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Identification of oncogenic long noncoding RNA SNHG12 and DUXAP8 in human bladder cancer through a comprehensive profiling analysis
Biomedicine & Pharmacotherapy 108 (2018) 500–507
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Identification of oncogenic long noncoding RNA SNHG12 and DUXAP8 in human bladder cancer through a comprehensive profiling analysis
T
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Bin Jianga,1, Hailong Sub,1, Jun Yuana, Hu Zhaoa, Wenkai Xiac, Zhenlei Zhaa, Bin Wua, , ⁎ Zhili Liud, a
Department of Urology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, People’s Republic of China Department of General and Pediatric Surgery, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, People’s Republic of China c Department of Nephrology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, People’s Republic of China d Department of Oncology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, Shoushan road 163, 214400, People's Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bladder cancer Long noncding RNA Bio-marker DUXAP8 SNHG12
Bladder cancer is a common urological malignancies world-wide. Recently, a growing number of evidence have highlighted the importance of long noncoding RNAs (lncRNAs) in multiple cancers development and progression. However, the expression pattern of lncRNAs in bladder cancer and their underlying function remain poorly understood. To identify lncRNAs profile alterations and uncover valuable lncRNA candidates for bladder cancer diagnostic, we conducted a comprehensively lncRNAs profiling analyses and explored their clinical relevance using The Cancer Genome Atlas (TCGA) data and three independent microarray profiling data from the Gene Expression Omnibus (GEO). After annotation and analyses of these data, we found that lots of lncRNAs were dysregulated in bladder specimen when compared with normal control specimen. In addition, we found that differential expression of these lncRNAs is accompanied by genomic variations, including genome loci copy number deletion or amplification. Importantly, a part of these lncRNAs are related to bladder cancer patients outcome, such as SNHG10, SNHG12 and LINC00115. Finally, we validated two of these lncRNAs' (DUXAP8 and SNHG12) function in bladder cancer cells by down-regulating their expression with siRNAs, and found that down-regulation of DUXAP8 and SNHG12 could inhibit bladder cancer cells proliferation in vitro. In summary, this study demonstrated that a lot of lncRNAs are dysregulated in bladder cancer, and might provide useful lncRNAs resource for potential prognostic or diagnostic markers for this disease.
1. Introduction Bladder cancer is the ninth common malignancy and represents one of the common urological carcinomas world-wide [1]. As a highly heterogeneous cancer, low-grade non-invasive papillary tumors constitute ∼70% of all bladder carcinomas cases, while invasive highgrade tumors constitute ∼30% of all bladder carcinomas [2,3]. Although great improvement have been made on the diagnostic and surgical techniques, chemotherapy and radiotherapy, the improve of bladder cancer patients survival rates remain unsatisfactory. Hence, identify novel molecular biomarkers for bladder cancer early diagnosis is still urgent. Meanwhile, a better knowledge of the molecular
mechanisms of bladder cancer tumorigenesis and metastasis is important for the development of new treatment method. Over the past decades, a great number of studies have uncovered that long non-coding RNAs (lncRNAs) are widely expressed in almost of human organ tissue and cells, and emerging as new layer of gene regulation [4,5]. lncRNAs are longer than 200 nt in length, and lacking of protein coding probability. However, accumulating studies reveal that lncRNAs participate in various cellular process, including X chromatin imprinting, glucose metabolism, immune response, muscle cell differentiation, tumor cells proliferation and chemotherapy resistance [6–10]. Moreover, lncRNAs differential profiling analyses in multiple cancers using RNA sequencing or microarray data indicate that lots of
⁎ Corresponding author at: Department of Oncology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, Shoushan road 163, 214400, People’s Republic of China. Tel.: +86 0510 80615151; fax: +86 0510 80615151 ⁎⁎ Corresponding author at: Department of Urology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, Shoushan road 163, 214400, People’s Republic of China E-mail addresses: [email protected] (W. Bin), [email protected] (Z. Liu). 1 These authors contributed equally to this work and should be regarded as joint first authors.
https://doi.org/10.1016/j.biopha.2018.09.025 Received 19 July 2018; Received in revised form 31 August 2018; Accepted 4 September 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. Differentially expressed lncRNAs in human bladder cancer tissues and normal tissues. (A) A heatmap was drawn to show the differentially expressed lncRNAs in bladder cancer and normal tissues by analyzing the TCGA RNA sequencing data. (B–D) Heatmaps were drawn to show the dysregulated lncRNAs in bladder cancer specimen compared with non-tumor tissues using the GSE89006, GSE51493 and GSE45184 datasets. (E) Overlap analyses of the altered lncRNAs profile (consistently up-regulated or down-regulated in at least two datasets, fold change) in TCGA, GSE89006, GSE51493 and GSE45184 datasets. (F) Venn diagram of differentially expressed lncRNAs in TCGA, GSE89006, GSE51493 and GSE45184 datasets.
related to patients poor outcome and promotes cell metastasis by binding with SUZ12 and inhibiting E-cadherin expression [18]. Although, a small part of lncRNAs have been investigated in human bladder cancer, most of the other lncRNAs’ clinical significance and functional roles in bladder cancer remains unclear. In the present study, we conducted a genome-wide analyses to identify differentially expressed lncRNAs in bladder cancer by analyzing RNA sequencing and gene microarray data from TCGA and GEO. The current study may provide a valuable resource of lncRNAs to further investigate and identify novel useful diagnostic biomarker for bladder cancer.
lncRNAs are dysregulated in human cancers and many of these dysregulated lncRNAs are associated with patients prognosis [11]. Additionally, experiment validation showed that those lncRNAs contribute to tumor development and progression through different mechanisms, such as histone modification, sponging microRNAs, regulation of protein translation and mRNA stability [12,13]. For examples, lncRNA HOXA11-AS is found to be over-expressed in human gastric carcinoma, and promotes cell growth and metastasis by acting as scaffold of LSD1and EZH2, and functioning as sponge for miR-1297. Moreover, lincRNA-UFC1 promotes hepatocellular carcinoma cells proliferation and inhibits apoptosis through interacting with HuR, and thereby increasing β-catenin mRNA and protein levels [14]. In case of bladder cancer, a few lncRNAs' biological function, clinical relevance and molecular mechanisms have been documented [15]. For example, LncRNA SPRY4-IT1 promotes cell proliferation and metastasis through acting as competing endogenous RNA of miR-101-3p and thereby up-regulating EZH2 expression in bladder cancer [16]. Liu and colleagues report that lncRNA H19 can function as a ceRNA of miR29b-3p and antagonize its repression of DNMT3B, resulted in promoting epithelial–mesenchymal transition and cell metastasis in bladder cancer [17]. Moreover, TGF-β induced lncRNA MALAT1 overexpression is
2. Methods and materials 2.1. lncRNA profiling analyses in TCGA and microarray data The RNA sequencing data of bladder cancer tissues and non-tumor tissues and relevant clinical information were downloaded from https://portal.gdc.cancer.gov/. Another three public bladder cancer gene microarray profiling data (GSE89006, GSE51493 and GSE45184 [19]) were obtained from the Gene Expression Omnibus (GEO). Differential profiling of lncRNAs in GSE89006, GSE51493 and GSE45184 501
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Fig. 2. Copy number variation analyses of lncRNAs' genome loci in bladder cancer. (A) The frequency of lncRNAs genome loci copy number gain (red) in bladder cancer tissues was shown. The rows are arranged according to the genomic locations of each lncRNAs. (B) The frequency of lncRNAs genome loci copy number loss (blue) in bladder cancer tissues was shown. The rows are arranged according to the genomic locations of each lncRNAs.
