|Ahead of print publication
Expression of oncolong noncoding RNA taurine-upregulated gene-1 in colon cancer: A clinical study supported by in silico analysis
Abdelrahman I Abushouk1, Shahad W Kattan2, Hanadi T Ahmedah3, Eshrag Baothman4, Sameerah Shaheen5, Eman A Toraih6, Manal Said Fawzy7
1 Department of Medicine, Harvard Medical School, Boston, MA, USA
2 Department of Medical Laboratory, College of Applied Medical Sciences, Taibah University, Yanbu, Saudi Arabia
3 Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Rabegh, Saudi Arabia
4 Department of Medicine, Batterjee Medical Technology College, Jeddah, Saudi Arabia
5 Anatomy Department and Stem Cell Unit, College of Medicine, King Saud University, Riyadh, Saudi Arabia
6 Department of Surgery, Tulane University, School of Medicine, New Orleans, Louisiana, USA; Genetics Unit, Department of Histology and Cell Biology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
7 Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt; Department of Biochemistry, Faculty of Medicine, Northern Border University, Saudi Arabia
|Date of Submission||18-Apr-2020|
|Date of Decision||01-Jun-2020|
|Date of Acceptance||15-Aug-2020|
|Date of Web Publication||20-Aug-2021|
Manal Said Fawzy,
Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Suez Canal University, P.O. Box: 41522, Ismailia
Source of Support: None, Conflict of Interest: None
Context: Recent studies confirmed that dysregulation of long noncoding RNAs (lncRNAs) is a potential contributor to the development and progression of colon cancer. However, the prognostic value of these RNA molecules remains controversial.
Aims: This study aimed to investigate the expression of taurine-upregulated gene-1 (TUG1) lncRNA in colon cancer and its clinical implications.
Subjects and Methods: A retrospective study on 47 formalin-fixed, paraffin-embedded samples of surgically resected primary colon cancer specimens was done. Total RNA purified from the colon cancer samples and noncancer adjacent tissue sections was quantified by real-time reverse transcription-polymerase chain reaction (qRT-PCR) to assess TUG1 relative expression levels normalized to GAPDH endogenous control. Also, in silico data analysis was applied.
Statistical Analysis Used: The relative expression levels were calculated using the LIVAK method. The survival rates were assessed using the Kaplan–Meier curves and the Cox proportional model. P < 0.05 was considered statistically significant.
Results: TUG1expression in the colon cancer specimens was significantly overexpressed (median = 21.50, interquartile range [IQR]: 7.0–209.2; P = 0.001) relative to the noncancerous tissues. In silico analysis confirmed TUG1 upregulation in colon carcinoma (median = 13.92, IQR: 13.5-1432). There were no significant associations between TUG1 expression and clinicopathological characteristics, such as the site, grade, stage, histopathological type, or the rates of lymphovascular invasion and relapse. Similarly, Kaplan–Meir and Cox multivariate regression analyses showed that TUG1 expression could not predict the overall survival and progression-free survival in colon cancer patients of our population.
Conclusions: This study confirms the overexpression of TUG1 lncRNA in colon cancer tissues. Larger sample size is warranted to further elucidate the specific role of TUG1 in colon cancer.
Keywords: Colon cancer, gene expression, long noncoding RNA, real-time quantitative polymerase chain reaction, taurine-upregulated gene-1
|How to cite this URL:|
Abushouk AI, Kattan SW, Ahmedah HT, Baothman E, Shaheen S, Toraih EA, Fawzy MS. Expression of oncolong noncoding RNA taurine-upregulated gene-1 in colon cancer: A clinical study supported by in silico analysis. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=324167
| > Introduction|| |
Colon cancer is a common malignancy with an annual incidence of almost one million cases and is a leading cause of cancer-related deaths. Despite the improvements in cancer management modalities, the outcomes of patients with advanced colon cancer remain unsatisfactory., Moreover, the genetic factors, implicated in the initiation and progression of colon cancer, remain elusive. This highlights the need for a better understanding of these molecular mechanisms to develop specific and more effective therapeutic approaches for colon cancer patients.
