Long non-coding RNA LINC 00987 may function as a tumor suppressor in lung adenocarcinoma

Long non-coding RNAs (lncRNAs) are a group of transcripts over 200 nucleotides in length that do not code for proteins. The association of the dysregulation of numerous lncRNAs with several malignancies, including lung cancer, has been frequently reported. This study aims to inspect the association of genomic and transcriptomic alterations to the lncRNA LINC00987 with lung adenocarcinoma, a subtype of lung cancer, using a bioinformatic approach. To this end, we used three publically available online databases, cBioPortal, the International Cancer Genome Consortium Data Portal and the GEPIA web server. In short, our results demonstrated that LINC00987 expression might have a tumor suppressive role in lung adenocarcinoma and levels of expression could be of prognostic value for this cancer type.


Introduction
Lung cancer is the leading cause of cancer-related death in the world among both men and women 1 .Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer and accounts for over a million cancer-related deaths each year 2,3 .Thus, the identification of molecules involved in the initiation and development of LUAD should be a high priority and could help the early diagnosis, prognosis, and treatment of this lethal cancer.
Long non-coding RNAs (lncRNAs) are a subclass of non-protein-coding transcripts over 200 nucleotides in length that are involved in several cancer-related biological processes, including cell growth, cell proliferation, and apoptosis 4,5 .It has been frequently reported that lncRNAs, due to their pivotal regulatory roles in the cellular events, especially cell cycle processes, can be used as cancer biomarkers 6 .HOTAIR, for example, is an oncogenic lncRNA that is being used as a biomarker for several cancers 7 .LINC00987 is a poorly known lncRNA that has been observed to be dysregulated in LUAD by microarray studies 8 .However, the genomic and transcriptomic alterations to LINC00987 lncRNA in LUAD have not yet been precisely examined.
In this study, we investigated the association of LINC00987 genomic and transcriptomic alterations with LUAD.Moreover, the relationship between the LINC00987 expression level and the overall survival (OS) of LUAD patients was inspected.To accomplish these goals, three well-known online databases, cBioPortal, the International Cancer Genome Consortium (ICGC) Data Portal and the GEPIA web server, were employed.

Genomic analysis
Copy number alterations in LINC00987 lncRNA across The Cancer Genome Atlas (TCGA) LUAD study (586 samples) were retrieved from the cBioPortal database 9,10 .To this end, the "Putative copy-number alterations from GISTIC" and "All Tumors (586)" were selected as the genomic profile and the patient/case set, respectively.The Gene-level Analysis tool of the TCGA Copy Number Portal (Analysis Version: 2015-06-01 stddata__ 2015_04_02 regular peel-off) 11 was used to determine whether LINC00987 is located within or near any peak region of somatic copy-number alterations in LUAD (516 samples).TCGA Copy Number Portal facilitates the understanding of high resolution copy number data amassed from cancer samples in the TCGA.Furthermore, the mutations to LINC00987 lncRNA in LUAD was analyzed by means of the ICGC Data Portal (https://dcc.icgc.org) To this purpose, the "LINC00987" term was searched in the ICGC Data Portal and the projects related to the LUAD were selected from the results.

Transcriptomic analysis
The GEPIA web server 12 was used to investigate the differential expression of LINC00987 lncRNA between LUAD tumor (#483) and normal (#347) samples.To this end, the Differential Expression Analysis and the Expression DIY Boxplot tools were employed with default options.The association of the differential expression of the LINC00987 lncRNA with the OS of LUAD patients was examined using the Survival Plots tool of the GEPIA web server with default parameters.

LINC00987 has genomic alterations in LUAD
Copy number alteration analysis of LINC00987 demonstrated that this lncRNA was deeply deleted in over 4 percent of LUAD sequenced cases/patients (Figure 1a).However, LINC00987 lncRNA was amplified in 5 out of 230 LUAD sequenced samples.Analysis of somatic copy-number alterations to LINC00987 demonstrated that this lncRNA is located within a focal peak region of deletion (chr12:2782810-17821101) in LUAD (overall frequency of deletion: 0.2578; q-value: 0.0802).In addition, our results indicated that LINC00987 was mutated in 6.06% of LUAD donors, including chr12:g.9392556T>C,chr12: g.9392654T>C, and chr12:g.9393053C>G,according to the LUNG CANCER -KR project.
LINC00987 is downregulated in LUAD Transcriptomic analysis of LINC00987 using the GEPIA web server showed that this lncRNA was significantly downregulated (log 2 (fold change) = −1.225;adjusted p-value = 1.17e −129 ) in LUAD (Figure 1b).Survival analysis indicated that the expression level of LINC00987 is significantly related (log-rank p-value = 0.0023) to the OS of LUAD patients (Figure 1c).

