Long non-coding RNA expression profiles predict clinical phenotypes of seminoma and yolk sac tumor

Abstract

Malignant germ cell tumors (GCTs) such as seminoma and yolk sac tumor cause serious health problems but with favorable prognosis if they were diagnosed timely. To investigate potential biomarkers used for GCTs diagnosis and phenotype distinguishment, we first applied a lncRNA classification pipeline to identify 368 lncRNAs represented on the Affymetrix Human Genome U133A Array. We then comprehensively analyzed the lncRNA expression patterns in a set of previously published gene expression profiles of seminoma and yolk sac tumor stratified by different age groups (children and adults). The lncRNAs expression signatures between children and adults in different GCTs phenotypic groups were identified respectively, five aberrantly expressed lncRNAs were shared by children and adults, indicating a role for them in distinguishing seminoma from yolk sac tumor regardless of age. In parallel, nine distinctive lncRNAs were also determined between seminoma and yolk sac tumor, which suggested that people may face a high risk of suffering from GCTs. Our findings may contribute to the early diagnosis and prognosis of GCTs regardless of patients' age and other diseases.

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Introduction

In recent years, cancer is emerging as a major public health problem worldwide. According to the statistical data released by the American Cancer Society and the National Cancer Center of China, cancer has become the second leading cause of death in both countries.^1,2 Over the past decades, the number of patients dying from cancer has substantially dropped, whereas cancer death numbers related to the brain and central nervous system (CNS) are increasing, for now, they have surpassed leukemia becoming the leading cause of cancer deaths in children and adolescents (aged birth to 19 years old).¹ Moreover, the 5 year survival rate (%) of brain and CNS cancers has risen from 57% to 74% among patients who were less than 20 years old, whereas the overall survival rate regardless of patient age was still under 35% and showed a decreasing trend.³

Germ cell tumors (GCTs), one phenotype of the brain and CNS cancer, accounted for 11.8% of pediatric tumors in China,⁴ and the malignant GCTs accounted for 2.9% of all malignant tumors in children who were younger than 15 years old worldwide.⁵ In general, GCTs are characterized by a high heterogeneity of their histological differentiation, but they show a similar histological pattern independent of their primary site or sex.⁶ As indicated by Teilum in 1965, the neoplastic cell that derived from gonadal or extragonadal germ cell was able to trans-differentiate into embryonal and exo-embryonal malignant carcinoma.⁷ The former includes mature/immature teratoma in embryo and choriocarcinoma (CHC) and yolk sac tumor (yolk sac tumor) outside the embryo. Meanwhile, the exo-embryonal carcinoma such as seminoma (testis), dysgerminoma (ovary) and germinoma (brain) are all malignant tumors (Fig. 1).

Fig. 1 Classification and development of germ cell tumors. Malignant tumors are labeled with pentagram.

In recent years, biomarkers including al-fetoprotein (AFP) and human choriogonadotropin (HCG) have been used for diagnosis of yolk sac tumor and CHC, and a moderate elevation of β-HCG was considered to occur in seminoma. Despite great progress achieved in the early diagnosis and distinguishment of different clinical phenotypes of GCTs, a great amount of misdiagnosis still occurred every year. For example, the reference value of HCG used to diagnosis seminoma/germinoma (<50 IU L⁻¹) was similar to syncytiotrophoblast-like giant cells.⁸ Additionally, in neonates and young infants, the AFP was born with a physiologically elevated level, but children older than two years old with a high AFP level (≥100 μg L⁻¹) can be considered as malignant GCTs.⁹ Nevertheless, in some liver diseases such as acute liver failure, hepatocellular carcinoma, and hepatoblastoma, the APF secretion is also elevated due to hepatocellular regeneration.¹⁰ Thus, it may lead to incorrect judgment to make clinical decisions only depending on images or molecular biomarkers, and it would be of great significance to find out more stable and accurate biomarkers that were used to diagnosis and distinguish different clinical phenotypes of GCTs regardless of patient age and the disturbance from other diseases.

