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Profiling and Bioinformatics Analyses of Hypoxia-Induced Differential Expression of Long Non-coding RNA in Glioblastoma Multiforme Cells

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Abstract

Hypoxic microenvironments are intricately linked to malignant characteristics of glioblastoma multiforme (GBM). Long non-coding ribonucleic acids (lncRNAs) have been reported to be involved in the progression of GBM and closely associated with hypoxia. Nevertheless, the differential expression profiles as well as functional roles of lncRNAs in GBM cells under hypoxic conditions remain largely obscure. We explored the expression profiles of lncRNAs in hypoxic U87 cells as well as T98G cells using sequencing analysis. The effect of differentially expressed lncRNAs (DElncRNAs) was assessed through bioinformatic analysis. Furthermore, the expression of lncRNAs significantly dysregulated in both U87 and T98G cells was further validated using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Relevant cell functional experiments were also conducted. We used predicted RNA-binding proteins (RBPs) to construct an interaction network via the interaction prediction module. U87 and T98G cells showed dysregulation of 1115 and 597 lncRNAs, respectively. Gene Ontology (GO) analysis indicated that altered lncRNA expression was associated with nucleotide-excision repair and cell metabolism in GBM cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed the association between dysregulated lncRNAs and the Hippo signaling pathway under hypoxia. The dysregulation of six selected lncRNAs (ENST00000371192, uc003tnq.3, ENST00000262952, ENST00000609350, ENST00000610036, and NR_046262) was validated by qRT-PCR. Investigation of lncRNA-microRNA (miRNA)-mRNA networks centered on HIF-1α demonstrated cross-talk between the six validated lncRNAs and 16 related miRNAs. Functional experiments showed the significant inhibition of GBM cell proliferation, invasion, and migration by the knockdown of uc003tnq.3 in vitro. Additionally, uc003tnq.3 was used to construct a comprehensive RBP-transcription factor (TF)-miRNA interaction network. The expression of LncRNAs was dysregulated in GBM cells under hypoxic conditions. The identified six lncRNAs might exert important effect on the development of GBM under hypoxic microenvironment.

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The raw data supporting the conclusion of this article have been uploaded to Supplementary Appendix 4.

Abbreviations

GBM:

Glioblastoma multiforme

lncRNA:

Long non-coding ribonucleic acid

HIF-1α:

Hypoxia-induced factor-1α

qRT-PCR:

Quantitative reverse-transcription polymerase chain reaction

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

ncRNA:

Non-coding ribonucleic acid

ceRNA:

Competing endogenous RNA

miRNA:

MicroRNA

DElncRNA:

Differentially expressed lncRNA

siRNA:

Small interfering RNA

CCK-8 assay:

Cell counting kit-8 solution assay

OD:

Optical density

one-way ANOVA:

One-way analysis of variance

FC:

Fold change

BP:

Biological process

CC:

Cellular component

MF:

Molecular function

UHRF1:

Ubiquitin-like with PHD and RING finger domains 1

UPAT:

UHRF1-protein associated transcript

HIE:

Hypoxic ischemic encephalopathy

IGFBP3:

Insulin-like growth factor binding protein 3

RBP:

RNA-binding protein

TF:

Transcription factor

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Acknowledgments

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (grant number: 81902521), Shanghai Sailing Program (grant number: 19YF1432800), and Research Project of **nhua Hospital (grant number: XH1936).

Author information

Author notes

  1. **aomin Cai, Mengshu Qian, Kui Zhang and Yanzhen Li have contributed equally to this work.

Authors and Affiliations

  1. Department of Neurosurgery, **nhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Yangpu District, Shanghai, 200092, China

    **aomin Cai, Yanzhen Li & Ming Chen

  2. Department of Emergency and Critical Care Medicine, Kong Jiang Hospital of Yangpu District, Shanghai, 200082, China

    Mengshu Qian

  3. Department of Plastic Surgery, Xuzhou Medical University Affiliated Xuzhou City Hospital, Xuzhou, 221000, Jiangsu, China

    Kui Zhang

  4. Department of Neurosurgery, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, 17 Lu Jiang Road, Luyang District, Hefei, 230001, Anhui, China

    Bowen Chang

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  1. **aomin Cai
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Contributions

MC and BWC designed and supervised the project. XMC, MSQ, KZ, and YZL conducted the experiments and performed data analysis. BWC and XMC wrote and revised the manuscript.

Corresponding authors

Correspondence to Bowen Chang or Ming Chen.

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Supplementary Information

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Supplementary file1 (ZIP 98 KB)

Supplementary file2 (ZIP 2 KB)

Supplementary file3 (ZIP 11 KB)

Supplementary file4 (ZIP 60432 KB)

Supplementary file5 Table 1. The primers of selected lncRNAs (DOCX 16 KB)

Supplementary file6 Table 2. Top 10 upregulated and downregulated lncRNAs in U87 under hypoxia (DOCX 20 KB)

Supplementary file7 Table 3. Top 5 upregulated and top 10 downregulated lncRNAs in T98G under hypoxia (DOCX 18 KB)

10528_2023_10597_MOESM8_ESM.docx

Supplementary file8 Table 4. Top 10 BP, CC and MF terms corresponding to upregulated and downregulated lncRNAs in U87 (DOCX 24 KB)

Supplementary file9 Table 5. Top 10 pathways corresponding to upregulated and downregulated lncRNAs in U87 (DOCX 17 KB)

10528_2023_10597_MOESM10_ESM.docx

Supplementary file10 Table 6. Top 10 BP, CC and MF terms corresponding to upregulated and downregulated lncRNAs in T98G(DOCX 23 KB)

10528_2023_10597_MOESM11_ESM.docx

Supplementary file11 Table 7. Top 10 pathways corresponding to upregulated and downregulated lncRNAs in T98G(DOCX 17 KB)

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Cai, X., Qian, M., Zhang, K. et al. Profiling and Bioinformatics Analyses of Hypoxia-Induced Differential Expression of Long Non-coding RNA in Glioblastoma Multiforme Cells. Biochem Genet (2023). https://doi.org/10.1007/s10528-023-10597-1

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