The relevance of the nonprotein coding genome was ignored for years, and it was even referred to as “Junk” DNA. Noncoding RNAs (ncRNAs) are classified based on their size into long ncRNA (lncRNAs), more than 200 nucleotides, and short ncRNAs (as microRNAs) that do not exceed 20–30 nucleotides., lncRNAs have been shown to play several roles in human cells as key regulators of gene expression, epigenetic modifications, chromatin remodeling, splicing, and cellular differentiation., In colon cancer, some lncRNAs as activated by transforming growth factor beta (ATB), colon cancer-associated transcript 1 (CCAT1), zinc finger NFX1-type containing 1 (ZNFX1)-antisense RNA 1 (ZFAS1), urothelial cancer-associated 1 (UCA1), and HOX transcript antisense RNA (HOTAIR) have been implicated as oncogenes that manipulate the biological properties of colon cells. However, the evidence remains inconclusive about the role of other lncRNAs in this cancer type.
Taurine-upregulated gene-1 (TUG1), also named as long intergenic non-protein coding RNA 80 (LINC00080 or NCRNA00080) or FLJ20618, is located along the long arm of chromosome 22q12.2 (maps to 22:30,969,245–30,979,395 on the forward strand according to the genome reference consortium human genome build 38), spanning 10.18 Kb long. This gene is composed of four exons that can encode for twenty different transcripts due to alternative splicing. However, none of them is translated into protein (www.ensembl.org). TUG1 lncRNA was initially discovered in mouse retinal cells as a transcript, upregulated by taurine. Later studies confirmed the dysregulation of this gene in various cancers, being upregulated in bladder carcinoma, osteosarcoma, and esophageal squamous cell carcinomas., However, it was found to be downregulated in non-small cell lung cancer, gliomas and multiple myelomas, highlighting the tissue-specific role in oncogenesis. The knockdown of TUG1 repressed cell proliferation and inhibited the migration and invasiveness of malignant cells. Although previous studies reported the upregulation of TUG1 expression in colorectal cancer patients,, the prognostic role of this lncRNA in colon cancer remains unconfirmed particularly in the current population. To this end, this study was aimed to evaluate the lncRNA TUG1 expression profile and explore its prognostic value in colon cancer tissues.
| > Subjects And Methods|| |
This retrospective study was carried out on an eligible sample of 47 formalin-fixed, paraffin-embedded (FFPE) tissues retrieved from surgically resected primary colon cancer specimens archived in the Pathology lab of the Hospital and the Oncology Center, between January 2013 and January 2017. All patients received no radiotherapy or chemotherapy before the surgery. Clinical and pathological data were obtained from medical records, including age, sex, site, and the size of the tumor, and lymph node status, with follow-up till January 2019 (minimum 24 months). Tumors were staged clinically according to the TNM classification of colon cancer. Samples with incomplete clinical data or follow-up period, history of receiving any type of treatment before surgery, and/or diagnosis with malignant disease primarily arising from other organs, as well as samples without available paired non-cancer tissue, very small size tissue specimen available for molecular work, and those with low concentration or without enough quality of the extracted total RNA to proceed in the downstream quantitative real-time polymerase chain reaction (qRT-PCR) were excluded. The noncancer adjacent tissues confirmed by two independent pathologists were used for molecular data normalization. The study was conducted according to the ethical and legal guidelines adopted by the Declaration of Helsinki 2008. Ethical approval for this study was granted by the local Medical Research Ethics Committee, and the patient consent was waived as the included samples in the present study were archived samples as mentioned above.
Sections (4-μm thickness) were prepared for routine hematoxylin and eosin staining. The histopathological subtype and features were evaluated blindly by two independent pathologists. Other sections were sliced and stored in autoclaved Eppendorf tubes for gene expression analysis.