The emerging role of long non-coding RNA (lncRNA) as promising biomarker and critical therapeutic target has drawn considerable attentions. However, the role of lncRNA in GCTs has not been investigated. Typically, lncRNAs are non-protein coding transcripts longer than 200 nucleotides which were involved in numerous critical biological processes such as X chromosome silencing, genomic imprinting, chromosome modification, transcriptional activation, transcriptional interference, and nuclear transport.¹¹ HOTAIR, for example, a well-studied lncRNA, was found aberrantly expressed in different subtypes of breast cancer, which highlighted the role of lncRNA in distinguishing breast cancer from different subtypes for the first time.¹² In glioma and colorectal cancer, lncRNAs such as HOXA-AS, MALAT1, and NEAT1 were all found to be specifically distributed.^13,14 Hence it may be a new way to distinguish malignant GCTs from embryonal to exo-embryonal via lncRNA profiling. Favorably, microarray datasets shared by previous studies can be achieved from the Gene Expression Omnibus (GEO) and used to investigate our hypothesis.

Herein, we aimed at profiling lncRNA expression signatures in embryonal malignant carcinoma (yolk sac tumor) and exo-embryonal malignant carcinoma (seminoma) by analyzing a cohort of previously published microarray datasets that achieved from the GEO. The distinctive lncRNAs were identified through comparison between groups of different age and GCTs phenotypes respectively. Our findings provide novel information on lncRNA expression profiles that may help to distinguish GCTs from different phenotypes regardless of the limitation of age and disturbance from other diseases, and the results also provided potential diagnostic biomarkers and therapeutic targets for yolk sac tumor and seminoma.

Materials and methods

GEO seminoma and yolk sac tumor expression data

All experiments were performed in compliance with the guidelines approved by the ethics committee at the Memorial Sloan-Kettering Cancer Center (New York, NY) between 1987 and 1999. Informed consents were obtained from human participants of this study. The microarray datasets of seminoma and yolk sac tumor related to children and adult were obtained from the GEO. To compare the lncRNA expression signatures according to patient of different age and GCT phenotypes, two panels of adult and pediatric GCT gene expression datasets were included in this study: GSE3218 and GSE10615. The raw files of these two datasets which were based on the platform of Affymetrix Human Genome U133A Array were downloaded from the GEO, the data quality control process including quartile normalization, background adjustment, and summarization was processed using the Robust Multichip Average software (RMA, 1.2.0 In Development), which has been proved to be more efficient in estimating lncRNA expression fold changes than other software. Also, samples with a median expression value that exceeded the control limit line in plots of normalized unscaled standard error (NUSE) and relative log expression (RLE) were excluded from the downstream analysis. With this, a set of probe ID-centric gene expression values was obtained.

lncRNA classification pipeline

To evaluate lncRNA expressions in the microarray datasets that were obtained from the above step, we adopted the lncRNA classification pipeline which had been previously described to identify lncRNAs represented on the Affymetrix Genome array.¹⁵ In brief, we first mapped the ID-centric gene expression matrix to the NetAffx Annotation File (HG-U133A Annotations, CSV format, Release 35, 7 MB, 10/7/2014), which was available on the Affymetrix official website (http://www.affymetrix.com). Next, we only retained probes that labeled as “NR_” in the column of RefSeq transcripts IDs. While in the Ensembl gene IDs column, we selected probes that labeled as “lincRNA,” “processed_transcript,” “macro_lncRNA” or “misc_RNA.” Lastly, we filtered the extracted annotated lncRNAs to exclude pseudogenes, rRNAs, microRNAs or other short RNAs (tRNAs, snRNAs, and snoRNAs).

Differentially expressed lncRNAs screening

Gene-e software was used to determine the differentially expressed lncRNAs between seminoma and yolk sac tumor in adult and children. Similarly, the distinctive lncRNAs between adult and children stratified by GCTs phenotypes (seminoma or yolk sac tumor) were also investigated. Conditions used to screen the differentially expressed lncRNAs were set as follows: false discovery rate (FDR) < 20%, fold change ≥ 2, permutation time 1000 and p-value < 0.01. The co-existed differentially expressed lncRNAs between seminoma and yolk sac tumor or adult and children were intersected using Venn diagram. In order to investigate the effectiveness of the pack mode, the principal component analysis (PCA) was adopted using MeV (available at http://mev.tm4.org).

Validation of differentially expressed lncRNAs

The Oncomine database which was hosted by Thermo Fisher Scientific Inc. provided more than 715 datasets and 86 733 samples with expertly curated data. Thus we took advantage of this database to validate the expression of lncRNAs shared by adults and children, the comparison mode was selected as cancer vs. normal after uploading lncRNAs, and values of fold change, t test, and p statistics were recorded and used for further analysis.

