Department of Oncology and Radiotherapy, Wuhan General Hospital of Guangzhou Military Command, Wuhan, 430070, Hubei Province, China.
Laboratory of Human Health and Genome Regulation, Wuhan, 430075, Hubei, China.
BMC Cancer. 2019 May 27;19(1):499. doi: 10.1186/s12885-019-5671-8.
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.
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.
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.
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.
异常剪接的异构体与癌症的进展和转移特别相关。细胞质衔接蛋白 CRKL（v-crk 禽肉瘤病毒 CT10 癌基因同源物样）是一种 CRK 样原癌基因，编码一个含有 SH2 和 SH3（src 同源）结构域的衔接蛋白。CRKL 通过其结合伙伴 BCR-ABL 和 TEL-ABL 与白血病紧密相关，在多种类型的人类癌症中上调，并诱导癌细胞增殖和侵袭。然而，目前尚不清楚信号衔接蛋白如 CRKL 是否可以调节可变剪接。
我们分析了 TCGA 数据库中 305 例宫颈癌组织样本中 CRKL 的表达水平，然后选择两组 CRKL 差异表达的癌症样本，以分析潜在的 CRKL 调节的可变剪接事件（ASEs）。通过 shRNA 敲低 CRKL，进一步研究 CRKL 调节的可变剪接和 HeLa 细胞中 SR 蛋白激酶的活性，使用 RNA-Seq 和 Western blot 技术。我们使用 HeLa 细胞样本的 RT-qPCR 分析和两个临床宫颈癌样本的 RNA-Seq 数据，验证了在 HeLa 细胞中通过 RNA-seq 检测到的 43 个 CRKL 调节的 ASE。
CRKL 的表达在Ⅰ期宫颈癌样本中大多上调。CRKL 敲低导致细胞增殖减少。CRKL 调节的大量基因的可变剪接富集在癌症相关的功能途径中，其中 DNA 修复和 G2/M 有丝分裂细胞周期、GnRH 信号通路是通过临床样本和 HeLa 细胞模型的结果得到的前 10 个富集 GO 术语和 KEGG 途径中共同的。我们表明，通过 ABLas 软件在 HeLa 细胞中进行计算分析显示的 CRKL 调节的 ASE 高度通过 RT-qPCR 验证，也通过宫颈癌临床样本验证。
这是第一个报告 CRKL 调节与肿瘤发生和癌症进展相关的许多基因的可变剪接的报告，这与 CRKL 作为信号衔接蛋白和激酶的作用一致。我们的结果强调了信号衔接蛋白 CRKL 可能整合外部和内在的细胞信号，并协调包括可变剪接调节在内的细胞信号通路的动态激活。