Taurine-upregulated gene-1 gene expression profiling
Qiagen RNeasy FFPE kits (Cat #74404, Qiagen, Hilden, Germany) were applied to purify total RNA from the obtained samples. Treatment of samples with RNase free DNase I for 2 h at 37°C was followed according to the supplier's instructions. RNA concentration and purity at the absorbance ratio of 260/280 nm were assessed, followed by an agarose gel electrophoresis to check RNA integrity. The extracted total RNA ranged between 20 and 65 ng/μL. The RNA samples were reverse-transcripted according to the supplier instructions., Detection of TUG1 and the endogenous control GAPDH expression levels was carried out using qRT-PCR following the Minimum Information for publication of qRT-PCR experiments (MIQE) guidelines. The TaqMan assays (Applied Biosystems, Thermo Fisher Scientific Inc., assay ID Hs05579214_s1 for TUG1 and Hs02786624_g1 for GAPDH) were used as previously described. Triplicate PCR runs were performed and average values were estimated, excluding >2 standard deviations. Consistent values were obtained with a nearly close cycle of quantification values of the same specimen. The relative expression levels were calculated using the LIVAK method.
In silico data analysis
Taurine-upregulated gene-1 gene analysis
Ensembl (www.ensembl.org), NCBI (www.ncbi.nlm.nih.gov), and Genecards (www.genecards.org) were used for gene structure and sequence analysis. Subcellular localization was determined using the Compartments database (https://compartmentsjensenlab.org/).
Taurine-upregulated gene-1-cancer interactions
The LncRNA and Disease Database (LncRNADisease) version 2.0 database (http://www.cuilab.cn) and MalaCards (malacards.org) were utilized to identify diseases associated with TUG1 gene based on previously published experimental and clinical studies. Cancer hallmarks analytic tools (chat.lionproject.net/) were used to determine the strength of association by text mining between TUG1 and each domain of cancer hallmarks.
Deregulation pattern of taurine-upregulated gene-1 in cancer
A Genevestigator search engine for gene expression (https://genevestigator.com) was used, which integrates manually curated microarray and RNA-Seq experiments across different biological contexts as diseases, drugs, tissues, cell lines, or genotypes. The colon datasets were retrieved from 1985 colon cancer patients, and TUG1 gene expression was explored.
KM plotter web application (http://kmplot. com/analysis/index.php?p = service&cancer = pancancer_mirna) and Prognoscan (http://dna00.bio.kyutech.ac.jp/PrognoScan/) were used to evaluate the prognostic performance of TUG1 expression in different types of cancer.
Data analyses were conducted using SPSS version 24 for Windows (IBM, Chicago, IL, USA). Mann–Whitney and Kruskal–Wallis tests were applied, when appropriate. A two-tailed P value was considered significant at <0.05. Binary logistic and linear regression methods were applied for the overall survival (OS) defined as “the duration from the date of primarily receiving treatment at the time of death from any causes or the date of the last follow-up,” and the progression-free survival (PFS) defined as “the interval between the date of patients primarily receiving treatment and the date when radiological evidence of recurrence was observed” to adjust for clinicopathological covariates. The survival rates were assessed using the Kaplan–Meier (KM) curves and the COX proportional model to calculate hazard ratios (HR) and 95% confidence interval (CI).
| > Results|| |
Demographic and clinical data
The mean age of patients was 58.9 ± 12.6 years. For patients whose follow-up data were available, 35.7% had lymphovascular invasion and 31.7% have experienced a relapse. The median OS was 48 (interquartile range [IQR]: 15) months, while the DFS had a median of 45 (IQR: 11) months. The detailed distribution of cancer site, grade, stage, TNM classification, and histopathological type is illustrated in [Table 1].
|Table 1: Association between taurine-upregulated gene-1 expression levels and the clinicopathological parameters in colon carcinoma|
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Taurine-upregulated gene-1 expression in physiological and pathological conditions including colon cancer patients
The lncRNA TUG1 is ubiquitously expressed in testis, endometrium, and thyroid along with other tissues including the colon. TUG1 was confined to the nucleus and extracellular space [Figure 1]a. In cancer, text mining by Cancer Hallmarks Analytic Tools revealed that TUG1 is involved mainly in resisting cell death, sustaining proliferative signaling, inducing angiogenesis, invasion, and metastasis [Figure 1]b. Neoplastic and nonneoplastic disorders associated with TUG1 are demonstrated in [Figure 1]c.
