 (47.2%)

PVT1 upregulation

9 (50%) 17 (53.1%)

17 (53.1%) 9 (50%)

7 (63.6%) 19 (48.7%)

10 (55.6%) 16 (50%)

11 (44%) 15 (60%)

16 (44.4%) 10 (71.4%)

5 (35.7%) 21 (58.3%)

NEAT1 upregulation

10 (55.6%) 20 (62.5%)

20 (62.5%) 10 (55.6%)

4 (36.4%) 26 (66.7%)

7 (38.9%) 19 (59.4%)

17 (68%) 13 (52%)

23 (63.9%) 7 (50%)

11 (78.6%) 19 (52.8%)

PVT1 downregulation

9 (50%) 15 (46.9%)

15 (46.9%) 9 (50%)

4 (36.4%) 20 (51.3%)

8 (44.4%) 16 (50%)

14 (56%) 10 (40%)

20 (55.6%) 4 (28.6%)

9 (64.3%) 15 (41.7%)

NEAT1 downregulation

0.63

0.63

0.09

0.9

0. 25

0.36

0.11

P value

0.83

0.83

0.38

0.7

0. 25

0.08

0.15

7 (38.9%) 18 (56.3%)

17 (53.1%) 10 (55.6%)

8 (72.7%) 17 (43.6%)

11 (61.1%) 14 (43.8%)

12 (48%) 13 (52%)

16 (44.4%) 9 (64.3%)

6 (42.9%) 19 (52.8%)

TUG1 upregulation

7 (38.9%) 17 (53.1%)

18 (56.3%) 6 (33.3%)

2 (18.2%) 22 (56.4%)

9 (50%) 15 (46.9%)

12 (48%) 12 (48%)

15 (41.7%) 9 (64.3%)

7 (50%) 17 (47.2%)

GHET1 upregulation

P value

11 (61.1%) 15 (46.9%)

14 (43.8%) 12 (66.7%)

9 (81.8%) 17 (43.6%)

9 (50%) 17 (53.1%)

13 (52%) 13 (52%)

21 (58.3%) 5 (35.7%)

7 (50%) 19 (52.8%)

GHET1 downregulation

11 (61.1%) 14 (43.8%)

15 (46.9%) 8 (44.4%)

3 (27.3%) 22 (56.4%)

7 (38.9%) 18 (56.2%)

13 (52%) 12 (48%)

20 (55.6%) 5 (35.7%)

8 (57.1%) 17 (47.2%)

TUG1 downregulation

0.33

0.11

0.04

0.83

1

0.15

0.86

P value

0.23

0.55

0.47

0.23

0.77

0.2

0.52

6 (33.3%) 13 (40.6%)

11 (28.1%) 8 (44.4%)

7 (63.6%) 12 (30.8%)

7 (38.9%) 12 (37.5%)

8 (32%) 11 (44%)

11 30.6%) 8 (57.1%)

3 (21.4%) 16 (44.4%)

THRIL upregulation

8 (44.4%) 16 (50%)

16 (50%) 8 (44.4%)

6 (54.5%) 18 (46.2%)

10 (55.6%) 14 (43.8%)

11 (44%) 13 (52%)

16 (44.4%) 8 (57.1%)

8 (57.1%) 16 (44.4%)

HOTAIRM1 upregulation

P value

10 (55.6%) 16 (50%)

16 (50%) 10 (55.6%)

5 (45.5%) 21 (53.8%)

8 (44.4%) 18 (56.2%)

14 (56%) 12 (48%)

20 (69.4%) 6 (42.9%)

6 (42.9%) 20 (55.6%)

HOTAIRM1 down-regulation

12 (66.7%) 19 (59.4%)

21 (71.9%) 10 (55.6%)

4 (36.4%) 27 (69.2%)

11 (61.1%) 20 (62.5%)

17 (68%) 14 (56%)

25 (69.4%) 6 (42.9%)

11 (78.6%) 20 (55.6%)

THRIL downregulation

0.7

0.7

0.62

0.42

0.0.57

0.42

0.42

P value

0.61

0.48

0.07

0.92

0.0.38

0.08

0.19

P value

Table 4 Associations between relative expression of lncRNAs and patients' data (Up-/down-regulation of genes were defined based on the relative expression of each gene in tumor tissue compared with its corresponding ANCT.)