Fig. 3. Analyses of bladder cancer patients' OS and RFS related lncRNAs. (A) The heatmaps were drawn to show the Hazard ratio value and log rank P value of bladder cancer patients' OS and RFS related lncRNAs. (B) The Kaplan–Meier curves for bladder cancer patients' OS in high- SNHG10 or LINC00115 and low- SNHG10 or LINC00115 groups in the TCGA set. (C) The Kaplan–Meier curves for bladder cancer patients' RFS in high-SNHG10 or SNHG12 and low- SNHG10 or SNHG12 groups in the TCGA set.
and microarray data.
was conducted using the Agilent-045997 Arraystar human lncRNA microarray V3, Agilent-028004 SurePrint G3 Human GE 8 x 60 K Microarray and Arraystar Human LncRNA microarray V2.0 platforms. R software and packages were used to preprocess the RNA sequencing 502
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Fig. 4. Down-regulation of DUXAP8 and SNHG12 inhibits cell proliferation in bladder cancer. (A) The fold-change of DUXAP8 and SNHG12 expression levels between bladder cancer tissues and normal tissues in TCGA and GSE89006 datasets. (B) The relative expression of DUXAP8 and SNHG12 in negative control or DUXAP8 and SNHG12 siRNAs transfected UMUC3 and SW780 cells was detected by qRT-PCR. (C) Growth curves of UMUC3 and SW780 cells after transfection with DUXAP8 and SNHG12 siRNAs or negative control were determined by CCK8 assays. Values represent the mean ± s.d. from three independent experiments. (E) The colony formation ability of UMUC3 and SW780 cells after transfection with DUXAP8 and SNHG12 siRNAs or negative control were determined by colony formation assays. ** P < 0.01; * P < 0.05.
2.2. Copy number variation analyses
2.4. Cell culture and transfection
The raw gene copy number variations data of each bladder cancer specimen were down-loaded from Broad GDAC (https://portal.gdc. cancer.gov/). Then, GISTIC 2.0 [20] pipeline was used to evaluate the significancee of each lncRNAs copy number deletions or amplifications in their genome locus. All lncRNAs genomic regions were aligned and mapped according to GISTIC peaks, and the peaks of deletion or amplification with q values ≤ 0.25 were defined as significant. The peak q values, numbers and focal/broad frequencies of each lncRNAs were integrated at gene level.
Bladder cancer cell lines UMUC3 and SW780 were obtained from the American Type Culture Collection (ATCC), and cultured in RPMI 1640 medium (GIBCO, USA) containing 10% fetal bovine serum (FBS). The cells were maintained in a humidified incubator with a 5% CO2 atmosphere at 37℃. The small interference RNAs (siRNAs) target DUXAP8 and SNHG12 (Invitrogen, Carlsbad, CA) were transiently transfected into UMUC3 and SW780 cells by using RNAiMAX (Invitrogen, USA) based on the manufacturer’s manual. 2.5. RNA extraction and qRT-PCR
2.3. Analyses of bladder cancer survival related lncRNAs
Total RNA of UMUC3 and SW780 cells was extracted with RNeasy Purification Kit (QIAGEN) following the manufacturer’s instructions. Then, 1 μg of total RNA was reverse-transcribed to cDNA by using the Reverse Transcriptase kit (Takara, Dalian, China) according to manufacturer’s manual. The Quantitative RT-PCR assays were implemented with SYBR Select Master Mix (Applied Biosystems) on ABI 7900 system (Applied Biosystems, CA, USA). The primers used were: DUXAP8, forward 5'-AGGATGGAGTCTCGCTGTATTGC-3', reverse 5'- GGAGGTTTG TTTTCTTCTTTTTT-3'; SNHG12, forward 5'-TCTGGTGATCGAGGACT TCC-3', reverse 5'-ACCTCCTCAGTATCACACACT-3';GAPDH, forward 5'AGAAGGCTGGGGCTCATTTG-3', reverse 5'- AGGGGCCATCCACAGTC TTC-3'. The 2−ΔΔCT method was performed to determine DUXAP8 and SNHG12 expression levels.
Univariable Cox regression analyses using Bio-conductor and R software was conducted to explore the significance of lncRNAs for bladder cancer patients’ overall survival (OS) or recurrence free survival (RFS) prediction. Next, multivariable Cox regression analysis using Bio-conductor and R software was performed to determine whether lncRNA could be used as dependent variable factor. Then, each lncRNA’s risk score was calculated. Next, the patients with bladder cancer were classified into high- or low-lncRNAs expression group based on the chose lncRNAs median expression levels. Finally, lncRNA with log rank P value < 0.05 between high-lncRNA expression and lowlncRNA expression groups were considered significant. 503
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Fig. 5. Down-regulation of DUXAP8 and SNHG12 inhibits cell invasion and induces cell apoptosis in bladder cancer. (A, B) Apoptosis ratio of negative control, DUXAP8 or SNHG12 siRNAs transfected SW780 cells was observed using flow cytometry assays. (C, D) Transwell assays were performed to evaluate the invasive ability of negative control, DUXAP8 or SNHG12 siRNAs transfected SW780 cells. * P < 0.05.
fixed with 4% Paraformaldehyde, and stained by 0.1% crystal violet. Next, the stained cells were imaged, and counted from six random vision(Olympus, Tokyo, Japan).
2.6. Cell proliferation and colony formation assay The CCK8 assays were conducted to evaluate UMUC3 and SW780 cells viability after transfection with SNHG12, DUXAP8 or scrambled siRNAs. 48 h after transfection, UMUC3 and SW780 cells (2000/well) were plated into 96 well plate The cells in 96-well plate were cultured, and 10 μl CCK8 (Dojindo, Japan) was added into each well, and the cells were incubated for 2 h. The relative numbers of cell in each well were determined by measuring absorbance at 450 nm using the ELx600 microplate reader (BioTek, Winooski, Vermont). For colony formation assay, UMUC3 and SW780 cells transfected with SNHG12, DUXAP8 or scrambled siRNAs were seeded into six-well plates and cultured for two weeks. Then, the cells were fixed with 4% paraformaldehyde and stained with purple crystal. The number of colonies were calculated under the microscope.
