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Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Published on: April 26, 2017
CRKL regulates alternative splicing of cancer-related genes in cervical cancer samples and HeLa cell
Qingling Song1, Fengtao Yi2, Yuhong Zhang3,4
1Department of Oncology and Radiotherapy, Wuhan General Hospital of Guangzhou Military Command, Wuhan, 430070, Hubei Province, China.
Background:
Aberrant spliced isoforms are specifically associated with cancer progression and metastasis. The cytoplasmic adaptor CRKL (v-crk avian sarcoma virus CT10 oncogene homolog-like) is a CRK like proto-oncogene, which encodes a SH2 and SH3 (src homology) domain-containing adaptor protein. CRKL is tightly linked to leukemia via its binding partners BCR-ABL and TEL-ABL, upregulated in multiple types of human cancers, and induce cancer cell proliferation and invasion. However, it remains unclear whether signaling adaptors such as CRKL could regulate alternative splicing.
Methods:
We analyzed the expression level of CRKL in 305 cervical cancer tissue samples available in TCGA database, and then selected two groups of cancer samples with CRKL differentially expressed to analyzed potential CRKL-regulated alternative splicing events (ASEs). CRKL was knocked down by shRNA to further study CRKL-regulated alternative splicing and the activity of SR protein kinases in HeLa cells using RNA-Seq and Western blot techniques. We validated 43 CRKL-regulated ASEs detected by RNA-seq in HeLa cells, using RT-qPCR analysis of HeLa cell samples and using RNA-seq data of the two group of clinical cervical samples.
Results:
The expression of CRKL was mostly up-regulated in stage I cervical cancer samples. Knock-down of CRKL led to a reduced cell proliferation. CRKL-regulated alternative splicing of a large number of genes were enriched in cancer-related functional pathways, among which DNA repair and G2/M mitotic cell cycle, GnRH signaling were shared among the top 10 enriched GO terms and KEGG pathways by results from clinical samples and HeLa cell model. We showed that CRKL-regulated ASEs revealed by computational analysis using ABLas software in HeLa cell were highly validated by RT-qPCR, and also validated by cervical cancer clinical samples.
Conclusions:
This is the first report of CRKL-regulation of the alternative splicing of a number of genes critical in tumorigenesis and cancer progression, which is consistent with CRKL reported role as a signaling adaptor and a kinase. Our results underline that the signaling adaptor CRKL might integrate the external and intrinsic cellular signals and coordinate the dynamic activation of cellular signaling pathways including alternative splicing regulation.
Insights
Aberrant splicing is linked to cancer. This study reveals that CRKL (v-crk avian sarcoma virus CT10 oncogene homolog-like) signaling adaptor regulates alternative splicing in cancer, impacting cell proliferation and DNA repair pathways.
Area of Science:
- Molecular Biology
- Cancer Research
- Genomics
Background:
- Aberrant spliced isoforms are hallmarks of cancer progression and metastasis.
- Cytoplasmic adaptor CRKL (v-crk avian sarcoma virus CT10 oncogene homolog-like) is upregulated in various cancers and promotes proliferation and invasion.
- The role of signaling adaptors like CRKL in regulating alternative splicing remains largely unknown.
Purpose of the Study:
- To investigate whether the signaling adaptor CRKL regulates alternative splicing in cancer.
- To identify CRKL-regulated alternative splicing events (ASEs) in cervical cancer.
- To elucidate the functional consequences of CRKL-mediated alternative splicing.
Main Methods:
- Analysis of CRKL expression in 305 cervical cancer samples from TCGA.
- CRKL knockdown in HeLa cells using shRNA, followed by RNA-Seq and Western blot.
- Validation of CRKL-regulated ASEs using RT-qPCR and clinical sample data.
Main Results:
- CRKL expression was predominantly upregulated in stage I cervical cancer.
- CRKL knockdown reduced cervical cancer cell proliferation.
- CRKL-regulated ASEs were enriched in cancer-related pathways, including DNA repair and cell cycle regulation.
- Computational analysis of ASEs in HeLa cells was validated by RT-qPCR and clinical samples.
Conclusions:
- This study is the first to report CRKL's regulation of alternative splicing in genes critical for tumorigenesis.
- CRKL acts as a signaling adaptor that integrates cellular signals to coordinate pathways, including alternative splicing.
- CRKL's role in alternative splicing contributes to its function in cancer progression.
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