Roles Of SRSF6 In Colorectal Cancer Progression

It has been found that about 23,000 human genes produce exceedingly more than 300,000 proteins.As a main cause for this amplification,more than 90%of human genes are predicted to undergo alternative splicing.Through fine-tuning of RNA splicing,organism can make response to the environmental stress with maintenance of biological balance,which may further provide material basis for biological evolution.Aberrant alternative splicing facilitates cancer development and progression through both increasing oncogenic isoforms and reducing normal isoforms.With the popularization of next-generation sequencing,more and more genes with aberrant splice were identified to be involved in multiple oncological processes,including angiogenesis,invasion and metastasis,immune destruction and cellular energetics.Serine/arginine-rich(SR)RNA binding protein family is one major class of splicing factors,which are concentrated in nuclear speckles and function critical roles in constitutive and alternative splicing.SR proteins could recruit spliceosome assembly via binding to specific RNA sequence,regulating the alternative splicing of exons or introns.With abnormal splicing of RNA,deregulation of SR proteins could promote cancer proliferation and metastasis and inhibit apoptosis.As an main member of SR proteins,SRSF6 genome copy number was found to be ampified in multiple tumors.Especially,it has been identified that overexpression of SRSF6 in skin was associated with TNC aberrant splicing,which mediate skin cancer proliferation.Our previous study showed that SRSF6 is the target of the long noncoding RNA,LINC01133,that antagonizes SRSF6 to inhibit epithelial-mesenchymal transition(EMT)and metastasis in colorectal cancer(CRC).However,the biological role of SRSF6 in colorectal cancer and the specific underlying mechanism of SRSF6-regulated splicing need to be further elucidated.In the current study,we systematically identified SRSF6-regulated AS events and the specific recognition motif for SRSF6 via next-generation RNA-sequencing(RNA-seq).As a master regulator of ZO-1 AS,SRSF6 promotes CRC progression.More importantly,we screened and identified indacaterol,a β2 adrenergic receptor agonist approved for COPD treatment,as candidate therapeutic pharmaceutical for CRC through targeting SRSF6 to modulate AS.To our knowledge,this is the first candidate drug to regulate AS via targeting SR protein in cancer treatment.1.Altered SRSF6 expression promote colorectal cancer progression.The gene copy number of SRSF6 is significantly increased in MSS and MSI-L subtypes of CRC samples from TCGA databases.Moreover SRSF6 mRNA expression was positively correlated with copy number variations in TCGA database.We detected the expression of SRSF6 in a cohort of 311 colorectal cancer and matched paired normal mucosa tissues by qRT-PCR.The SRSF6 expression was significantly increased in tumor compared with matched normal tissues.And patients with higher SRSF6 expression were found to have a significantly reduced overall survival time when compared with patients with lower SRSF6 expression.To test the biological roles of SRSF6 in CRC,we stably knocked down SRSF6 in six colorectal cancer cell lines.With CCK8,colony formation assay and transwell assay,we found that proliferation ability,anchorage independent growth and motility and invasive properties in all six colorectal cancer cell lines were significantly reduced as a result of SRSF6 knock-down.Further rescue assay showed that regaining SRSF6 in the knockdown cells could recover these abilities.Moreover,we have investigated the biological function of SRSF6 in CRC with multiple in vivo models,including subcutaneous tumor growth in nude mice,mouse tail-vein assay and mouse colitis-associated CRC model.2.SRSF6 regulates alternative RNA splicing via binding to specific motif.To further elucidate the biological roles of SRSF6 in CRC,we performed transcriptional sequencing.By sequencing three pairs of independent RNA samples from scrambled control and SRSF6-knockdown SW620 cells,1158 SRSF6-regulated AS events were identified and which were further validated by RT-PCR.In addition,we next identified SRSF6-bind RNA through RNA-immunoprecipitation sequencing(RIP-seq),and discovered the SRSF6 RNA binding motif in accompanied with data from transcriptional sequencing.Gel shift assay and splicing reporter assay were performed to further validate the SRSF6 RNA binding motif.3.SRSF6 promotes tumor progression through regulating ZO-1 alternative splicing.As a downstream target of SRSF6,tight junction protein 1(ZO-1)contains two alternative splicing variants,ZO-1 Exon23 inclusion(E23+)and ZO-1 Exon23 skipping(E23-).We knocked down SRSF6 in SW620 cells and found ZO-1 E23+ was increased and cell mobility was decreased.Furthermore,when ZO-1 E23+ was knocked down in SW620 with SRSF6 knock-down,their mobility was patially rescued.To further explore the exact function of these specific ZO-1 variants,we measured the expression of ZO-1 variants in six CRC cell lines,and found ZO-1 E23+ knocked down could reduce mobility ability of CRC cell lines while ZO-1 E23-could not.More importantly,we found there was a negative correlation between the PSI of ZO-1 and the expression of SRSF6 in CRC tissues and mice models,and patients with lower PSI index displayed poorer survival rate.4.Indacaterol as pharmacological inhibitor of SRSF6 blocked CRC progression.To further explore the specific function of SRSF6 domains,we overexpressed SRSF6 deletion mutants in SRSF6 knocked down SW620 cells and explored their biological function.Specifically,RRM2 domain was indispensable for SRSF6 mediated tumor progression,which was further validated by the higher motif affinity measured by gel shift assay.We proposed that RRM2 may serve as a potential target for SRSF6 inhibitor design.However,for the crystal structure of SRSF6 protein has not been reported yet,we performed homology modeling of SRSF6 protein to predict the 3D structure of SRSF6 protein.Targeting the predicted RNA binding pocket with RRM2 domain,the first inhibitor targeting SRSF6 protein was screened within 4855 FDA approved drugs from Drugbank database.Indacaterol,known as a long-actingβ2-agonists(LABAs)used in treatment for COPD,was found to inhibit SRSF6-regulated AS events and inhibit CRC proliferation and metastasis in vivo and in vitro.In summary,the following conclusions were drawn according to the study.(1)Excessive SRSF6 expression promotes CRC proliferation and metastasis,and may serve as a hall marker to predict poor prognosis.(2)SRSF6 was identified to be related with plenty of RNA alternative splicing.Specially,with directly binding to its binding motif,SRSF6 could initiate the splicing of ZO-1 exon23 to promote CRC metastasis.The patients with higher ratio of exon23 inclusion ZO-1 variant were accompanied with a better prognosis,suggesting the splicing pattern of ZO-1 may serve as a new hall marker for CRC prognosis.(3)As an example of conventional drug in new use,we screened that indacaterol could inhibit CRC in vitro and in vivo.Our result repositioned indacaterol as an potential anti-tumor drug through targeting SRSF6 and modulating its function in alternative splicing.