Our analysis of the current samples revealed statistically significant overexpression (P = 0.001) of TUG1 in the colon cancer group (median: 21.50, IQR: 7-209.2) in comparison to the control group. The expression of TUG1 in both groups is illustrated in [Figure 2].
|Figure 2: Relative expression level of taurine-upregulated gene-1 in colon cancer specimens was normalized to the GAPDH expression level for each sample and calculated using the delta-delta cycle of quantification method (=2[−ΔΔCq]) compared to noncancerous tissues. Values are presented as median (Q1-Q3). P values were calculated by the Mann–Whitney U-test|
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By searching public repositories for relevant experiments on colon carcinoma, 1985 patients, profiled on the Affymetrix Human Genome 133 plus2 array platform, were selected. The analysis revealed overexpression of the lncRNA TUG1 in colon carcinoma (median and quartiles = 13.92 (13.47–1432), mean and SD = 13.90 ± 0.64) [Figure 3].
|Figure 3: Expression of taurine-upregulated gene-1 gene across colon tissues and cell types. Data source: Genevestigator.com|
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Taurine-upregulated gene-1 expression and clinicopathological characteristics
There was no significant association between TUG1 expression and age, gender, the site, grade, stage, histopathological type, or the rates of lymphovascular invasion and relapse in the study samples. The details of TUG1 associations with different clinicopathological variables are illustrated in [Table 1].
Kaplan–Meier survival and COX multiple regression analyses were applied to identify the independent predictors of survival in patients with colon cancer. The log-rank of OS was significantly predicted by cancer type (P = 0.02, survival being worst in anaplastic and mucinous carcinoma) and metastasis (P = 0.04). However, they both became insignificant in the multiple Cox regression analysis. The Spearman's correlation test showed no correlation between TUG1 expression and OS (P = 0.64) [Table 2].
|Table 2: Multivariable analysis of overall survival in colon cancer patients|
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Similarly, the expression of TUG1 could not predict OS in KM analysis (P = 0.6). The correlation and Kaplan-Meir survival curves of the OS are illustrated in [Figure 4].
|Figure 4: Association between taurine-upregulated gene-1 expression level and survival. (a) Correlation between expression level and overall survival, Spearman's correlation test was used. (b) Kaplan–Meier survival curve. Log rank test was applied|
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For PFS, the Kaplan–Meier curves showed that cancer type (P = 0.04, being worse in anaplastic and signet cell carcinomas), stage (P = 0.007), lymph node involvement (P = 0.02), and distant metastasis (P = 0.01) could predict PFS in colon cancer patients. Multivariate Cox regression showed that LN involvement and metastasis could serve as independent predictors for PFS [Table 3].
|Table 3: Prognostic factors and Cox proportional hazard models for progression-free survival|
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To identify the association of TUG1 with the survival of other cancers, a total of 396 datasets including TUG1 probe ID and clinical data from 12 types of cancer (bladder, blood, brain, breast, colorectal, esophagus, eye, head and neck, lung, ovarian, skin, and soft tissue) were analyzed. Out of them, 18 platforms showed association with OS, disease-specific survival, relapse-free survival, distant metastasis-free survival, and distant recurrence-free survival. Upregulation of TUG1 gene was associated with poor survival in breast cancer, uveal melanoma, acute myeloid leukemia, lung adenocarcinoma, multiple myeloma, liposarcoma, and head-and-neck cancer (squamous cell carcinoma). In contrast, prolonged survival was significantly associated with colorectal cancer, glioma, and ovarian cancer [Figure 5]a. We could not find datasets on colon cancer in KM plotter, however, an analysis of 160 rectal adenocarcinoma patients revealed no association with survival (log-rank P = 0.137) [Figure 5]b.