2.9. Statistical analysis All in vitro validation data were analyzed from three independent experiments. SPSS version 18.0 (SPSS Inc.Chicago, IL, USA) and GraphPad Prism 5.0 software were utilized for the independent and paired samples’ t-test or ANOVA to analyze qRT-PCR and in vitro experiment data as indicated.A p-value < 0.05 was deemed statistically significant. 3. Results 3.1. Identify dysregulated lncRNAs in human bladder cancer
2.7. Cell apoptosis analysis To identify dysregulated lncRNAs in human bladder tissues, we firstly downloaded the RNA sequencing data of bladder cancer and normal specimen from TCGA and three microarray profiling data (GSE89006, GSE51493 and GSE45184) from the GEO. The TCGA dataset includes 414 bladder cancer specimen and 19 normal specimen; GSE89006 dataset includes 4 paired bladder cancer and para-cancer tissues; GSE51493 consists of 12 bladder carcinoma primary tumors and 3 normal tissues; GSE45184 consists of 3 paired bladder cancer and adjacent normal tissues. Re-annotation and differential lncRNAs profiling analyses revealed that 862 lncRNAs expression were dysregulared (281 upregulated and 581 downregulated) in bladder cancer from the TCGA dataset; 268 lncRNAs expression was dysregulated (116 upregulated and 152 downregulated) in GSE45184 dataset; 839 lncRNAs were differentially expressed (240 upregulated and 599 downregulated) in the GSE51493 dataset; and 2526 lncRNAs were dysregulated in the GSE89006 dataset (1138 increased and 1388 decreased) (Fig. 1A–D, and Table S1). Furthermore, overlap analyses showed that
SW780 cells were cultivated in 6-well plate and transfected DUXAP8, SNHG12 or negative control siRNAs. 48 h after transfection, all the cells were harvested and double stained with FITC-Annexin V and PI with FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) based on the manufacturers’ manual. Flow cytometry (EPICS, XL-4, Beckman, CA, USA) was used to examine the cell apoptosis. 2.8. Cell invasion assays To evaluate bladder cancer cell invasive ability, transwell assays were conducted. For invasion assays, 5 × 104 cells in 300 μl medium without serum were added into the upper culture chamber with Matrigel coated 8-mm membrane (Millipore, Billerica, MA, USA). Then, 700 μl medium containing 10% fetal bovine serum was added into the lower wells of 12-well plate. 24 h after incubation, the bladder cancer cells on upper membrane of chamber were removed by cotton wool, 504
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Fig. 6. DUXAP8 and SNHG12 co-expressed protein coding genes and enriched pathways. (A) The circle diagram shows the top 50 DUXAP8 and SNHG12 co-expressed protein coding genes in bladder cancer in TCGA dataset. (B, C) The “DUXAP8” and “SNHG12” co-expressed protein coding genes enriched pathways and their interacted network in bladder cancer. (D) The DUXAP8 expression was positively related to MAGEA4 and MAGEA6 expression in bladder cancer tissues. (E) The SNHG12 expression was positively related to HDAC10 and AGER expression, but negatively related to LATS2 and PCDH7 expression in bladder cancer tissues.
that 24 over-expressed lncRNAs and 17 decreased lncRNAs are significantly related to bladder cancer patients shorter OS (log rank P < 0.05), and 16 increased lncRNAs and 22 down-regulated lncRNAs are significantly related to bladder cancer patients shorter RFS (log rank P < 0.05) (Fig. 3A, and Table S3). Taken SNHG10, LINC00115, and SNHG12 for example, bladder cancer patients with high SNHG10 or LINC00115 expression had shorter OS time, while bladder cancer patients with high SNHG10 or SNHG12 expression had shorter RFS time (Fig. 3B–C). These data suggest that those bladder cancer survival related lncRNAs might be useful candidates for patients outcome prediction.
137 lncRNAs were consistently up-regulated and 309 lncRNAs were down-regulated in at least two of these datasets (Fig. 1E–F, Table S2). These findings suggest that lots of lncRNAs are differentially expressed in human bladder cancer, and these lncRNAs may be useful diagnosis biomarkers. 3.2. lncRNAs genome loci copy number variations in bladder cancer Recently, a growing number of studies demonstrate that genomic alterations as well as transcription factors contribute to lncRNAs alterations in various tumor cells. To further explore whether genomic alterations are involved in lncRNAs dysregulation, we downloaded the gene copy number variations data in all bladder cancer samples from the Broad GDAC. Next, genomic loci copy number frequency of each differentially lncRNAs was calculated, and the alterations in each bladder cancer specimen with q value less than 0.25 was defined as significant. As a result, a lot of lncRNAs genome loci had copy number gain (such as PVT1, IFNG-AS1 and CALML3-AS1) or frequency loss (such as LINC01198, PTPRD-AS1 and DLEU7-AS1) in bladder cancer (Fig. 2A-B, and Table S3). These findings indicate that a part of dysregulated lncRNAs in bladder cancer might be affected by copy number variations in their genomic loci.
3.4. Knockdown of DUXAP8 and SNHG12 inhibit bladder cancer cells proliferation To further evaluate the analysis results, we focused on these overexpressed lncRNAs which may be suitable biomarkers. Among those increased lncRNAs, DUXAP8 and SNHG12 expression are up-regulated in TCGA and GSE89006 datasets (Fig. 4A). Moreover, the survival analyses results also revealed that higher SNHG12 expression is related to bladder cancer patients' shorter RFS. DUXAP8 has also been found to be up-regulated in gastric cancer [21], non small cell lung cancer [22] and renal cell carcinoma [23], while SNHG12 also exerts oncogenic function in multiple cancers [24,25]. However, their functional roles in bladder cancer remain unclear, hence, we chose these two lncRNAs for further investigation. Then, we analyzed DUXAP8 and SNHG12 expression in bladder cancer cell lines using the CCLE RNA sequencing data [26]. The relative expression levels of DUXAP8 and SNHG12 (FPKM value) in each bladder cancer cell lines were calculated, and the value of FPKM ≥ 10 was defined as "relative high expression level". We
3.3. Identification of bladder cancer survival associated lncRNAs Accumulating evidence has demonstrated that lots of lncRNAs are related to several cancers patients outcome and could be novel predictors for tumor patients prognosis. To identify bladder cancer overall survival (OS) and recurrence free survival (RFS) related lncRNAs, we conducted univariable Cox regression analysis. As a result, we found 505
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sequencing and lncRNA microarray technique enables us to analyze large-scale differentially expressed lncRNAs in human cancers. In the present study, we conducted a comprehensively differential profiling analyses of lncRNAs in human bladder cancer and adjacent normal specimen to identify more dysregulated lncRNAs. The findings is this study uncovered that hundreds of lncRNAs' expression are dysregulated in baldder cancer, such as up-regulated DUXAP8, AC005281.1 and LINC01424, and down-regulated HAND2-AS1, FENDRR, LINC00473. Moreover, copy number variation analysis of those dysregulated lncRNAs’ genome loci revealed that a small part of those dysregulated lncRNAs had copy number amplification or deletion in their genome loci, such as PVT1, IFNG-AS1, CALML3-AS1, LINC01198, PTPRD-AS1 and DLEU7-AS1. These findings suggest that genomic alterations may involve in lncRNAs’ alteration in bladder cancer. Furthermore, we found that a few of lncRNAs are related to bladder cancer patients’ OS or RFS, including SNHG10, LINC00115 and SNHG12, indicating that lncRNAs might be valuable predictor for bladder cancer patients’ outcome. To evaluate our analysis findings and determine whether those dysregulated lncRNAs, we chose two up-regulated lncRNAs DUXAP8 and SNHG12 and investigated their function in bladder cancer. Our findings showed that down-regulation of DUXAP8 and SNHG12 significantly impaired bladder cancer cells in vitro growth, colony formation capacity, invasive ability and induced cell apoptosis. Moreover, GO and pathway analyses revealed that SNHG12 and DUXAP8 co-expressed protein coding genes are enriched in cell cell cycle, focal adhesion and PI3K-Akt signaling pathways, which are important for bladder tumorigenesis and tumor progression. As well as our findings, DUXAP8 was also reported to be up-regulated in human esophageal squamous cell carcinoma [32], gastric carcinoma [21] and NSCLC [22]. Over-expressed DUXAP8 promotes NSCLC cells growth and invasion through epigenetically inhibiting EGR1 and RHOB transcription via interacting with EZH2 and LSD1 [22]. Moreover, several studies have revealed that SNHG12 are over-expressed in multiple cancers, including papillary thyroid carcinoma [24], gastric cancer [25], and Cervical Cancer [33]. Tian and colleagues found that up-regulated SNHG12 promotes tumorigenesis and hepatocellular carcinoma cells metastasis through functioning as a ceRNA for miR-199a/b-5p to modulate MLK3 expression and regulate NF-κB pathway [34]. These findings suggest that those dysregulated lncRNAs (such as DUXAP8 and SNHG12) might play key roles in human bladder cancer tumorigenesis and tumor progression.
