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Cited by 35Cancer-related molecular mechanisms researchTesticular diseases and treatmentsRNA modifications and cancer

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Integrated analysis of high-throughput sequencing data reveals the key role of LINC00467 in the invasion and metastasis of testicular germ cell tumors

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Bo, Hao, Zhu, Fang, Liu, Zhizhong, Deng, Qi, Liu, Guangmin, Li, Ruixue, Zhu, Wenbing, Tan, Yueqiu, Liu, Gang, Fan, Jingyu, Fan, Liqing·2021·Cell Death Discovery
DOI 10.1038/s41420-021-00588-9PMC PMC8346510
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Abstract

Long noncoding RNAs (lncRNAs) are involved in various physiological and pathological processes. However, the role of lncRNAs in testicular germ cell tumor (TGCT) has been rarely reported. Our purpose is to comprehensively survey the expression and function of lncRNAs in TGCT. In this study, we used RNA sequencing to construct the lncRNA expression profiles of 13 TGCT tissues and 4 paraneoplastic tissues to explore the function of lncRNAs in TGCT. The bioinformatics analysis showed that many lncRNAs are differentially expressed in TGCT. GO and KEGG enrichment analyses revealed that the differentially expressed lncRNAs participated in various biological processes associated with tumorigenesis in cis and trans manners. Further, we found that the expression of LINC00467 was positively correlated with the poor prognosis and pathological grade of TGCT using WGCNA analysis and GEPIA database data mining. In vitro experiments revealed that LNC00467 could promote the migration and invasion of TGCT cells by regulating the expression of AKT3 and influencing total AKT phosphorylation. Further analysis of TCGA data revealed that the expression was negatively correlated with the infiltration of immune cells and the response to PD1 immunotherapy. In summary, this study is the first to construct the expression profile of lncRNAs in TGCT. It is also the first study to identify the metastasis-promoting role of LNC00467, which can be used as a potential predictor of TGCT prognosis and immunotherapeutic response to provide a clinical reference for the treatment and diagnosis of TGCT metastasis.