Statistical analysis

All statistical analyses were processed using SAS version 9.2 for windows (SAS Institute Inc., Cary, North Carolina, USA). Differentially expressed lncRNAs were investigated using Gene-e software, PCA analysis was adopted using MeV online version. The age of children and adult with normal distribution were shown as mean ± standard deviation (SD). A p value less than 0.05 was considered as statistically significant unless otherwise specified.

Results

Datasets characteristics

The gene expression data of pediatric and adult seminoma and yolk sac tumors were included in this study: GES10615 and GSE3218A. The gene chip GSE10615 contained 28 pediatric samples, among which 18 samples were malignant yolk sac tumors, while 10 samples were malignant seminomas, including 1 seminoma sample which was excluded after quality control (Fig. 2 and 3). In parallel, a total number of 21 adult samples including 9 yolk sac tumors and 12 seminomas were included in GES3218A. Each sample involved in this study was purely one phenotype of tumor specified, not mixed with others. The age of the children represented by GSE10615 was 8.3 ± 5.6 years old, while the related indicator was not obtained in the adult group.

Fig. 2 Density plots of log₂ PM by array. Pediatric samples were draw in blue, adult samples were shown in yellow, the abnormal sample which was excluded from the downstream analysis was shown in red.

Fig. 3 NUSE and RLE median plots by array. The median of the normalized unscaled standard error (NUSE) and relative log expression (RLE) of every sample were evaluated in the expression quality control process, samples that exceeded the control limits (blue) were considered as low quality and would be excluded from the downstream analysis.

lncRNA expression profiles on Affymetrix Human Genome U133A Array

With the lncRNA classification pipeline, 398 probe sets corresponding to 368 lncRNA genes were identified. Of these, 49 probe sets (40 genes) were annotated as lncRNAs by both RefSeq and Ensembl database, 267 probe sets (216 genes) were annotated by RefSeq database, and 180 probe sets (192 genes) were annotated by Ensembl database. In addition, probe sets that were annotated by both databases but had controversial definitions were excluded from this study (Tables S1 and S2†).

Distinctive lncRNA expressions between seminoma and yolk sac tumor

We compared the lncRNA expression patterns between seminoma and yolk sac tumor stratified by age. A total number of 13 probe sets corresponding to 11 lncRNA genes were identified in adults. Meanwhile, 13 probe sets corresponding to 12 genes were found aberrantly expressed in children, and 6 probe sets (5 genes) including XIST, C17orf86, FAM182A, FLJ11235, and C12orf47 were shared by children and adult. Moreover, 19 probe sets corresponding to 16 lncRNA genes were identified between seminoma and yolk sac tumor when the effect of age was excluded (Table 1). Interestingly, five lncRNAs shared by adult and children were all involved in this cluster, and there were 10 differentially expressed lncRNAs that were overlapped in adult and pediatric lncRNA clusters. The PCA results of seminoma and yolk sac tumor were shown in Fig. 4, the cumulative component percentage reached up to 90% when the component number was set as 3. However, it was much higher (more than 90%) when the component number was set as 2 regardless of age, and dots with different colors of the same disease tightly clustered together.