found that DUXAP8 and SNHG12 expression levels were high in most of the bladder cancer cell lines. According to the CCLE data, UMUC3 and SW780 cells was chosen for further experiments. Next, DUXAP8 and SNHG12-specific siRNAs were tranfected into UMUC3 and SW780 cells to down-regulate their expression. The results of qRT-PCR revealed that DUXAP8 and SNHG12 expression were significantly decreased in two siRNAs transfected UMUC3 and SW780 cells compared with control cells (Fig. 4B). Next, CCK8 assays revealed that down-regulation of SNHG12 and DUXAP8 expression impaired UMUC3 and SW780 cells proliferation when compared with control cells (Fig. 4C–F). Moreover, down-regulation of DUXAP8 or SNHG12 inhibited UMUC3 and SW780 cells colony formation capacity. 3.5. Down-regulation of DUXAP8 and SNHG12 inhibits bladder cancer cells invasion and induces cell apoptosis To determine whether down-regulation of DUXAP8 and SNHG12 mediated suppression of cell proliferation by enhancing apoptosis, we performed flow cytometry cell apoptosis assay. The results showed that the ratios of apoptosis in si-DUXAP8 or si-SNHG12 group were increased compared with control cells (Fig. 5A-B). Moreover, the results of transwell assay illustrated that down-regulation of DUXAP8 and SNHG12 could impair the invasive ability of SW780 cells compared with control cells (Fig. 5C–D). 3.6. SNHG12 and DUXAP8 co-expressed genes and enriched pathways in bladder cancer To determine SNHG12 and DUXAP8 associated protein coding genes (PCGs) and their involved pathways in bladder cancer, we analyzed SNHG12 and DUXAP8 co-expressed PCGs using TCGA data. The results showed that lots of oncogenes expression are positively associated with SNHG12 and DUXAP8 expression, while some tumor suppressors expression are negatively associated with SNHG12 and DUXAP8 expression in bladder cancer tissues (Fig. 6A). Meanwhile, GO and pathway analysis revealed that DUXAP8 and SNHG12 co-expressed PCGs are enriched in focal adhesion, cell cycle and PI3K-Akt signaling pathway etc (Fig. 6B–C). Interestingly, these biological process and pathways are important for regulating cancer cells growth and invasion. Furthermore, DUXAP8 expression is positively related to MAGEA6 and MAGEA4 in bladder cancer; SNHG12 expression is positively associated with HDAC10 and AGER, and negatively associated with PCDH7 and LATS2 in bladder cancer (Fig. 6D–E).
5. Conclusion 4. Discussion Taken together, this study found that lots of lncRNAs were dysregulated in human bladder cancer specimen compared with normal tissues. And, copy number amplification or deletion may involve in part of those lncRNAs’ alteration in bladder cancer. Moreover, a small portion of the dysregulated lncRNAs are associated with patients OS and RFS time in bladder cancer, which might be independent prediction factors of bladder cancer patients’ survival. The findings in this study highlight the importance of lncRNAs in bladder cancer, and our analyses data could provide valuable lncRNA candidates for bladder cancer diagnosis or target therapy. However, there are a few limitations in this study, such as only SNHG12 and DUXAP8 function were validated and their underlying molecular mechanisms are not documented, which would be further explored in our future study.
Recently, sequencing and annotation of the whole human genome have uncovered that thousands of lncRNAs are transcribed from these non-coding regions. To date, a great number of studies have documented that lnRNAs' dysregulation play critical roles in human cancers development and tumor progression [27]. For example, Chen et al. Reported that up-regulated LNIC01234 promotes gastric cancer cells growth through functioning as competing endogenous RNA (CeRNA) for miR-204-5p, thereby antagonize its’ suppression of CBFB [28]. Moreover, lncRNA miR503HG is markedly down-regulated in hepatocellular carcinoma, while over-expression of miR503HG could inhibit cell invasion and metastasis through interacting with HNRNPA2B1 and promoting its’ degradation [29]. Simultaneously, a few lncRNAs’ functions in bladder cancer have been identified, and their underlying molecular mechanisms were clarified. For instance, Wang et al. found that GAS5 expression was decreased in bladder cancer, and GAS5 overexpression could induce cell apoptosis by inhibiting EZH2 transcription [30]. In addition, lncRNA MALAT1 promotes bladder cancer cells growth and metastasis in vivo by acting as a sponge for miR-124 to release its’ repression of FOXQ1 [31]. Over the past decade, the rapid improvement of next generation
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Conflicts of interest The authors have no actual or potential conflicts of interest to 506
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declare.
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Acknowledgments This work was supported by the National Scientific Foundation of China (No.81402554 to LZL), Jiangsu Provincial Medical Youth Talent (QNRC2016134) and the Scientific Foundation of Wuxi City of Jiangsu (Q201403 to LZL, Q201754 to XWK) Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.09.025. References [1] R. Siegel, D. Naishadham, A. Jemal, Cancer statistics, 2013, CA Cancer J. Clin. 63 (1) (2013) 11–30. [2] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA Cancer J. Clin. 64 (1) (2014) 9–29. [3] W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He, Cancer statistics in China, 2015, CA Cancer J. Clin. 66 (2) (2016) 115–132. [4] T. Derrien, R. Johnson, G. Bussotti, A. Tanzer, S. Djebali, H. Tilgner, G. Guernec, D. Martin, A. Merkel, D.G. Knowles, J. Lagarde, L. Veeravalli, X. Ruan, Y. Ruan, T. Lassmann, P. Carninci, J.B. Brown, L. Lipovich, J.M. Gonzalez, M. Thomas, C.A. Davis, R. Shiekhattar, T.R. Gingeras, T.J. Hubbard, C. Notredame, J. Harrow, R. Guigo, The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression, Genome Res. 22 (9) (2012) 1775–1789. [5] T. Nagano, P. Fraser, No-nonsense functions for long noncoding RNAs, Cell 145 (2) (2011) 178–181. [6] C.P. Ponting, P.L. Oliver, W. Reik, Evolution and functions of long noncoding RNAs, Cell 136 (4) (2009) 629–641. [7] M. Huarte, LncRNAs have a say in protein translation, Cell Res. 23 (4) (2013) 449–451. [8] M. Kretz, Z. Siprashvili, C. Chu, D.E. Webster, A. Zehnder, K. Qu, C.S. Lee, R.J. Flockhart, A.F. Groff, J. Chow, D. Johnston, G.E. Kim, R.C. Spitale, R.A. Flynn, G.X. Zheng, S. Aiyer, A. Raj, J.L. Rinn, H.Y. Chang, P.A. Khavari, Control of somatic tissue differentiation by the long non-coding RNA TINCR, Nature 493 (7431) (2013) 231–235. [9] R.A. Flynn, H.Y. Chang, Long noncoding RNAs in cell-fate programming and reprogramming, Cell Stem Cell 14 (6) (2014) 752–761. [10] Q.N. Chen, C.C. Wei, Z.X. Wang, M. Sun, Long non-coding RNAs in anti-cancer drug resistance, Oncotarget (2016). [11] X. Yan, Z. Hu, Y. Feng, X. Hu, J. Yuan, S.D. Zhao, Y. Zhang, L. Yang, W. Shan, Q. He, L. Fan, L.E. Kandalaft, J.L. Tanyi, C. Li, C.X. Yuan, D. Zhang, H. Yuan, K. Hua, Y. Lu, D. Katsaros, Q. Huang, K. Montone, Y. Fan, G. Coukos, J. Boyd, A.K. Sood, T. Rebbeck, G.B. Mills, C.V. Dang, L. Zhang, Comprehensive genomic characterization of long non-coding RNAs across human cancers, Cancer Cell 28 (4) (2015) 529–540. [12] A.A. Yarmishyn, I.V. Kurochkin, Long noncoding RNAs: a potential novel class of cancer biomarkers, Front. Genet. 