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Fig. 1 Overview of lncRNA Expression in TGCT Samples. A RNA sequencing flowchart. B Abundance of known lncRNAs to novel lncRNAs (top) and the ratio of known mRNAs to novel mRNAs (bottom). C Type abundance diagrams of known (left) and novel (right) lncRNAs. D Box plots of transcript expression levels in each group. E Length distribution density diagrams of known mRNAs and new mRNAs (pink and blue) and known lncRNAs and new lncRNAs (green and purple). F Distribution of the number of known lncRNAs and novel lncRNAs (top) and the number of known mRNAs and novel mRNAs (bottom) in each group. G Pie charts of the distribution of lncRNA types in the different groups of samples. H Wayne diagrams of known lncRNAs (left) and new lncRNAs (right) in each group.
Fig. 1 Overview of lncRNA Expression in TGCT Samples. A RNA sequencing flowchart. B Abundance of known lncRNAs to novel lncRNAs (top) and the ratio of known mRNAs to novel mRNAs (bottom). C Type abundance diagrams of known (left) and novel (right) lncRNAs. D Box plots of transcript expression levels in each group. E Length distribution density diagrams of known mRNAs and new mRNAs (pink and blue) and known lncRNAs and new lncRNAs (green and purple). F Distribution of the number of known lncRNAs and novel lncRNAs (top) and the number of known mRNAs and novel mRNAs (bottom) in each group. G Pie charts of the distribution of lncRNA types in the different groups of samples. H Wayne diagrams of known lncRNAs (left) and new lncRNAs (right) in each group.CC BY · PubMed Central
Fig. 2 LncRNAs are significantly differentially expressed in TGCT. A Hierarchical clustering plots based on sample lncRNA expression (left) and sample mRNA expression (right). B PCA analysis plots based on sample lncRNA expression (left) and sample mRNA expression (right). C Histogram of DElncRNAs number between groups. D Upset plots of the distribution of upregulated lncRNAs (left) and downregulated lncRNAs (right) in each group. E Heatmap showed the common differentially expressed lncRNAs of seminoma and non-seminoma compared with adjacent tissues. F Verification of the common DElncRNAs in GEPIA database.
Fig. 2 LncRNAs are significantly differentially expressed in TGCT. A Hierarchical clustering plots based on sample lncRNA expression (left) and sample mRNA expression (right). B PCA analysis plots based on sample lncRNA expression (left) and sample mRNA expression (right). C Histogram of DElncRNAs number between groups. D Upset plots of the distribution of upregulated lncRNAs (left) and downregulated lncRNAs (right) in each group. E Heatmap showed the common differentially expressed lncRNAs of seminoma and non-seminoma compared with adjacent tissues. F Verification of the common DElncRNAs in GEPIA database.CC BY · PubMed Central
Fig. 3 Functional enrichment analysis of the common DElncRNAs. A Circos plots show the distribution of differentially expressed lncRNAs on each chromosome. Upregulated lncRNAs are shown in red, while downregulated lncRNAs are shown in blue. B Bubble plots of the transcription factor enrichment analysis of co-differentially expressed lncRNAs. The darker the yellow color, the higher the transcription factor expression, while the bubble size indicates the number of lncRNAs. C Histogram of type distribution of common differentially expressed lncRNAs. D, G Plots showing the results of the GO and KEGG enrichment of the antisense lncRNA target genes. E, H Plots showing the results of the GO and KEGG enrichment of the lncRNA cis-action target genes. F, I Plots showing the results of the GO and KEGG enrichment of the lncRNA trans-action target genes.
Fig. 3 Functional enrichment analysis of the common DElncRNAs. A Circos plots show the distribution of differentially expressed lncRNAs on each chromosome. Upregulated lncRNAs are shown in red, while downregulated lncRNAs are shown in blue. B Bubble plots of the transcription factor enrichment analysis of co-differentially expressed lncRNAs. The darker the yellow color, the higher the transcription factor expression, while the bubble size indicates the number of lncRNAs. C Histogram of type distribution of common differentially expressed lncRNAs. D, G Plots showing the results of the GO and KEGG enrichment of the antisense lncRNA target genes. E, H Plots showing the results of the GO and KEGG enrichment of the lncRNA cis-action target genes. F, I Plots showing the results of the GO and KEGG enrichment of the lncRNA trans-action target genes.CC BY · PubMed Central
Fig. 4 LINC00467 expression is significantly correlated with clinicopathological features, metastasis, and the prognosis of TGCT. A, B WGCNA analysis of the clinical phenotype module correlation using self-tested RNA sequencing data (A) and TGCT data from TCGA database (B). C MMdarkGreen module, Meturquoise module, and Wayne diagram of the differential lncRNAs. D, E Quantitative real-time PCR (D) and GEPIA database TCGA data (E) were used to verify the differential expression of LNC00467 in TGCT. F Expression level of LINC00467 in the TGCT dataset of the GEPIA database was associated with the pathological grade of TGCT. G, H, TGCT data from the Kaplan-Meier Plotter database were used to analyze the correlation between LINC00467 and the prognosis of TGCT patients.
Fig. 4 LINC00467 expression is significantly correlated with clinicopathological features, metastasis, and the prognosis of TGCT. A, B WGCNA analysis of the clinical phenotype module correlation using self-tested RNA sequencing data (A) and TGCT data from TCGA database (B). C MMdarkGreen module, Meturquoise module, and Wayne diagram of the differential lncRNAs. D, E Quantitative real-time PCR (D) and GEPIA database TCGA data (E) were used to verify the differential expression of LNC00467 in TGCT. F Expression level of LINC00467 in the TGCT dataset of the GEPIA database was associated with the pathological grade of TGCT. G, H, TGCT data from the Kaplan-Meier Plotter database were used to analyze the correlation between LINC00467 and the prognosis of TGCT patients.CC BY · PubMed Central
Fig. 5 LINC00467 promotes TGCT cell migration and invasion. A qRT-PCR assay was used to detect LINC00467 overexpression in the cell lines. B qRT-PCR assay was used to detect the silencing efficiency of LINC00467 siRNA in the cell lines. The relative expression of LINC00467 in each group was calculated using β-actin as an internal reference and normalized to 1 in the NC siRNA group. C, E Transwell cell migration assays were used to determine the effects of LINC00467 overexpression (C) and the silencing of LNC00467 (E) on the migration ability of TGCT cells. D, F Transwell cell invasion assays were used to determine the effects of LINC00467 overexpression (D) and the silencing of LNC00467 (F) on the migration ability of TGCT cells. G, H Clone formation assay to detect the effect of LINC00467 overexpression (G) and the silencing of LNC00467 (H) on the colony-formation ability of TGCT cells. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. no significance. All experiments were performed in triplicate. Scale bar = 100 μm.
Fig. 5 LINC00467 promotes TGCT cell migration and invasion. A qRT-PCR assay was used to detect LINC00467 overexpression in the cell lines. B qRT-PCR assay was used to detect the silencing efficiency of LINC00467 siRNA in the cell lines. The relative expression of LINC00467 in each group was calculated using β-actin as an internal reference and normalized to 1 in the NC siRNA group. C, E Transwell cell migration assays were used to determine the effects of LINC00467 overexpression (C) and the silencing of LNC00467 (E) on the migration ability of TGCT cells. D, F Transwell cell invasion assays were used to determine the effects of LINC00467 overexpression (D) and the silencing of LNC00467 (F) on the migration ability of TGCT cells. G, H Clone formation assay to detect the effect of LINC00467 overexpression (G) and the silencing of LNC00467 (H) on the colony-formation ability of TGCT cells. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. no significance. All experiments were performed in triplicate. Scale bar = 100 μm.CC BY · PubMed Central
Fig. 6 LINC00467 regulates AKT3-dependent AKT phosphorylation to promote the migration and invasion abilities of TGCT cells. A KEGG enrichment analysis of the genes co-expressed with LNC00467 in the Meturquoise module is shown in Fig. 4B. B, C Western blotting analysis of the change in the p-AKT level after the overexpression of LNC00467 (B) and LINC00467 silencing (C). D Data from the GEPIA database were used to determine the correlation between LINC00467 expression and AKT3 expression. E qRT-PCR and western blotting were used to determine the mRNA and protein levels of AKT3 after LINC00467 silencing. F Transwell cell migration and invasion assays were used to determine the effect of silencing on the migration and invasion abilities of TGCT cells. G Transwell cell migration and invasion assays were used to detect the effect of LINC00467 overexpression and simultaneous silencing of AKT3 on the migration and invasion abilities of TGCT cells. **P < 0.01; ***P < 0.001. All assays were performed in triplicate. Scale bar = 100 μm.
Fig. 6 LINC00467 regulates AKT3-dependent AKT phosphorylation to promote the migration and invasion abilities of TGCT cells. A KEGG enrichment analysis of the genes co-expressed with LNC00467 in the Meturquoise module is shown in Fig. 4B. B, C Western blotting analysis of the change in the p-AKT level after the overexpression of LNC00467 (B) and LINC00467 silencing (C). D Data from the GEPIA database were used to determine the correlation between LINC00467 expression and AKT3 expression. E qRT-PCR and western blotting were used to determine the mRNA and protein levels of AKT3 after LINC00467 silencing. F Transwell cell migration and invasion assays were used to determine the effect of silencing on the migration and invasion abilities of TGCT cells. G Transwell cell migration and invasion assays were used to detect the effect of LINC00467 overexpression and simultaneous silencing of AKT3 on the migration and invasion abilities of TGCT cells. **P < 0.01; ***P < 0.001. All assays were performed in triplicate. Scale bar = 100 μm.CC BY · PubMed Central
Fig. 7 LINC00467 is negatively correlated with TGCT immune infiltration. A Correlation of LNC0040067 expression with estimate score, immune score, and stromal score. B Correlation between LNC00467 expression and the degree of immune cell infiltration. C Correlation between LNC00467 expression and immune checkpoint gene expression. D Expression of LNC00467 in samples from the response and non-response to immunotherapy groups. R: response, NS: non-response.
Fig. 7 LINC00467 is negatively correlated with TGCT immune infiltration. A Correlation of LNC0040067 expression with estimate score, immune score, and stromal score. B Correlation between LNC00467 expression and the degree of immune cell infiltration. C Correlation between LNC00467 expression and immune checkpoint gene expression. D Expression of LNC00467 in samples from the response and non-response to immunotherapy groups. R: response, NS: non-response.CC BY · PubMed Central