Table 1 Differentially expressed lncRNAs between seminoma and yolk sac tumor

Probesets	RefSeq transcript ID	Ensembl gene ID	Gene symbol	Regulation^a	Gene title
a Compared with seminoma.
Adult
205677_s_at	NR_002605	ENSG00000273541	DLEU1	Down	Deleted in lymphocytic leukemia 1
221728_x_at	NR_001564	ENSG00000274655	XIST	Down	X (inactive)-specific transcript
214218_s_at	NR_001564	ENSG00000274655	XIST	Down	X (inactive)-specific transcript
207698_at	NR_026773	ENSG00000146521	C6orf123	Down	Chromosome 6 open reading frame 123
222001_x_at	NR_024510	ENSG00000277147	LOC728855	Up	Hypothetical LOC728855
205677_s_at	NR_002605	ENSG00000176124	DLEU1	Down	Deleted in lymphocytic leukemia 1
221621_at	NR_027058	ENSG00000234912	C17orf86	Down	Chromosome 17 open reading frame 86
220904_at	NR_026780	ENSG00000231690	C6orf208	Up	Chromosome 6 open reading frame 208
222001_x_at	NR_024510	ENSG00000226067	LOC728855	Down	Hypothetical LOC728855
220505_at	NR_024274	ENSG00000224854	C9orf53	Down	Chromosome 9 open reading frame 53
220904_at	NR_026780	ENSG00000281305	C6orf208	Down	Chromosome 6 open reading frame 208
214218_s_at	NR_001564	ENSG00000229807	XIST	Down	X (inactive)-specific transcript
221728_x_at	NR_001564	ENSG00000229807	XIST	Down	X (inactive)-specific transcript
216053_x_at	NR_026713	ENSG00000125804	FAM182A	Down	Family with sequence similarity 182
216053_x_at	NR_026713	ENSG00000175170	FAM182A	Down	Family with sequence similarity 182
220364_at	NR_027706	ENSG00000278921	FLJ11235	Down	Hypothetical FLJ11235
215283_at	NR_015389	ENSG00000263753	LOC339290	Up	Hypothetical LOC339290
217506_at	NR_015389	ENSG00000263753	LOC339290	Up	Hypothetical LOC339290
219817_at	NR_015404	ENSG00000234608	C12orf47	Down	Chromosome 12 open reading frame 47
Child
221728_x_at	NR_001564	ENSG00000274655	XIST	Down	X (inactive)-specific transcript
214218_s_at	NR_001564	ENSG00000274655	XIST	Down	X (inactive)-specific transcript
206478_at	NR_026800	ENSG00000226777	KIAA0125	Down	KIAA0125
221621_at	NR_027058	ENSG00000234912	C17orf86	Down	Chromosome 17 open reading frame 86
206819_at	NR_003714	ENSG00000197210	POM121L9P	Down	POM121 membrane glycoprotein-like 9
209917_s_at	NR_015381	ENSG00000182165	TP53TG1	Down	TP53 target 1
206819_at	NR_003714	ENSG00000161103	POM121L9P	Down	POM121 membrane glycoprotein-like 9
214218_s_at	NR_001564	ENSG00000229807	XIST	Down	X (inactive)-specific transcript
221728_x_at	NR_001564	ENSG00000229807	XIST	Down	X (inactive)-specific transcript
216053_x_at	NR_026713	ENSG00000125804	FAM182A	Down	Family with sequence similarity 182
220399_at	NR_024321	ENSG00000225880	NCRNA00115	Down	Non-protein coding RNA 115
216053_x_at	NR_026713	ENSG00000175170	FAM182A	Down	Family with sequence similarity 182
214839_at	NR_024281	ENSG00000253230	LOC157627	Down	Hypothetical LOC157627
206819_at	NR_003714	ENSG00000128262	POM121L9P	Down	POM121 membrane glycoprotein-like 9
206478_at	NR_026800	ENSG00000277059	KIAA0125	Down	KIAA0125
220364_at	NR_027706	ENSG00000278921	FLJ11235	Down	Hypothetical FLJ11235
219817_at	NR_015404	ENSG00000234608	C12orf47	Down	Chromosome 12 open reading frame 47
220399_at	NR_024321	ENSG00000272812	NCRNA00115	Down	Non-protein coding RNA 115
219442_at	NR_024034	ENSG00000276867	C16orf67	Up	Chromosome 16 open reading frame 67
219442_at	NR_024034	ENSG00000131797	C16orf67	Up	Chromosome 16 open reading frame 67
214983_at	NR_001545	ENSG00000233864	TTTY15	Up	Testis-specific transcript, Y-linked 15

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Fig. 4 Distinctive lncRNAs between seminoma and yolk sac tumor. Differentially expressed lncRNAs between seminoma and yolk sac tumor stratified by different age groups were investigated (adult: (A), children: (B)), the lncRNAs that were relatively high expressed were shown in red, the lower expressed ones were shown in green. (C) was a Venn diagram representing for the lncRNAs shared by adult and children. The results of principal component analysis were shown in (D–F). The percentage of every component was shown in (D), the lower control limit (10%) for the principal component analysis were dotted in yellow, and two classes of samples corresponding to the dots of different colors in the 3D images were clustered ((E) adult, (F) children, seminoma: cyan, yolk sac tumor: red).

