Full-length Transcriptomics Helps to Reveal The Mechanism of Cancer

In recent years, with the development of high-throughput sequencing technology, transcriptome sequencing has become the main means of studying gene expression regulation. Full-length transcriptome is based on the long-read sequencing platform of PacBio and Nanopore. It can directly obtain the full-length sequence and complete structural information of mRNA containing 5'UTR, 3'UTR and polyA tails without interrupting splicing, so as to accurately analyze the structural information such as alternative splicing and fusion genes of species with reference genome, and overcome the problem of short splicing and incomplete information of transcripts of species without reference genome. It has been widely used in exploring the pathogenesis of cancer or other diseases.

Identification of alternative splicing using full length transcriptomics

An organism's genome is constantly attacked by various stress signals, resulting in DNA damage. Excessive DNA damage impairs genomic integrity and blocks DNA replication and transcription. More and more evidence show that DNA damage is enhanced in many cardiovascular diseases, it has been well established that Ca²⁺/calmodulin-dependent kinase II (CaMKII) plays a central role in the regulation of cardiomyocyte death and excessive CaMKII activation contributes to myocardial infarction, cardiomyopathy and heart failure. Researchers performed full-length transcriptome analysis on the heart tissues of mice, rats, rhesus monkeys and humans and successfully portrayed the cutting panorama of CaMKII-δ for cracking the pathogenesis mediated by CaMKII-δ. Subsequently, the author confirmed the expression of mRNA and protein by means of RNA-seq, western blot and mass spectrometry, the results indicated that CaMKII-δ9 potently promotes cardiomyocyte death, which is the most abundant CaMKII-δ splice variant in human heart. Additionally, CaMKII-δ9 tar-gets the ubiquitin-conjugating enzyme E2T (UBE2T) for phosphorylation and degradation, disrupting UBE2T-dependent DNA repair and leading to the accumulation of DNA damage and genome instability. This study not only revealed the pathogenesis of cardiomyopathy mediated by CaMKII-δ9, but also emphasized that CaMKII-δ9 can be a potentially important target for the treatment of myocardial injury and heart failure.

Exploring cancer mechanisms using full-length transcriptomics

Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, accounting for 75%–85% of cases. Hepatocellular carcinoma (HCC) has a high recurrence rate and a low 5-year survival rate. Studying the single-cell expression profile of recurrent patients may be of great significance for designing effective anticancer therapies, including immunotherapy. The researchers first screened immune cells and non-immune cells from tumor tissues and adjacent tissues of 12 primary and 6 early recurrent HCC patients through CD45+ to obtain nearly 17000 cells, then sequenced the full-length transcripts, and identified the cell subtypes and genes of the data. Early-relapse tumors have reduced levels of regulatory T cells, increased dendritic cells (DCs), and increased infiltrated CD8+ T cells, compared with primary tumors, in two independent cohorts. CD8+ T cells in RT, characterized by overexpression of KLRB1 (CD161), primarily presented an innate-like, low-cytotoxic, and low-clonal-expansion state, with the low expression of co-stimulatory and checkpoint molecules. Patients with more CD161+ CD8+ T cells showed a worse prognosis. These findings in two additional cohorts of paired PT and RT samples were confirmed by using scRNA-seq or immunohisto-chemical (IHC) staining. Differential gene analysis showed that malignant cells in RT had higher immune escape characteristics, which might inhibit the ability of DC to activate CD8+ T cells. This study systematically analyzed the immune micro-ecological differences between primary and recurrent hepatocellular carcinoma from the single cell level and polychromatic fluorescence immunohistochemistry technology, revealed the characteristic immune atlas and immune escape mechanism of early recurrent hepatocellular carcinoma, and provided more theoretical basis and experimental evidence for further improving the efficacy of immunotherapy for hepatocellular carcinoma and finding effective new strategies for the prevention and treatment of recurrence and metastasis of hepatocellular carcinoma.

Full-length transcriptomics has significant advantages in identifying alternative splicing. Variable transcripts may serve as a new potential molecular target for the treatment of some cancers, and provide new ideas and clues for further revealing the molecular mechanism of cancer metastasis in the future. In a word, it provides important insights into the pathogenesis of tumorigenesis and plays an important guiding role in the development of targeted treatment strategies.

References:

1. Sun, Yunfan et al. "Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma." Cell vol. 184,2 (2021): 404-421.e16.
2. Kiyose, Hiroki et al. "Comprehensive analysis of full-length transcripts reveals novel splicing abnormalities and oncogenic transcripts in liver cancer." PLoS genetics vol. 18,8 e1010342. 4 Aug. 2022
3. Zhang, Mao et al. "CaMKII-δ9 promotes cardiomyopathy through disrupting UBE2T-dependent DNA repair." Nature cell biology vol. 21,9 (2019): 1152-1163.