Mechanisms Of DDX27 Promoting The Proliferation Of Colorectal Cancer Through Regulating GOLT1B Alternative Splicing

Objectives: To investigate the expression levels and biological functions of DDX27 as a novel alternative splicing regulator in CRC,identify and evaluate potential mechanisms and regulatory networks of DDX27,and assess its potential value as a therapeutic target for CRC.Methods: DDX27 expression levels in CRC tissues and adjacent tissues were detected using quantitative real-time polymerase chain reaction(q RT-PCR)and western blotting(WB).DDX27 expression levels were also evaluated in one normal colon epithelial cell line and five CRC cell lines using q RT-PCR and WB.Stable CRC cell lines with overexpression or low expression of DDX27 were constructed using a lentivirus carrying overexpression plasmids or short hairpin RNA(sh RNA),and the effects of DDX27 on CRC cell proliferation were evaluated using CCK-8,Ed U,and colony formation assays.The effects of DDX27 on CRC in vivo were evaluated using a subcutaneous transplantation tumor model in nude mice.The potential splicingregulated genes of DDX27 were explored using high-throughput RNA sequencing(RNA-seq),q RT-PCR,nucleocytoplasmic fractionation,gel electrophoresis,immunofluorescence,RNA immunoprecipitation,RNA pull-down.GOLT1 B was selected as a downstream gene,and stable CRC cell lines with low expression of GOLT1 B were constructed using sh RNA lentivirus.The effects of GOLT1 B on CRC cell proliferation were evaluated using CCK-8,Ed U,and colony formation assays.Finally,the potential value of DDX27 and GOLT1 B as therapeutic targets for CRC was evaluated using patient-derived xenograft(PDX)models.Results:(1)DDX27 expression levels were significantly lower in network database tissue samples and 48 pairs of CRC tissues than in adjacent tissues,while DDX27 expression levels were significantly higher in most CRC cell lines than in normal colon epithelial cells.The expression level of DDX27 was significantly correlated with the size of patients’ tumors(p=0.035).In addition,DDX27 was significantly associated with poor survival prognosis(p=0.041,p=0.037).(2)DDX27 knockdown effectively inhibited CRC cell proliferation in vitro,and significantly suppressed the growth level and rate of subcutaneous transplantation tumors in vivo.(3)DDX27 was mainly distributed in the nucleus and could bind to the pre-m RNA of the transport protein GOLT1 B,thereby regulating the alternative splicing of GOLT1 B pre-m RNA,promoting the upregulation of GOLT1B-EI3 m RNA(including exon 3)and downregulation of GOLT1B-ES3 m RNA(exon 3 skipping),and ultimately increasing the functional protein of GOLT1 B,which further promoted the transport of p65 into the nucleus and activated the NF-κB signaling pathway.(4)Exogenous inhibition of DDX27 or GOLT1 B significantly reduced the growth of CRC PDX tumors.Conclusions:(1)DDX27 is highly expressed in colorectal cancer and negatively correlated with poor prognosis in colorectal cancer patients.(2)DDX27 promotes functional protein production of GOLT1 B through selective splicing,promotes the nuclear location of p65,and activates the NF-κB signaling pathway.(3)Both DDX27 and GOLT1 B may serve as potential therapeutic targets for colorectal cancer.