Distinctive lncRNA expressions between children and adult

We also compared the lncRNA expression patterns between children and adult stratified by seminoma and yolk sac tumor. In seminoma, 16 probe sets corresponding to 14 lncRNAs were identified, while in yolk sac tumor, 17 probe sets corresponding to 16 lncRNAs were shown to be aberrantly expressed (Table 2). A total number of 9 lncRNAs such as PART1, NCRNA00230A, POM121L9P, MEG3, TP53TG1, LOC157627, LOC339290, DKFZP434L187, and TTTY15 were shared by seminoma and yolk sac tumor (Table 3). The PCA results of children and adult were shown in Fig. 5 with a validity ≥90%, and 2 components were clustered, dots of the same age group were clustered together.

Table 2 Differentially expressed lncRNAs between children and adult

Probesets	RefSeq transcript ID	Ensembl gene ID	Gene symbol	Regulation^a	Gene title
a Compared with children.
Seminoma
221728_x_at	NR_001564	ENSG00000274655	XIST	Down	X (inactive)-specific transcript
214218_s_at	NR_001564	ENSG00000274655	XIST	Down	X (inactive)-specific transcript
205833_s_at	NR_024617	ENSG00000152931	PART1	Down	Prostate androgen-regulated transcript 1
205833_s_at	NR_024617	ENSG00000273701	PART1	Down	Prostate androgen-regulated transcript 1
205833_s_at	NR_024617	ENSG00000275634	PART1	Down	Prostate androgen-regulated transcript 1
216786_at	NR_002161	ENSG00000233522	NCRNA00230A	Down	Non-protein coding RNA 230A
207161_at	NR_022006	ENSG00000122548	KIAA0087	Down	KIAA0087
206819_at	NR_003714	ENSG00000197210	POM121L9P	Down	POM121 membrane glycoprotein-like 9
210794_s_at	NR_002766	ENSG00000214548	MEG3	Down	Maternally expressed 3
212732_at	NR_002766	ENSG00000214548	MEG3	Down	Maternally expressed 3
216722_at	NR_001559	ENSG00000230265	VENTXP1	Down	VENT homeobox (Xenopus laevis) 1
210241_s_at	NR_015381	ENSG00000182165	TP53TG1	Down	TP53 target 1
206819_at	NR_003714	ENSG00000161103	POM121L9P	Down	POM121 membrane glycoprotein-like 9
207259_at	NR_024626	ENSG00000167117	C17orf73	Down	Chromosome 17 open reading frame 73
214218_s_at	NR_001564	ENSG00000229807	XIST	Down	X (inactive)-specific transcript
221728_x_at	NR_001564	ENSG00000229807	XIST	Down	X (inactive)-specific transcript
220399_at	NR_024321	ENSG00000225880	NCRNA00115	Down	Non-protein coding RNA 115
214839_at	NR_024281	ENSG00000253230	LOC157627	Down	Hypothetical LOC157627
206819_at	NR_003714	ENSG00000128262	POM121L9P	Down	POM121 membrane glycoprotein-like 9
216786_at	NR_002161	ENSG00000230663	NCRNA00230A	Down	Non-protein coding RNA 230A
217506_at	NR_015389	ENSG00000263753	LOC339290	Down	Hypothetical LOC339290
216722_at	NR_001559	ENSG00000259849	VENTXP1	Down	VENT homeobox (Xenopus laevis) 1
216596_at	NR_026771	ENSG00000225930	DKFZP434L187	Down	Hypothetical LOC26082
220399_at	NR_024321	ENSG00000272812	NCRNA00115	Down	Non-protein coding RNA 115
214983_at	NR_001545	ENSG00000233864	TTTY15	Up	Testis-specific transcript, Y-linked 15
216596_at	NR_026771	ENSG00000282096	DKFZP434L187	Down	Hypothetical LOC26082
Yolk sac
206478_at	NR_026800	ENSG00000226777	KIAA0125	Up	KIAA0125