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Table 5 Correlations between expression levels of lncRNAs (R2 values are shown. * indicates P < .05. **indicates P < .01).

HOTAIR

Tumor ANCT Normal Tumor ANCT Normal Tumor ANCT Normal Tumor ANCT Normal Tumor ANCT Normal Tumor ANCT Normal Tumor ANCT Normal Tumor ANCT Normal Tumor ANCT Normal

DLEU2 NEAT1 TUG1 THRIL FAS-AS1 DSCAM-AS1 PVT1 GHET1

control control control control control control control control control

HOTAIRM1

GHET1

PVT1

DSCAM-AS1

FAS-AS1

THRIL

TUG1

NEAT1

DLEU2

0.37** 0.36** 0. 42** 0.43** 0.26** 0.49** 0.36** 0.5** 0. 45** 0.51** 0.39** 0.02 0.21* 0.38** 0.21* 0.35** 0.29* 0.29* 0.47** 0.56** 0.02 0.32** 0.43** 0. 34* 0.2* 0.04 0.001

0.26* 0.04 0.04 0.21** 0.18* 0.09 0.28** 0.01 0.005 0.27** 0.002 0.09 0.12* 0.003 0.13* 0.23** 0.23** 0.12 0.21* 0.02 0.09 0.2* 0.001 0.05

0.62** 0.51** 0.51** 0.47** 0.31** 0.38** 0.54** 0.82** 0.43** 0.45** 0.6** 0.06 0.62** 0.68** 0.24** 0.22* 0.2* 0.4** 0.42** 0.7** 0.006

0.6** 0.4** 0.01 0.73** 0.23** 0.006 0.48** 0.8** 0.01 0.71** 0.8** 0.001 0.24** 0.7** 0.004 0.42** 0.26** 0.007

0.37** 0.2* 0.44** 0.41** 0.22** 0.33** 0.36** 0.31** 0.41** 0.37** 0.24** 0.13* 0.15* 0.17* 0.15*

0.37** 0.2* 0.44** 0.41** 0.22** 0.33** 0.36** 0.31** 0.41** 0.37** 0.24** 0.13*

0.59** 0.27** 0.04 0.77** 0.13* 0.09 0.56** 0.7** 0.09

0.55** 0.52** 0.57** 0.66** 0.32** 0.56**

0.71** 078** 0.72**

100

PVT1 100

80

80 Sensitivity

Sensitivity

60

60

40

HOTAIR NEAT1 FAS-AS1 PVT1 GHET1 HOTAIM1

40 20

20

0 0

20

40 60 100-Specificity

80

100

Fig. 2. ROC curve analysis of PVT1 for assessment of diagnostic power of this lncRNA in tumor tissues vs. ANCTs.

PVT1

AUC

Ja

Sensitivity

Specificity

P-valueb

≤4.15

0.6

0.26

86

40

0.07

80

100

observation is consistent with the previous studies regarding the similar expression patterns between bladder tumors, carcinoma in situ (CIS) lesions and histologically normal biopsies adjacent to the CIS lesions. These results indicated the existence of a CIS expression profile in the urothelium of patients regardless of the organization of urothelial cells in tumors, or as histologically normal-appearing cells [17]. Based on the accuracy of this panel in differentiation of tumor tissues from normal tissues, we suggest further assessment of this panel in urine samples of bladder cancer patients and healthy subjects to appraise their potential in non-invasive methods of cancer diagnosis. In the current study, we reported down-regulation of PVT1 in tumor tissues compared with both ANCTs and normal controls. In contrast with our report, a previous study has demonstrated up-regulation of this lncRNA in bladder cancer tissues compared with ANCTs. Authors also demonstrated the role of PVT1 in enhancement of cell proliferation