6 (2015) 145. [13] H. Ling, K. Vincent, M. Pichler, R. Fodde, I. Berindan-Neagoe, F.J. Slack, G.A. Calin, Junk DNA and the long non-coding RNA twist in cancer genetics, Oncogene (2015). [14] C. Cao, J. Sun, D. Zhang, X. Guo, L. Xie, X. Li, D. Wu, L. Liu, The long intergenic noncoding RNA UFC1, a target of MicroRNA 34a, interacts with the mRNA stabilizing protein HuR to increase levels of beta-catenin in HCC cells, Gastroenterology 148 (2) (2015) 415–426 e18. [15] S. Peter, E. Borkowska, R.M. Drayton, C.P. Rakhit, A. Noon, W. Chen, J.W. Catto, Identification of differentially expressed long noncoding RNAs in bladder cancer, Clin. Cancer Res. 20 (20) (2014) 5311–5321. [16] D. Liu, Y. Li, G. Luo, X. Xiao, D. Tao, X. Wu, M. Wang, C. Huang, L. Wang, F. Zeng, G. Jiang, LncRNA SPRY4-IT1 sponges miR-101-3p to promote proliferation and metastasis of bladder cancer cells through up-regulating EZH2, Cancer Lett. 388 (2017) 281–291.
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Identification of oncogenic long noncoding RNA SNHG12 and DUXAP8 in human bladder cancer through a comprehensive profiling analysis
T
⁎⁎
Bin Jianga,1, Hailong Sub,1, Jun Yuana, Hu Zhaoa, Wenkai Xiac, Zhenlei Zhaa, Bin Wua, , ⁎ Zhili Liud, a
Department of Urology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, People’s Republic of China Department of General and Pediatric Surgery, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, People’s Republic of China c Department of Nephrology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, People’s Republic of China d Department of Oncology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, Shoushan road 163, 214400, People's Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bladder cancer Long noncding RNA Bio-marker DUXAP8 SNHG12
Bladder cancer is a common urological malignancies world-wide. Recently, a growing number of evidence have highlighted the importance of long noncoding RNAs (lncRNAs) in multiple cancers development and progression. However, the expression pattern of lncRNAs in bladder cancer and their underlying function remain poorly understood. To identify lncRNAs profile alterations and uncover valuable lncRNA candidates for bladder cancer diagnostic, we conducted a comprehensively lncRNAs profiling analyses and explored their clinical relevance using The Cancer Genome Atlas (TCGA) data and three independent microarray profiling data from the Gene Expression Omnibus (GEO). After annotation and analyses of these data, we found that lots of lncRNAs were dysregulated in bladder specimen when compared with normal control specimen. In addition, we found that differential expression of these lncRNAs is accompanied by genomic variations, including genome loci copy number deletion or amplification. Importantly, a part of these lncRNAs are related to bladder cancer patients outcome, such as SNHG10, SNHG12 and LINC00115. Finally, we validated two of these lncRNAs' (DUXAP8 and SNHG12) function in bladder cancer cells by down-regulating their expression with siRNAs, and found that down-regulation of DUXAP8 and SNHG12 could inhibit bladder cancer cells proliferation in vitro. In summary, this study demonstrated that a lot of lncRNAs are dysregulated in bladder cancer, and might provide useful lncRNAs resource for potential prognostic or diagnostic markers for this disease.
1. Introduction Bladder cancer is the ninth common malignancy and represents one of the common urological carcinomas world-wide [1]. As a highly heterogeneous cancer, low-grade non-invasive papillary tumors constitute ∼70% of all bladder carcinomas cases, while invasive highgrade tumors constitute ∼30% of all bladder carcinomas [2,3]. Although great improvement have been made on the diagnostic and surgical techniques, chemotherapy and radiotherapy, the improve of bladder cancer patients survival rates remain unsatisfactory. Hence, identify novel molecular biomarkers for bladder cancer early diagnosis is still urgent. Meanwhile, a better knowledge of the molecular
mechanisms of bladder cancer tumorigenesis and metastasis is important for the development of new treatment method. Over the past decades, a great number of studies have uncovered that long non-coding RNAs (lncRNAs) are widely expressed in almost of human organ tissue and cells, and emerging as new layer of gene regulation [4,5]. lncRNAs are longer than 200 nt in length, and lacking of protein coding probability. However, accumulating studies reveal that lncRNAs participate in various cellular process, including X chromatin imprinting, glucose metabolism, immune response, muscle cell differentiation, tumor cells proliferation and chemotherapy resistance [6–10]. Moreover, lncRNAs differential profiling analyses in multiple cancers using RNA sequencing or microarray data indicate that lots of
⁎ Corresponding author at: Department of Oncology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, Shoushan road 163, 214400, People’s Republic of China. Tel.: +86 0510 80615151; fax: +86 0510 80615151 ⁎⁎ Corresponding author at: Department of Urology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Wuxi, Shoushan road 163, 214400, People’s Republic of China E-mail addresses: [email protected] (W. Bin), [email protected] (Z. Liu). 1 These authors contributed equally to this work and should be regarded as joint first authors.
https://doi.org/10.1016/j.biopha.2018.09.025 Received 19 July 2018; Received in revised form 31 August 2018; Accepted 4 September 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. Differentially expressed lncRNAs in human bladder cancer tissues and normal tissues. (A) A heatmap was drawn to show the differentially expressed lncRNAs in bladder cancer and normal tissues by analyzing the TCGA RNA sequencing data. (B–D) Heatmaps were drawn to show the dysregulated lncRNAs in bladder cancer specimen compared with non-tumor tissues using the GSE89006, GSE51493 and GSE45184 datasets. (E) Overlap analyses of the altered lncRNAs profile (consistently up-regulated or down-regulated in at least two datasets, fold change) in TCGA, GSE89006, GSE51493 and GSE45184 datasets. (F) Venn diagram of differentially expressed lncRNAs in TCGA, GSE89006, GSE51493 and GSE45184 datasets.
related to patients poor outcome and promotes cell metastasis by binding with SUZ12 and inhibiting E-cadherin expression [18]. Although, a small part of lncRNAs have been investigated in human bladder cancer, most of the other lncRNAs’ clinical significance and functional roles in bladder cancer remains unclear. In the present study, we conducted a genome-wide analyses to identify differentially expressed lncRNAs in bladder cancer by analyzing RNA sequencing and gene microarray data from TCGA and GEO. The current study may provide a valuable resource of lncRNAs to further investigate and identify novel useful diagnostic biomarker for bladder cancer.