215972_at	NR_024617	ENSG00000152931	PART1	Down	Prostate androgen-regulated transcript 1
222001_x_at	NR_024510	ENSG00000277147	LOC728855	Up	Hypothetical LOC728855
221129_at	NR_026770	ENSG00000267496	C17orf88	Down	Chromosome 17 open reading frame 88
222021_x_at	NR_003264	ENSG00000281237	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000242086	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000281794	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000215837	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000280912	SDHAP1	Down	Succinate dehydrogenase complex
216786_at	NR_002161	ENSG00000233522	NCRNA00230A	Down	Non-protein coding RNA 230A
206819_at	NR_003714	ENSG00000197210	POM121L9P	Down	POM121 membrane glycoprotein-like 9
210794_s_at	NR_002766	ENSG00000214548	MEG3	Down	Maternally expressed 3
212732_at	NR_002766	ENSG00000214548	MEG3	Down	Maternally expressed 3
222021_x_at	NR_003264	ENSG00000280512	SDHAP1	Down	Succinate dehydrogenase complex
222001_x_at	NR_024510	ENSG00000226067	LOC728855	Up	Hypothetical LOC728855
222021_x_at	NR_003264	ENSG00000281687	SDHAP1	Down	Succinate dehydrogenase complex
210241_s_at	NR_015381	ENSG00000182165	TP53TG1	Down	TP53 target 1
206819_at	NR_003714	ENSG00000161103	POM121L9P	Down	POM121 membrane glycoprotein-like 9
222021_x_at	NR_003264	ENSG00000281334	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000073578	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000282953	SDHAP1	Down	Succinate dehydrogenase complex
216053_x_at	NR_026713	ENSG00000125804	FAM182A	Down	Family with sequence similarity 182
222021_x_at	NR_003264	ENSG00000281915	SDHAP1	Down	Succinate dehydrogenase complex
216053_x_at	NR_026713	ENSG00000175170	FAM182A	Down	Family with sequence similarity 182
214839_at	NR_024281	ENSG00000253230	LOC157627	Down	Hypothetical LOC157627
206819_at	NR_003714	ENSG00000128262	POM121L9P	Down	POM121 membrane glycoprotein-like 9
216786_at	NR_002161	ENSG00000230663	NCRNA00230A	Down	Non-protein coding RNA 230A
206478_at	NR_026800	ENSG00000277059	KIAA0125	Up	KIAA0125
222021_x_at	NR_003264	ENSG00000280521	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000280993	SDHAP1	Down	Succinate dehydrogenase complex
215283_at	NR_015389	ENSG00000263753	LOC339290	Up	Hypothetical LOC339290
222021_x_at	NR_003264	ENSG00000280909	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000281060	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000281603	SDHAP1	Down	Succinate dehydrogenase complex
222021_x_at	NR_003264	ENSG00000281418	SDHAP1	Down	Succinate dehydrogenase complex
216596_at	NR_026771	ENSG00000225930	DKFZP434L187	Down	Hypothetical LOC26082
64432_at	NR_015404	ENSG00000234608	C12orf47	Down	Chromosome 12 open reading frame 47
220324_at	NR_026807	ENSG00000233237	C6orf155	Down	Chromosome 6 open reading frame 155
222021_x_at	NR_003264	ENSG00000185485	SDHAP1	Down	Succinate dehydrogenase complex
214983_at	NR_001545	ENSG00000233864	TTTY15	Up	Testis-specific transcript, Y-linked 15
216596_at	NR_026771	ENSG00000282096	DKFZP434L187	Down	Hypothetical LOC26082