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Citations

Cites

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Cited by

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Metadata

Authors
Bo, H., Zhu, F., Liu, Z., Deng, Q., Liu, G., Li, R., Zhu, W., Tan, Y., Liu, G., Fan, J., & Fan, L.
Affiliations
Central South University (CN)
Corresponding author
Fan, Liqing
ORCID
Venue
Cell Death Discovery
Publisher
Springer Science and Business Media LLC
Published
2021-08-06
Volume
7
Issue
1
Pages
206-206
ISSN
2058-7716 (ISSN-L)
DOI
10.1038/s41420-021-00588-9
PMC
PMC8346510
OpenAlex
W3188659136
Cited by
35
References
1
Citations by year
2026: 3 · 2025: 4 · 2024: 5 · 2023: 9 · 2022: 13 · 2021: 1
FWCI
2.78
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death Discovery (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • the National Key Research and Development Program of China
  • the Special Fund of Clinical Medicine of the Chinese Medical Association
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
  • Health Sciences › Medicine › Surgery › Testicular diseases and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA modifications and cancer
Keywords
long non-coding RNA, testicular germ cell tumors, tumor invasion, metastasis, AKT3, Akt phosphorylation, cell migration, lncRNA expression profiling, prognosis, immune cell infiltration, tumor suppressor lncRNA

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