lncRNAs are dysregulated in human cancers and many of these dysregulated lncRNAs are associated with patients prognosis [11]. Additionally, experiment validation showed that those lncRNAs contribute to tumor development and progression through different mechanisms, such as histone modification, sponging microRNAs, regulation of protein translation and mRNA stability [12,13]. For examples, lncRNA HOXA11-AS is found to be over-expressed in human gastric carcinoma, and promotes cell growth and metastasis by acting as scaffold of LSD1and EZH2, and functioning as sponge for miR-1297. Moreover, lincRNA-UFC1 promotes hepatocellular carcinoma cells proliferation and inhibits apoptosis through interacting with HuR, and thereby increasing β-catenin mRNA and protein levels [14]. In case of bladder cancer, a few lncRNAs' biological function, clinical relevance and molecular mechanisms have been documented [15]. For example, LncRNA SPRY4-IT1 promotes cell proliferation and metastasis through acting as competing endogenous RNA of miR-101-3p and thereby up-regulating EZH2 expression in bladder cancer [16]. Liu and colleagues report that lncRNA H19 can function as a ceRNA of miR29b-3p and antagonize its repression of DNMT3B, resulted in promoting epithelial–mesenchymal transition and cell metastasis in bladder cancer [17]. Moreover, TGF-β induced lncRNA MALAT1 overexpression is
2. Methods and materials 2.1. lncRNA profiling analyses in TCGA and microarray data The RNA sequencing data of bladder cancer tissues and non-tumor tissues and relevant clinical information were downloaded from https://portal.gdc.cancer.gov/. Another three public bladder cancer gene microarray profiling data (GSE89006, GSE51493 and GSE45184 [19]) were obtained from the Gene Expression Omnibus (GEO). Differential profiling of lncRNAs in GSE89006, GSE51493 and GSE45184 501
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Fig. 2. Copy number variation analyses of lncRNAs' genome loci in bladder cancer. (A) The frequency of lncRNAs genome loci copy number gain (red) in bladder cancer tissues was shown. The rows are arranged according to the genomic locations of each lncRNAs. (B) The frequency of lncRNAs genome loci copy number loss (blue) in bladder cancer tissues was shown. The rows are arranged according to the genomic locations of each lncRNAs.
Fig. 3. Analyses of bladder cancer patients' OS and RFS related lncRNAs. (A) The heatmaps were drawn to show the Hazard ratio value and log rank P value of bladder cancer patients' OS and RFS related lncRNAs. (B) The Kaplan–Meier curves for bladder cancer patients' OS in high- SNHG10 or LINC00115 and low- SNHG10 or LINC00115 groups in the TCGA set. (C) The Kaplan–Meier curves for bladder cancer patients' RFS in high-SNHG10 or SNHG12 and low- SNHG10 or SNHG12 groups in the TCGA set.
and microarray data.
was conducted using the Agilent-045997 Arraystar human lncRNA microarray V3, Agilent-028004 SurePrint G3 Human GE 8 x 60 K Microarray and Arraystar Human LncRNA microarray V2.0 platforms. R software and packages were used to preprocess the RNA sequencing 502
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Fig. 4. Down-regulation of DUXAP8 and SNHG12 inhibits cell proliferation in bladder cancer. (A) The fold-change of DUXAP8 and SNHG12 expression levels between bladder cancer tissues and normal tissues in TCGA and GSE89006 datasets. (B) The relative expression of DUXAP8 and SNHG12 in negative control or DUXAP8 and SNHG12 siRNAs transfected UMUC3 and SW780 cells was detected by qRT-PCR. (C) Growth curves of UMUC3 and SW780 cells after transfection with DUXAP8 and SNHG12 siRNAs or negative control were determined by CCK8 assays. Values represent the mean ± s.d. from three independent experiments. (E) The colony formation ability of UMUC3 and SW780 cells after transfection with DUXAP8 and SNHG12 siRNAs or negative control were determined by colony formation assays. ** P < 0.01; * P < 0.05.
2.2. Copy number variation analyses
2.4. Cell culture and transfection
The raw gene copy number variations data of each bladder cancer specimen were down-loaded from Broad GDAC (https://portal.gdc. cancer.gov/). Then, GISTIC 2.0 [20] pipeline was used to evaluate the significancee of each lncRNAs copy number deletions or amplifications in their genome locus. All lncRNAs genomic regions were aligned and mapped according to GISTIC peaks, and the peaks of deletion or amplification with q values ≤ 0.25 were defined as significant. The peak q values, numbers and focal/broad frequencies of each lncRNAs were integrated at gene level.
Bladder cancer cell lines UMUC3 and SW780 were obtained from the American Type Culture Collection (ATCC), and cultured in RPMI 1640 medium (GIBCO, USA) containing 10% fetal bovine serum (FBS). The cells were maintained in a humidified incubator with a 5% CO2 atmosphere at 37℃. The small interference RNAs (siRNAs) target DUXAP8 and SNHG12 (Invitrogen, Carlsbad, CA) were transiently transfected into UMUC3 and SW780 cells by using RNAiMAX (Invitrogen, USA) based on the manufacturer’s manual. 2.5. RNA extraction and qRT-PCR
2.3. Analyses of bladder cancer survival related lncRNAs
Total RNA of UMUC3 and SW780 cells was extracted with RNeasy Purification Kit (QIAGEN) following the manufacturer’s instructions. Then, 1 μg of total RNA was reverse-transcribed to cDNA by using the Reverse Transcriptase kit (Takara, Dalian, China) according to manufacturer’s manual. The Quantitative RT-PCR assays were implemented with SYBR Select Master Mix (Applied Biosystems) on ABI 7900 system (Applied Biosystems, CA, USA). The primers used were: DUXAP8, forward 5'-AGGATGGAGTCTCGCTGTATTGC-3', reverse 5'- GGAGGTTTG TTTTCTTCTTTTTT-3'; SNHG12, forward 5'-TCTGGTGATCGAGGACT TCC-3', reverse 5'-ACCTCCTCAGTATCACACACT-3';GAPDH, forward 5'AGAAGGCTGGGGCTCATTTG-3', reverse 5'- AGGGGCCATCCACAGTC TTC-3'. The 2−ΔΔCT method was performed to determine DUXAP8 and SNHG12 expression levels.
Univariable Cox regression analyses using Bio-conductor and R software was conducted to explore the significance of lncRNAs for bladder cancer patients’ overall survival (OS) or recurrence free survival (RFS) prediction. Next, multivariable Cox regression analysis using Bio-conductor and R software was performed to determine whether lncRNA could be used as dependent variable factor. Then, each lncRNA’s risk score was calculated. Next, the patients with bladder cancer were classified into high- or low-lncRNAs expression group based on the chose lncRNAs median expression levels. Finally, lncRNA with log rank P value < 0.05 between high-lncRNA expression and lowlncRNA expression groups were considered significant. 503
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Fig. 5. Down-regulation of DUXAP8 and SNHG12 inhibits cell invasion and induces cell apoptosis in bladder cancer. (A, B) Apoptosis ratio of negative control, DUXAP8 or SNHG12 siRNAs transfected SW780 cells was observed using flow cytometry assays. (C, D) Transwell assays were performed to evaluate the invasive ability of negative control, DUXAP8 or SNHG12 siRNAs transfected SW780 cells. * P < 0.05.
fixed with 4% Paraformaldehyde, and stained by 0.1% crystal violet. Next, the stained cells were imaged, and counted from six random vision(Olympus, Tokyo, Japan).