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Table 3 Differentially expressed lncRNAs between seminoma and yolk sac tumor despite of age

Probesets	RefSeq transcript ID	Ensembl gene ID	Gene symbol	Regulation^a	Gene title
a Compared with seminoma.
205677_s_at	NR_002605	ENSG00000273541	DLEU1	Down	Deleted in lymphocytic leukemia 1
221728_x_at	NR_001564	ENSG00000274655	XIST	Down	X (inactive)-specific transcript
214218_s_at	NR_001564	ENSG00000274655	XIST	Down	X (inactive)-specific transcript
206478_at	NR_026800	ENSG00000226777	KIAA0125	Down	KIAA0125
205834_s_at	NR_024617	ENSG00000152931	PART1	Down	Prostate androgen-regulated transcript 1
207698_at	NR_026773	ENSG00000146521	C6orf123	Down	Chromosome 6 open reading frame 123
222001_x_at	NR_024510	ENSG00000277147	LOC728855	Up	Hypothetical LOC728855
205677_s_at	NR_002605	ENSG00000176124	DLEU1	Down	Deleted in lymphocytic leukemia 1
205834_s_at	NR_024617	ENSG00000275634	PART1	Down	Prostate androgen-regulated transcript 1
221621_at	NR_027058	ENSG00000234912	C17orf86	Down	Chromosome 17 open reading frame 86
206819_at	NR_003714	ENSG00000197210	POM121L9P	Down	POM121 membrane glycoprotein-like 9
220904_at	NR_026780	ENSG00000231690	C6orf208	Up	Chromosome 6 open reading frame 208
222001_x_at	NR_024510	ENSG00000226067	LOC728855	Up	Hypothetical LOC728855
210886_x_at	NR_015381	ENSG00000182165	TP53TG1	Down	TP53 target 1
209917_s_at	NR_015381	ENSG00000182165	TP53TG1	Down	TP53 target 1
220904_at	NR_026780	ENSG00000281305	C6orf208	Up	Chromosome 6 open reading frame 208
206819_at	NR_003714	ENSG00000161103	POM121L9P	Down	POM121 membrane glycoprotein-like 9
214218_s_at	NR_001564	ENSG00000229807	XIST	Down	X (inactive)-specific transcript
221728_x_at	NR_001564	ENSG00000229807	XIST	Down	X (inactive)-specific transcript
216053_x_at	NR_026713	ENSG00000125804	FAM182A	Down	Family with sequence similarity 182
220399_at	NR_024321	ENSG00000225880	NCRNA00115	Down	Non-protein coding RNA 115
216053_x_at	NR_026713	ENSG00000175170	FAM182A	Down	Family with sequence similarity 182
206819_at	NR_003714	ENSG00000128262	POM121L9P	Down	POM121 membrane glycoprotein-like 9
206478_at	NR_026800	ENSG00000277059	KIAA0125	Down	KIAA0125
220364_at	NR_027706	ENSG00000278921	FLJ11235	Down	Hypothetical FLJ11235
215283_at	NR_015389	ENSG00000263753	LOC339290	Up	Hypothetical LOC339290
217506_at	NR_015389	ENSG00000263753	LOC339290	Up	Hypothetical LOC339290
219817_at	NR_015404	ENSG00000234608	C12orf47	Down	Chromosome 12 open reading frame 47
220399_at	NR_024321	ENSG00000272812	NCRNA00115	Down	Non-protein coding RNA 115
219442_at	NR_024034	ENSG00000276867	C16orf67	Up	Chromosome 16 open reading frame 67
219442_at	NR_024034	ENSG00000131797	C16orf67	Up	Chromosome 16 open reading frame 67

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Fig. 5 Distinctive lncRNAs between adult and children. The distinctive lncRNAs between adult and children stratified by different clinical phenotypes of malignant germ cell tumors were identified ((A) seminoma, (B) yolk sac tumor), the lncRNAs that were relatively high expressed were shown in red, the lower expressed ones were shown in green. The overlapped lncRNAs between seminoma and yolk sac tumor were described with the shadow region in the Venn diagram (C). The results of principal component analysis were shown in (D–F). The percentage of every component was shown in (D), the lower control limit (10%) for the principal component analysis were dotted in yellow, and two classes of the samples corresponding to the dots of different colors in 3D images were clustered ((E) seminoma, (F) yolk sac tumor, adult: green, children: yellow).

Validation of differentially expressed lncRNAs

The expression levels of lncRNAs shared by yolk sac tumor and seminoma were investigated using Oncomine database and compared between yolk sac tumor and seminoma, yolk sac tumor or seminoma and normal respectively. Apart from lncRNA FAM182A and FLJ11235 which cannot be found in Oncomine database, the expressions of lncRNA XIST and C17orf86 were all of statistical significance between cancer and normal except for C17orf86 in seminoma (Table 4), which were highly consistent with our assumptions.

Table 4 Comparison of lncRNA expressions between cancer and normal

lncRNA	Seminoma			Yolk sac tumor
	Fold change	t value	p value	Fold change	t value	p value
XIST	12.12	8.34	9.28 × 10^−7	2.19	2.03	0.04
C17orf86	1.23	2.90	0.01	1.00	0.02	0.49

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Discussion

To date, different phenotypes of malignant GCTs were determined mainly depending on histological changes, but it may be misdiagnosed when facing patients of different ages or suffering from other diseases. As epidemiological and clinical evidence indicated, the histological changes of malignant GCTs among different clinical phenotypes were partly overlapped,^16,17 thus biomarkers that were currently used also led to misunderstandings under certain conditions. Therefore, the emerging role of lncRNAs as potential diagnostic biomarkers maybe shine insight on the antidiastole on GCTs phenotypes.