Table 6 The results of ROC curve analysis for assessment of diagnostic power of PVT1 in tumor tissues vs. ANCTs (a: Youden index, b: Significance level P (Area = 0.5), Estimate criterion: optimal cut-off point for gene expression). Estimate criterion

40 60 100-Specificity

Fig. 3. ROC curve analysis of lncRNAs for assessment of their diagnostic power in tumor tissues vs. normal controls.

0 0

20

could differentiate bladder cancer samples from normal bladder tissue with high accuracy. However, the diagnostic power of lncRNAs in discrimination of tumor tissues from ANCTs was not acceptable. This 5

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References

Table 7 The results of ROC curve analysis for assessment of diagnostic power of lncRNAs in tumor tissues vs. control tissues (a: Youden index, b: Significance level P (Area = 0.5), Estimate criterion: optimal cut-off point for gene expression).

HOTAIR NEAT1 TUG1 FAS-AS1 PVT1 GHET1 HOTAIRM1 Combination of seven genes

Estimate criterion

AUC

Ja

Sensitivity

Specificity

P-valueb

≤1.69 ≤0.86 ≤1.58 ≤0.33 ≤4.15 > 0.07 > 12.31 >0

0.63 0.65 0.65 0.78 0.75 0.76 0.69 1

0.33 0.32 0.35 0.54 0.42 0.46 0.46 1

50 56 42 68 86 76 60 100

83 76.7 93.3 86.7 56.7 70 86.7 100

0.03 0.01 0.01 < 0.0001 < 0.0001 < 0.0001 0.0009 < 0.0001

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and inhibition of cell apoptosis [18]. The discordance between our results and the results of mentioned study might be explained by the ethic-based differences or dissimilarities in age and sex ratio between these studies. Alternatively, the discrepancy might be due to incoherency in diagnosis of ANCTs. HOTAIR, NEAT1, TUG1 and FAS-AS1 were significantly downregulated in tumor tissues compared with normal controls but their expressions were similar between tumoral tissues and ANCTs. Such expression patterns are not consistent with the previously proposed roles for NEAT1 [19], HOTAIR [20] and TUG1 [7]. So, future functional studies are needed to explore the significance of these lncRNAs in the tumorigenesis of bladder cancer. Besides, GHET1 and HOTAIRM1 were significantly up-regulated in tumor tissues compared with normal controls. Li et al. have detected up-regulation of GHET1 in bladder cancer tissues compared to ANCTs. Moreover, they have shown correlations between elevated levels of this lncRNA and determinants of aggressive behavior of tumors [21]. In line with these observations, GHET1 silencing inhibited the proliferation and invasion of bladder cancer cells and suppressed the epithelial-mesenchymal-transition [21]. We also detected significant associations between relative expression levels of DLEU2 and patients' age, GHET1 and hematuria and FAS-AS1 and tumor recurrence. Such associations may imply the role of this lncRNAs in certain aspects of tumorigenesis process. Future studies are required to explore their net functions by recruiting larger cohorts of patients and implementation of functional studies. Finally, significant pairwise correlations were detected between expression levels of lncRNAs in certain tissue types. Such correlations tended to be more prominent in tumor tissues rather than normal controls which suggest the significance of interaction networks between lncRNAs in the context of cancer. Such network might be part of “Achilles' heel” the in carcinogenesis process. Consequently, the result of the present study implies the role of this lncRNA panel in the diagnosis of bladder cancer. However, based on the presence of some discrepancies between these results and the results of previous studies, future studies are necessary to appraise their role as therapeutic targets. Acknowledgement The current study was supported by a grant from Shahid Beheshti University of Medical Sciences. Conflict of interest The authors declare they have no conflict of interest.

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