2.6. Cell proliferation and colony formation assay The CCK8 assays were conducted to evaluate UMUC3 and SW780 cells viability after transfection with SNHG12, DUXAP8 or scrambled siRNAs. 48 h after transfection, UMUC3 and SW780 cells (2000/well) were plated into 96 well plate The cells in 96-well plate were cultured, and 10 μl CCK8 (Dojindo, Japan) was added into each well, and the cells were incubated for 2 h. The relative numbers of cell in each well were determined by measuring absorbance at 450 nm using the ELx600 microplate reader (BioTek, Winooski, Vermont). For colony formation assay, UMUC3 and SW780 cells transfected with SNHG12, DUXAP8 or scrambled siRNAs were seeded into six-well plates and cultured for two weeks. Then, the cells were fixed with 4% paraformaldehyde and stained with purple crystal. The number of colonies were calculated under the microscope.
2.9. Statistical analysis All in vitro validation data were analyzed from three independent experiments. SPSS version 18.0 (SPSS Inc.Chicago, IL, USA) and GraphPad Prism 5.0 software were utilized for the independent and paired samples’ t-test or ANOVA to analyze qRT-PCR and in vitro experiment data as indicated.A p-value < 0.05 was deemed statistically significant. 3. Results 3.1. Identify dysregulated lncRNAs in human bladder cancer
2.7. Cell apoptosis analysis To identify dysregulated lncRNAs in human bladder tissues, we firstly downloaded the RNA sequencing data of bladder cancer and normal specimen from TCGA and three microarray profiling data (GSE89006, GSE51493 and GSE45184) from the GEO. The TCGA dataset includes 414 bladder cancer specimen and 19 normal specimen; GSE89006 dataset includes 4 paired bladder cancer and para-cancer tissues; GSE51493 consists of 12 bladder carcinoma primary tumors and 3 normal tissues; GSE45184 consists of 3 paired bladder cancer and adjacent normal tissues. Re-annotation and differential lncRNAs profiling analyses revealed that 862 lncRNAs expression were dysregulared (281 upregulated and 581 downregulated) in bladder cancer from the TCGA dataset; 268 lncRNAs expression was dysregulated (116 upregulated and 152 downregulated) in GSE45184 dataset; 839 lncRNAs were differentially expressed (240 upregulated and 599 downregulated) in the GSE51493 dataset; and 2526 lncRNAs were dysregulated in the GSE89006 dataset (1138 increased and 1388 decreased) (Fig. 1A–D, and Table S1). Furthermore, overlap analyses showed that
SW780 cells were cultivated in 6-well plate and transfected DUXAP8, SNHG12 or negative control siRNAs. 48 h after transfection, all the cells were harvested and double stained with FITC-Annexin V and PI with FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) based on the manufacturers’ manual. Flow cytometry (EPICS, XL-4, Beckman, CA, USA) was used to examine the cell apoptosis. 2.8. Cell invasion assays To evaluate bladder cancer cell invasive ability, transwell assays were conducted. For invasion assays, 5 × 104 cells in 300 μl medium without serum were added into the upper culture chamber with Matrigel coated 8-mm membrane (Millipore, Billerica, MA, USA). Then, 700 μl medium containing 10% fetal bovine serum was added into the lower wells of 12-well plate. 24 h after incubation, the bladder cancer cells on upper membrane of chamber were removed by cotton wool, 504
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Fig. 6. DUXAP8 and SNHG12 co-expressed protein coding genes and enriched pathways. (A) The circle diagram shows the top 50 DUXAP8 and SNHG12 co-expressed protein coding genes in bladder cancer in TCGA dataset. (B, C) The “DUXAP8” and “SNHG12” co-expressed protein coding genes enriched pathways and their interacted network in bladder cancer. (D) The DUXAP8 expression was positively related to MAGEA4 and MAGEA6 expression in bladder cancer tissues. (E) The SNHG12 expression was positively related to HDAC10 and AGER expression, but negatively related to LATS2 and PCDH7 expression in bladder cancer tissues.
that 24 over-expressed lncRNAs and 17 decreased lncRNAs are significantly related to bladder cancer patients shorter OS (log rank P < 0.05), and 16 increased lncRNAs and 22 down-regulated lncRNAs are significantly related to bladder cancer patients shorter RFS (log rank P < 0.05) (Fig. 3A, and Table S3). Taken SNHG10, LINC00115, and SNHG12 for example, bladder cancer patients with high SNHG10 or LINC00115 expression had shorter OS time, while bladder cancer patients with high SNHG10 or SNHG12 expression had shorter RFS time (Fig. 3B–C). These data suggest that those bladder cancer survival related lncRNAs might be useful candidates for patients outcome prediction.
137 lncRNAs were consistently up-regulated and 309 lncRNAs were down-regulated in at least two of these datasets (Fig. 1E–F, Table S2). These findings suggest that lots of lncRNAs are differentially expressed in human bladder cancer, and these lncRNAs may be useful diagnosis biomarkers. 3.2. lncRNAs genome loci copy number variations in bladder cancer Recently, a growing number of studies demonstrate that genomic alterations as well as transcription factors contribute to lncRNAs alterations in various tumor cells. To further explore whether genomic alterations are involved in lncRNAs dysregulation, we downloaded the gene copy number variations data in all bladder cancer samples from the Broad GDAC. Next, genomic loci copy number frequency of each differentially lncRNAs was calculated, and the alterations in each bladder cancer specimen with q value less than 0.25 was defined as significant. As a result, a lot of lncRNAs genome loci had copy number gain (such as PVT1, IFNG-AS1 and CALML3-AS1) or frequency loss (such as LINC01198, PTPRD-AS1 and DLEU7-AS1) in bladder cancer (Fig. 2A-B, and Table S3). These findings indicate that a part of dysregulated lncRNAs in bladder cancer might be affected by copy number variations in their genomic loci.