In this study, two groups of patients (children and adult) which were stratified by two phenotypes of malignant GCTs (seminoma and yolk sac tumor) were involved. With the lncRNA classification pipeline, we firstly analyzed the differentially expressed lncRNAs between seminoma and yolk sac tumor. A set of 11 aberrantly expressed lncRNAs were identified in adult and 12 lncRNAs were determined in children. Five distinctive lncRNAs including XIST, C17orf86, FAM182A, FLJ11235, and C12orf47 were involved in the intersection of children and adult, indicating a potential role of these lncRNAs in distinguishing seminoma from yolk sac tumor regardless of age. As reported previously, XIST expressions were widely detected in seminomatous testicular germ cell tumors, and the presence of the unmethylated XIST were frequent in testicular germ cell tumors.¹⁸ To our best knowledge, the role of the other four lncRNAs in malignant GCTs has not been investigated. However, the lncRNA C17orf86 that was also known as SNHG20 was associated with the metastasis of hepatocellular carcinoma, and the elevated expression level of SNHG20 could promote carcinoma cellular invasion.^19,20 Besides, the function of the left three lncRNAs including FAM182A, FLJ11235, and C12orf47 has not been explored even in other diseases.

We also processed comparisons between children and adult, which were stratified by two phenotypes of GCTs (seminoma and yolk sac tumor). In total, 14 expressed lncRNAs were identified in seminoma and 16 lncRNAs were determined in yolk sac tumor. Of these, 9 lncRNAs including PART1, NCRNA00230A, POM121L9P, MEG3, TP53TG1, LOC157627, LOC339290, DKFZP434L187, and TTTY15 were overlapped between seminoma and yolk sac tumor, which suggested that people with one or a certain number of the overlapped lncRNAs may be facing high risk in suffering from seminoma or yolk sac tumor. Coincident with previous studies, MEG3, which has been widely found in many cancers, also regulated the growth of testicular germ cell tumor through PTEN/PI3K/AKT pathway.²¹ And the lncRNA POM121L9P was pointed out to be associated with male sterility via binding to Piwi proteins in mammalian.²² Another lncRNA, PART1, is a novel human prostate-specific and androgen-regulated gene that loci in chromosome 5q12, the available studies have proved that the expression level of this lncRNA was elevated by approximate 73.1% detected using specimens of stage I–III non-small cell lung cancer.²³ Moreover, TP53TG1 was an important regulator of cellular homeostasis, which could undergo cancer-specific promoter hypermethylation-associated silencing and inhibit the occurrence and development of cancer.²⁴ Another lncRNA, TTTY15, was highly cited in prostate cancer, the fusion action mode of this gene with USP9Y was identified in a large cohort study of prostate cancer.^25–27 The function of the left lncRNAs is still lack of annotations.

To further explore identical biomarkers that were unaffected by age and GCTs phenotypes, we collaborated lncRNAs shared by groups of different ages or GCTs phenotypes, but none lncRNA fed back when we loaded these two datasets into the statistical software to compare for the overlapped region. However, in the validation process conducted by investigating Oncomine database, we found lncRNA XIST was differentially expressed between cancer and normal, indicating a lncRNA screening criteria of high strict and accurate hold by this study.

In summary, five differentially expressed lncRNAs shared by adult and child were identified in comparison between seminoma and yolk sac tumor, while nine lncRNAs shared by seminoma and yolk sac tumor were determined in comparison between adult and child. The lncRNAs identified in this study may be of great potential in distinguishing GCTs of different phenotypes (seminoma and yolk sac tumor), and they can also be used as promising biomarkers in indicating risk levels from which patients of seminoma or yolk sac tumor may suffer regardless of age. Although some of the lncRNAs had been validated, the majority of them have not been investigated, further studies are still needed.

Conflicts of interest

There are no conflicts of interests.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (No. 81273039, 81472954 and 81773404). We thank those who helped us a lot in School of Public Health, Zhengzhou University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra12131h

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