Discussion
The aim of this study was to examine the association of genotranscriptomic alterations of LINC00987 lncRNA with LUAD using a bioinformatic approach.There is an increasing number of publications revealing the key roles of lncRNAs in cancer development and their potential as cancer biomarker.
According to our results, LINC00987 lncRNA is deleted in a significant proportion of the LUAD cases.LINC00987 was mutated in over 6 percent of LUAD patients.Frequent deletion and functional mutations are characteristic of dysregulated tumor suppressor genes in cancer [13][14][15] .LINC00987 lncRNA is underexpressed in LUAD samples compared with normal samples.Furthermore, survival analysis indicated that downregulation of LINC00987 lncRNA is associated with a lower OS period in LUAD patients.These cues, (that is, the under-expression of LINC00987 in cancer and the positive association of its expression level with OS of cancer patients) are characteristic of tumor suppressor genes in cancer.diagnosis and/or prognosis of patients with this cancer type.However, further bioinformatic and experimental studies are required to decipher the precise function of LINC00987 in lung tissue and evaluate its application as a biomarker for LUAD.

2.
3. The authors found that LINC00987 is a new long non-coding RNA which is poorly known in lung adenocarcinoma.Using genomic and transcriptomic databases, the authors discovered that LINC00987 is a tumor suppressive non-coding RNA and its expression levels correlate with patients' survival.This finding is a good clue to discovering potential roles of LINC00987 in clinical diagnosis and prognosis in lung cancer patients.However, additional data are needed to claim that LINC00987 is a tumor suppressor.

My comments are as below.
Could any known lung relevant lncRNAs (such as MALAT1) be applied and validated using the database (GEPIA)?
Could the author reproduce the data shown in Fig 1b and 1c using a different database to demonstrate differential expressions of LINC00987 in lung adenocarcinoma and normal tissues and survival curve?
Could the author validate the differential expression levels for LINC00987 in several lung adenocarcinoma and normal lung epithelial cell lines?Furthermore, could plasmids for LINC00987 be applied to validate the tumor suppressive roles in lung adenocarcinoma cell lines?

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Partly 1.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment.A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: Tumor initiation/progression, cancer metabolism, non-coding RNAs I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Antonio Marco
School of Biological Sciences, University of Essex, Colchester, UK The authors explored major databases of genomic alterations in cancer samples and evaluated a potential role of LINC00987 in Lung Adenocarcinoma.They concluded that their results "demonstrate that LINC00987 expression might have a tumor suppressive role in lung adenocarcinoma".
As an investigation evaluating the relationship between a non-coding gene an a human disease it is an interesting piece of work.However, I believe that the results are not supported and the work lacks a more appropriate statistical framework.
The choice of LINC00987 as a potential gene involved in this cancer is based on a paper by Xu et After screening the paper and the supplementary annotation I haven't found any mention to it.It al. may be due to a change in nomenclature, but then it must be clearly stated.
The deletion of LINC00987 is associated to a deletion of half on an arm of chromosome 12.There are hundreds of genes in the region so any association to LINC00987 must be clearly differentiated.
Likewise, the expression levels can be associated to this deletion.Hundreds of other genes will be 3.

3.
Likewise, the expression levels can be associated to this deletion.Hundreds of other genes will be also underexpressed.This also affects the analysis of Figure 1C.From my point of view, and considering the evidence shown, there is no evidence of a causal connection between LINC00987 and Lung Adenocarcinoma.A convincing analysis should take into account the expected mutation/deletion/expression/survival profile of other genes (expectations) including those in the frequently deleted arm of chromosome 12.

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? No
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: Non-coding RNAs, Gene expression, Evolution, Genomics, Computational Biology I have read this submission.I believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

3.
other genes might affect the overall survival of LUAD patients, we specifically investigated whether the expression level of LINC00987 is associated with the overall survival of LUAD patients or not.This is a common and approved strategy and statistical analysis.Actually, this is a research note, not a comprehensive research paper, which could be an inspiring viewpoint for the conduction of further in-silico and in-vitro assays on the association of LINC00987 and LUAD.
Overall, thank you for your precious time and consideration.
No competing interests were disclosed.

Competing Interests:
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Figure 1 .
Figure 1.Genomic and transcriptomic alterations of LINC00987 long non-coding RNA (lncRNA) in lung adenocarcinoma (LUAD).(a) Copy number alterations of LINC00987 in LUAD according to the The Cancer Genome Atlas data (230 sequenced samples).Red, blue, and gray colors are indicative of amplified (n=10), deleted (n=5), and non-altered (n=215) samples, respectively.Cumulatively, 7% (n=15) of the sequenced samples had copy number alterations.The figure was retrieved from the cBioPortal database.(b) Dysregulation of LINC00987 lncRNA in LUAD.The asterisk indicates the extreme outliers.The figure was created using Boxplot function in Expression DIY of the GEPIA web server.(c) The association of LINC00987 expression level with the overall survival of LUAD patients.The figure was created using the GEPIA web server.
/doi.org/10.5256/f1000research.16089.r36973© 2018 Zhang W. This is an open access peer review report distributed under the terms of the Creative Commons , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution Licence work is properly cited.Wen Cai Zhang Department of Pathology, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA, USA /doi.org/10.5256/f1000research.16089.r34549© 2018 Marco A. This is an open access peer review report distributed under the terms of the Creative Commons , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution Licence work is properly cited.