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Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic?related genes and pathways in HeLa cells.

ABSTRACT: Cytokeratin 18 (CK18), one of the major components of intermediate filaments (IF) in simple epithelial cells, undergoes caspase?mediated cleavage upon epithelial cell necrosis and apoptosis. CK18 has been used as a biomarker of several cancers and has been reported to be dysregulated in cervical cancers. The effects of dysregulated expression of CK18 at a molecular level are, however, unclear. In the present study, the function of CK18 in HeLa cells, a cell line derived from a cervical cancer cells, was investigated using shRNA knockdown. Reduced levels of CK18 led to a significant decrease in cell apoptosis, compared with control cells. Notably, RNA?seq analysis of the transcriptomes of HeLa cells, with or without CK18 knockdown, revealed that genes in the NF??B pathway, and certain apoptosis pathways, were under global transcriptional and alternative splicing regulation. Quantitative RT?PCR confirmed the CK18?regulated transcription of apoptotic genes FAS and FADD, as well as immune genes CXCL2 and CD79B, in addition to alternative splicing of FAS and CTNNB1. Western blot analysis further revealed that CK18 knockdown led to reduced expression of CASP8. In conclusion, the present study indicated that CK18 played a role in apoptosis, which may be mediated via a feed?back regulation loop and may involve regulation of transcription and alternative splicing of a number of genes in apoptotic pathways.

SUBMITTER: Cheng Y

PROVIDER: S-EPMC6549092 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic‑related genes and pathways in HeLa cells.

Cheng Yi Y Qin Kai K Huang Nan N Zhou Zhipeng Z Xiong Huihua H Zhao Jing J Zhang Yi Y Yu Shiying S

Oncology reports 20190522 1

Cytokeratin 18 (CK18), one of the major components of intermediate filaments (IF) in simple epithelial cells, undergoes caspase‑mediated cleavage upon epithelial cell necrosis and apoptosis. CK18 has been used as a biomarker of several cancers and has been reported to be dysregulated in cervical cancers. The effects of dysregulated expression of CK18 at a molecular level are, however, unclear. In the present study, the function of CK18 in HeLa cells, a cell line derived from a cervical cancer ce ...[more]

PMID: 31115582

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Project description:Post‑transcriptional mechanisms are an important approach in the treatment of cancer, and may also be hijacked by tumor cells to help adapt to the local microenvironment. Filamin B (FLNB), an actin‑binding protein that provides crucial scaffolds for cell motility and signaling, has also been identified as an RNA‑binding protein. Recent studies demonstrated that FLNB might play an important role, not only in skeletal development, but also in regulating tumorigenesis; however, the effects of dysregulated expression of FLNB at the molecular level are not clear. In the present study, RNA‑sequencing was performed to analyze changes in overall transcriptional and alternative splicing between the knocked‑down FLNB and the control in HeLa cells. Decreased FLNB levels resulted in significantly lower apoptosis compared with control cells. FLNB knockdown extensively regulated the expression of genes in cell apoptosis, tumorigenesis, metastases, transmembrane transport and cartilage development. Moreover, FLNB regulated alternative splicing of a large number of genes involved in 'cell death' and the 'apoptotic process'. Some genes and alternative splicing related to skeletal development were enriched and regulated by FLNB. Reverse transcription‑quantitative‑PCR identified FLNB‑regulated transcription and alternative splicing of genes, such as NLR family apoptosis inhibitory protein, interleukin 23 subunit α, metastasis associated lung adenocarcinoma transcript 1, phosphofurin acidic cluster sorting protein 2, bone morphogenetic protein 7, matrix metallopeptidase 13, collagen type II α 1 chain, fibroblast growth factor receptor 2 and vitamin D receptor. The present study is the first study, to the best of the authors' knowledge, to provide transcriptome‑wide analysis of differential gene expression and alternative splicing upon FLNB silencing. The present results suggested that FLNB may play an important regulatory role in cervical cancer cell apoptosis via regulation of transcription and alternative splicing, which provide insight for the current understanding of the mechanisms of FLNB‑mediated gene regulation.

Project description:BackgroundAberrant 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.MethodsWe 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.ResultsThe 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.ConclusionsThis 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.

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Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic?related genes and pathways in HeLa cells.

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Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic‑related genes and pathways in HeLa cells.

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