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Identification of hypoxia-induced isoforms and novel genes with nanopore long reads sequencing

ABSTRACT: Long-read nanopore sequencing is capable of continuously reading thousands of base pairs. Here, we report the application of nanopore sequencing technology for analysing hypoxia-specific transcriptomes in normal liver and liver cancer cell lines, which help us to better understand the mechanism of cell survival and metastasis under hypoxia. We conducted transcriptomic sequencing of normal liver and liver cancer cell lines under normoxia or hypoxia, founding that long-read sequencing could detect thousands of hypoxia-specific isoforms and unannotated novel genes. At the cellular level, we have verified that the novel gene 644 is most up-regulated novel gene under hypoxic conditions. We observed that the novel gene 644 possesses classical mRNA characteristics, with its CDS region encoding 91 amino acids. Through protein structure predictions, we discovered that the novel gene 644 contains a transmembrane domain. Experimental validation confirmed that the novel gene 644 is a membrane protein, and knockdown of the novel gene 644 significantly suppresses the proliferative capacity of liver cancer cells under hypoxic conditions. Furthermore, we revealed the protein structure alteration of hypoxia specific isoforms with molecular dynamics simulation. The results indicate that our hypoxic dataset can serve as a foundation for the structural simulation of hypoxic-specific proteins. In summary, we have presented a hypoxic-specific third-generation sequencing dataset for further analysis, which provides sufficient details on cellular transcriptomes and is particularly valuable for understanding the survival and metastasis of tumor under hypoxic conditions. From this dataset, we have validated the importance of novel gene 644 for tumor survival under hypoxia and identified alterations in hypoxic-specific protein structures. These discoveries can bring us new knowledge about hypoxia, leading to the identification of potential tumor treatment targets and biomarkers.

ORGANISM(S): Homo sapiens

PROVIDER: GSE263126 | GEO | 2024/05/28

REPOSITORIES: GEO

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Project description:Purpose: Due to its high metastatic proclivity, pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly types of cancer. Therefore, it is imperative to better understand how the disease spreads as it progresses. Using a novel genetically engineered mouse model that allows us to isolate a subpopulation of cancer cells with superior metastatic capacity, we show that this aggressive phenotype correlates exclusively with a strong hypoxia signature. We subsequently identified the novel hypoxia-inducible gene Blimp1, which appears to play a critical role in regulating the hypoxic response upon its induction. Furthermore, genetic ablation of Blimp1 greatly reduces the level of metastasis in a PDAC mouse model. The nature of this Blimp1-regulated hypoxia signature is very unstable, since the seeded metastatic lesions mostly re-adopt similar transcriptomic profiles as the primary tumors. In conclusion, our results offer a potential mechanistic insight into how hypoxia drives metastasis in PDAC. Methods: The liver metastasis cell line 688M was subjected to control or knockdown of Blimp1 before assay. Cells ere validated for knockdown efficiency and then cultured under normoxia and hypoxia (0.5% O2) for 24 hours before preparation for RNA-Seq (total of 4 groups with duplicates, overall 8 samples). Results: For the control knockdown group, 0.5% O2 culture(hypoxia) for 24 hours induced dramatic changes in global gene expression, and Blimp1 knockdown potently mitigated changes of significant amount of genes induced under hypoxia. In brief, cell cycle-related genes that are normally suppressed by hypoxia (which contributes to hypoxia-induced cell cycle arrest) are not suppressed under hypoxia in the Blimp1 knockdown cells. In contrast, ~35% of genes that are induced under hypoxia in the control knockdown cells are instead induced less than 50% (under hypoxia) upon Blimp1 knockdown. Conclusions: Blimp1 is a critical regulator of hypoxic response in pancreatic cancer.

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Identification of hypoxia-induced isoforms and novel genes with nanopore long reads sequencing

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