In silico RNA-seq and experimental analyses reveal the differential expression and splicing of EPDR1 and ZNF518B genes in relation to KRAS mutations in colorectal cancer cells

Abstract

Several drugs used for the treatment of colorectal cancer (CRC) are targeted at the epidermal growth factor receptor, but mutations in genes of the RAS family cause resistance to these drugs. Thus, extensive research is being carried out to counterbalance this resistance. The G13D mutation of KRAS is common in humans, and we previously reported that this mutation results in the epigenetic modification of hnRNP proteins, involved in RNA splicing. As aberrant splicing often results in oncogenicity, the present study aimed to identify the genes which show altered splicing patterns in connection with the G13D KRAS mutation. To accomplish this, we first carried out an in silico analysis of RNA-seq databases and found that the distribution of alternative splicing isoforms of genes RPL13, HSP90B1, ENO1, EPDR1 and ZNF518B was altered in human CRC cell lines carrying the G13D KRAS mutation when compared to cell lines carrying wild-type KRAS. The in silico results were experimentally validated by quantitative real‑time PCR. Expression of the genes EPDR1 and ZNF518B was negligible in the Caco2, RKO and SW48 cell lines, which possess wild-type KRAS, while the HCT116, DLD1 and D-Mut1 cell lines, harbouring the G13D mutation, expressed these genes. Moreover, in both genes, the ratio of isoforms was significantly different between the parental DLD1 (+/G13D) and D-Mut1 cells, in which the wild-type allele had been knocked out. DWT7m cells also expressed both genes. These cells, derived from DLD1, have spontaneously acquired a G12D mutation in their single KRAS allele in 20% of the population. The present data suggest a relationship between KRAS mutations, particularly G13D, and the expression of the EPDR1 and ZNF518B genes and expression of their isoforms and provide enhanced understanding of the molecular mechanisms involved in the resistance of CRC cells to anti‑EGF receptor therapies.

EPDR1 correlates with immune cell infiltration in hepatocellular carcinoma and can be used as a prognostic biomarker

“…Furthermore, genetic variants in EPDR1 have been reported to be linked to several diseases, including Dupuytren's disease [21,25,26] and primary angle closure glaucoma [23,27], however these observations do not provide obvious insight into the molecular functions of the protein. In 2016 F. Valiente et al reported that EPDR1 and its spliced isoforms are differentially expressed in human CRC cell lines, and the up-regulation of EPDR1 in human colorectal cancer was found to promote cell growth, proliferation, and invasiveness [28]. Genome-wide DNA methylation profiling studies revealed that methylation-mediated epigenetic silencing of EPDR1 may play an important role in preventing CRC progression [29].…”

Identification of novel alternative splicing isoform biomarkers and their association with overall survival in colorectal cancer

“…Aberrant splicing events in CRC and/or other cancer types were identified using either microarray or RNA-seq techniques in the past [14][15][16][17]. The roles of AS in CRC were reported for individual genes: ITGA6 [18], MAP4K4 [19], EPDR1, ZNF518B [20], FIR [21], BRAF [22], Rac1 [23], OCT4 24], RON [25], CD44 [26,27], KRAS [28], ZNF148 [29], FAK [30]. Global AS analysis in CRC or Pan-Cancer has also been performed using large number of RNA-seq data (i.e.…”

Identification of novel alternative splicing isoform biomarkers and their association with overall survival in colorectal cancer

“…ITGA6 [18], MAP4K4 [19], EPDR1, ZNF518B [20], FIR [21], BRAF [22], Rac1 [23], OCT4 24], RON [25], CD44 [26,27], KRAS [28], ZNF148 [29], FAK [30]. Global AS analysis in CRC or Pan-Cancer has also been performed using large number of RNA-seq data (i.e.…”