3.4. Knockdown of DUXAP8 and SNHG12 inhibit bladder cancer cells proliferation To further evaluate the analysis results, we focused on these overexpressed lncRNAs which may be suitable biomarkers. Among those increased lncRNAs, DUXAP8 and SNHG12 expression are up-regulated in TCGA and GSE89006 datasets (Fig. 4A). Moreover, the survival analyses results also revealed that higher SNHG12 expression is related to bladder cancer patients' shorter RFS. DUXAP8 has also been found to be up-regulated in gastric cancer [21], non small cell lung cancer [22] and renal cell carcinoma [23], while SNHG12 also exerts oncogenic function in multiple cancers [24,25]. However, their functional roles in bladder cancer remain unclear, hence, we chose these two lncRNAs for further investigation. Then, we analyzed DUXAP8 and SNHG12 expression in bladder cancer cell lines using the CCLE RNA sequencing data [26]. The relative expression levels of DUXAP8 and SNHG12 (FPKM value) in each bladder cancer cell lines were calculated, and the value of FPKM ≥ 10 was defined as "relative high expression level". We
3.3. Identification of bladder cancer survival associated lncRNAs Accumulating evidence has demonstrated that lots of lncRNAs are related to several cancers patients outcome and could be novel predictors for tumor patients prognosis. To identify bladder cancer overall survival (OS) and recurrence free survival (RFS) related lncRNAs, we conducted univariable Cox regression analysis. As a result, we found 505
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sequencing and lncRNA microarray technique enables us to analyze large-scale differentially expressed lncRNAs in human cancers. In the present study, we conducted a comprehensively differential profiling analyses of lncRNAs in human bladder cancer and adjacent normal specimen to identify more dysregulated lncRNAs. The findings is this study uncovered that hundreds of lncRNAs' expression are dysregulated in baldder cancer, such as up-regulated DUXAP8, AC005281.1 and LINC01424, and down-regulated HAND2-AS1, FENDRR, LINC00473. Moreover, copy number variation analysis of those dysregulated lncRNAs’ genome loci revealed that a small part of those dysregulated lncRNAs had copy number amplification or deletion in their genome loci, such as PVT1, IFNG-AS1, CALML3-AS1, LINC01198, PTPRD-AS1 and DLEU7-AS1. These findings suggest that genomic alterations may involve in lncRNAs’ alteration in bladder cancer. Furthermore, we found that a few of lncRNAs are related to bladder cancer patients’ OS or RFS, including SNHG10, LINC00115 and SNHG12, indicating that lncRNAs might be valuable predictor for bladder cancer patients’ outcome. To evaluate our analysis findings and determine whether those dysregulated lncRNAs, we chose two up-regulated lncRNAs DUXAP8 and SNHG12 and investigated their function in bladder cancer. Our findings showed that down-regulation of DUXAP8 and SNHG12 significantly impaired bladder cancer cells in vitro growth, colony formation capacity, invasive ability and induced cell apoptosis. Moreover, GO and pathway analyses revealed that SNHG12 and DUXAP8 co-expressed protein coding genes are enriched in cell cell cycle, focal adhesion and PI3K-Akt signaling pathways, which are important for bladder tumorigenesis and tumor progression. As well as our findings, DUXAP8 was also reported to be up-regulated in human esophageal squamous cell carcinoma [32], gastric carcinoma [21] and NSCLC [22]. Over-expressed DUXAP8 promotes NSCLC cells growth and invasion through epigenetically inhibiting EGR1 and RHOB transcription via interacting with EZH2 and LSD1 [22]. Moreover, several studies have revealed that SNHG12 are over-expressed in multiple cancers, including papillary thyroid carcinoma [24], gastric cancer [25], and Cervical Cancer [33]. Tian and colleagues found that up-regulated SNHG12 promotes tumorigenesis and hepatocellular carcinoma cells metastasis through functioning as a ceRNA for miR-199a/b-5p to modulate MLK3 expression and regulate NF-κB pathway [34]. These findings suggest that those dysregulated lncRNAs (such as DUXAP8 and SNHG12) might play key roles in human bladder cancer tumorigenesis and tumor progression.
found that DUXAP8 and SNHG12 expression levels were high in most of the bladder cancer cell lines. According to the CCLE data, UMUC3 and SW780 cells was chosen for further experiments. Next, DUXAP8 and SNHG12-specific siRNAs were tranfected into UMUC3 and SW780 cells to down-regulate their expression. The results of qRT-PCR revealed that DUXAP8 and SNHG12 expression were significantly decreased in two siRNAs transfected UMUC3 and SW780 cells compared with control cells (Fig. 4B). Next, CCK8 assays revealed that down-regulation of SNHG12 and DUXAP8 expression impaired UMUC3 and SW780 cells proliferation when compared with control cells (Fig. 4C–F). Moreover, down-regulation of DUXAP8 or SNHG12 inhibited UMUC3 and SW780 cells colony formation capacity. 3.5. Down-regulation of DUXAP8 and SNHG12 inhibits bladder cancer cells invasion and induces cell apoptosis To determine whether down-regulation of DUXAP8 and SNHG12 mediated suppression of cell proliferation by enhancing apoptosis, we performed flow cytometry cell apoptosis assay. The results showed that the ratios of apoptosis in si-DUXAP8 or si-SNHG12 group were increased compared with control cells (Fig. 5A-B). Moreover, the results of transwell assay illustrated that down-regulation of DUXAP8 and SNHG12 could impair the invasive ability of SW780 cells compared with control cells (Fig. 5C–D). 3.6. SNHG12 and DUXAP8 co-expressed genes and enriched pathways in bladder cancer To determine SNHG12 and DUXAP8 associated protein coding genes (PCGs) and their involved pathways in bladder cancer, we analyzed SNHG12 and DUXAP8 co-expressed PCGs using TCGA data. The results showed that lots of oncogenes expression are positively associated with SNHG12 and DUXAP8 expression, while some tumor suppressors expression are negatively associated with SNHG12 and DUXAP8 expression in bladder cancer tissues (Fig. 6A). Meanwhile, GO and pathway analysis revealed that DUXAP8 and SNHG12 co-expressed PCGs are enriched in focal adhesion, cell cycle and PI3K-Akt signaling pathway etc (Fig. 6B–C). Interestingly, these biological process and pathways are important for regulating cancer cells growth and invasion. Furthermore, DUXAP8 expression is positively related to MAGEA6 and MAGEA4 in bladder cancer; SNHG12 expression is positively associated with HDAC10 and AGER, and negatively associated with PCDH7 and LATS2 in bladder cancer (Fig. 6D–E).
5. Conclusion 4. Discussion Taken together, this study found that lots of lncRNAs were dysregulated in human bladder cancer specimen compared with normal tissues. And, copy number amplification or deletion may involve in part of those lncRNAs’ alteration in bladder cancer. Moreover, a small portion of the dysregulated lncRNAs are associated with patients OS and RFS time in bladder cancer, which might be independent prediction factors of bladder cancer patients’ survival. The findings in this study highlight the importance of lncRNAs in bladder cancer, and our analyses data could provide valuable lncRNA candidates for bladder cancer diagnosis or target therapy. However, there are a few limitations in this study, such as only SNHG12 and DUXAP8 function were validated and their underlying molecular mechanisms are not documented, which would be further explored in our future study.
Recently, sequencing and annotation of the whole human genome have uncovered that thousands of lncRNAs are transcribed from these non-coding regions. To date, a great number of studies have documented that lnRNAs' dysregulation play critical roles in human cancers development and tumor progression [27]. For example, Chen et al. Reported that up-regulated LNIC01234 promotes gastric cancer cells growth through functioning as competing endogenous RNA (CeRNA) for miR-204-5p, thereby antagonize its’ suppression of CBFB [28]. Moreover, lncRNA miR503HG is markedly down-regulated in hepatocellular carcinoma, while over-expression of miR503HG could inhibit cell invasion and metastasis through interacting with HNRNPA2B1 and promoting its’ degradation [29]. Simultaneously, a few lncRNAs’ functions in bladder cancer have been identified, and their underlying molecular mechanisms were clarified. For instance, Wang et al. found that GAS5 expression was decreased in bladder cancer, and GAS5 overexpression could induce cell apoptosis by inhibiting EZH2 transcription [30]. In addition, lncRNA MALAT1 promotes bladder cancer cells growth and metastasis in vivo by acting as a sponge for miR-124 to release its’ repression of FOXQ1 [31]. Over the past decade, the rapid improvement of next generation
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Conflicts of interest The authors have no actual or potential conflicts of interest to 506
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declare.
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