|Figure 5: Evaluation of taurine-upregulated gene-1 as a prognostic biomarker in cancer. (a) Hazard ratio and confidence interval for poor survival in the significant cancer platforms. A total of 396 microarray datasets were screened and significant Hazard ratios are only shown in the figure. (b) Kaplan–Meier curve for taurine-upregulated gene-1 expression level in rectal adenocarcinoma. Data source: Kaplan–Meier plotter web application and prognoscan|
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| > Discussion|| |
The current study detected significant TUG1 upregulation in colon cancer tissues in comparison to noncancer tissues. Similar results were obtained from in silico analysis. This finding confirms the results of previous studies in the literature that showed increased TUG1 expression in patients with colon and colorectal cancers.,, However, this study did not confirm the value of TUG1 as an independent predictor of OS and PFS in colon cancer patients. Although the relatively small sample size of the current population could participate in this negative finding, our in silico analyses were consistent with this result. Furthermore, Li et al. in their recent meta-analysis of 15 studies confirmed the insignificant association between high TUG1 expression and OS in their pooled results in various tumors. Otherwise, Zhou et al. reported an association of high TUG1 expression with poor OS in their meta-analysis of 9 studies, although they showed TUG1 upregulation was not associated with age, gender, smoking, tumor diameter, TNM stage, or lymph node metastasis, consistent with our findings. These contradictory results warrant further larger studies in different ethnic population to reach consensus.
Several studies concluded TUG1 upregulation in multiple cancers, however the exact mechanisms of this oncogenic role remain unconfirmed. These studies have shown that the upregulation of TUG1 expression increases cellular proliferation, tumor invasiveness, and radioresistance and inhibits apoptosis.,, A recent study by Xiao et al. showed that TUG1 overexpression increased the activity of the Wnt/β-catenin pathway, promoting tumor progression. The knockdown of TUG1 expression inhibited the nuclear translocation of β-catenin, which suppressed the expression of the oncogenic c-Myc protein.
In addition, Sun et al. showed that TUG1 promotes tumor metastasis and invasion of colorectal cancer cells by activating the expression of genes, involved in epithelial–mesenchymal transition. In this process, epithelial cells lose their polarity and biological properties and change into mesenchymal phenotypes. They showed that TUG1 overexpression suppressed E-cadherin expression and augmented the expression of fibronectin, vimentin, and N-cadherin. Other studies in other cancer types concluded that TUG1 promotes cell proliferation by silencing KLF2 and downregulating miR-145a.
The epigenetic regulation of TUG1 expression has been investigated in a few studies. One study by Zhai et al. found that the downregulation of p63 (a member of the p53 family) increases the expression of TUG1 in colon cancer cell lines. Moreover, Sun et al. reported that histone deacetylation is a key epigenetic controller of TUG1 expression in colorectal cancer cells. They showed that histone deacetylase inhibition could enhance the expression of TUG1 in colorectal cancer cell lines. Further research is needed to characterize the regulatory genes of TUG1 expression in colon cancer cells.
We applied two forms of analysis: in silico and evaluation of FFPE specimens. Moreover, we performed a long-term follow-up (minimum of 24 months and up to 66 months) in the current study. The majority of data in the literature are on colorectal cancer with few specific data on colon cancer. This study confirms the overexpression of TUG1 in colon cancer; however, further research is still needed to answer some questions. For example, the upstream event and downstream targets of TUG1 dysregulation should be identified, using recent techniques as OMICS integrative analysis. Further, the safety of systemic TUG1 expression is a matter of concern. In addition, large cohort multicenter studies are encouraged to validate the prognostic value of TUG1 expression in colon cancer patients.
| > Conclusions|| |
The results of this preliminary study showed TUG1 upregulation in colon cancer tissues relative to non-cancerous tissues. The overexpression of TUG1 could not predict OS or PFS in our population. Further research is needed to validate these findings in larger studies and characterize the mechanisms of TUG1 implication in tumor development and progression.
The authors thank the Center of Excellence in Molecular and Cellular Medicine and the Oncology Diagnostic Unit, Suez Canal University, Ismailia, Egypt, for providing the facilities for performing the molecular work. The authors acknowledge Dr. Afaf T. Ibrahiem, Ass. Prof. Pathology, Faculty of Medicine, Mansoura University, Egypt, for help in histopathological examination.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]