Molecular Mechanism Of SRSF1 Enhances The Inclusion Of Exon 3 In SRA1 Pre-mRNA To Promote Invasion And Metastasis In Liver Cancer Cells

Background: In 2018,liver cancer became the sixth largest cancer in the world and the fourth leading cause of cancer deaths worldwide.Prone to distant metastasis of liver cancer is one of the important reasons for poor prognosis of patients with liver cancer.Therefore,studying the occurrence and development mechanism of liver cancer metastasis is of great significance to treat liver cancer.Many genetic diseases,developmental defects,and tumor occurrence and metastasis are caused by abnormal alternative splicing.Through research,we found that steroid receptor RNA activator 1(SRA1)exon 3 has alternative splicing in liver cancer.Steroid receptor RNA activator 1(SRA1)described as a novel transcriptional coactivator,but the mechanism of alternative splicing of SRA1 remains unclear and further research is needed.Objective: The purpose of this study was to investigate the functional differences between Exon 3-including isoform of SRA1(SRA-L)and Exon 3-skipping isoform of SRA1(SRA1-S),as well as the specific molecular mechanism of alternative splicing of Exon 3 of SRA1.To clarify the influence of SRSF1-SRA1 signal axis on the invasion and metastasis ability of liver cancer cells can provide new ideas for the study of the metastasis mechanism of liver cancer cells.Methods: 1.Screening for genes with alternative splicing in Hep G2 and HCCLM3 by RT-PCR to determine the research target SRA1.2.Analyzing the difference in SRA1 expression levels in clinical samples of liver cancer through a public database(UALCAN)to further confirm whether SRA1 is related to liver cancer.3.We prove the difference of SRA1 isomer through the following experiment.Using polyribosome analysis experiments and the CPAT website to predict the ability of SRA1 isoforms to encode proteins;To study the effects of overexpression or knockdown of SRA1 isomers on the proliferation,migration and invasion ability of liver cancer cells through CCK8 experiments,scratch healing experiments and in vivo tumor metastasis experiments;Using Western-blot experiments to study the effect of SRA1 isoforms on downstream AKT and ERK signaling pathway proteins;Through MS2-GFP-IP(MS2-GFP-co-immunoprecipitation)to study the ability of SRA1-L,SRA1-S isomers to bind to PPARγ receptors;Detection of the effects of SRA1-L and SRA1-S isomers on the transcription of tumor metastasis related genes by dual luciferase reporter gene.4.In order to preliminarily verify what regulates the alternative splicing of SRA1 exon 3 in liver cancer cells,we made the following corresponding experiments.Using q PCR to analysis the expressed splicing factors in LO2,Hep G2,HCCLM3 cells by;Using the UALCAN database to research the expressed splicing factors in normal tissues and liver cancer patients;Using transcription sequencing to analysis the expressed splicing factors in Hep G2,HCCLM3 cells;Analyzing what can bind to SRA1 and their binding sites through the RBP map bioinformatics website;Using RT-PCR to detect Effects of changes in SRSF1,SRSF8 or SRSF11 expression levels on SRA1-L and SRA1-S expression levels;pc DNA3.1-SRA1-minigene plasmid was transfected into cells and primers that specifically detected exogenous SRA1-L /SRA1-S isomers were used to detect SRSF1 expression of exogenous SRA1-L /SRA1-S isomers by q PCR experiments Level of influence;Using MS2-GFP-IP experiments to verify whether SRA1-minigene,SRA1-L,SRA1-S can bind to SRSF1.5.In order to further verify the molecular mechanism of SRSF1 enhances the inclusion of Exon 3 in SRA1 pre-m RNA,we conducted the following experiments.Using CLRIP(ultraviolet cross-linked RNA co-immunoprecipitation)experiments to verify the sequence segments of SRSF1 and SRA1 pre-m RNA binding;synthesis of biotin-labeled SRA1 pre-m RNA fragments and mutated fragments that may bind to SRSF1 in vitro Determine the binding sequence of SRSF1 and SRA1 pre-m RNA by RNA pull-down experiment;6.In order to prove whether SRSF1 can also promote the invasion of liver cancer cells,we did the following experiment.Using CCK8 experiments,scratch healing experiments,and in vitro tumor metastasis models to investigate the effects of overexpression or knockdown of SRSF1 on the proliferation,migration,and invasion of liver cancer cells;Studing the effects of SRSF1 on downstream AKT and ERK signaling pathway proteins by Western-blot experiments;7.Using cell invasion experiments and in vitro tumor metastasis models to detect the effects of SRSF1 and SRA1 isoform expression changes on the invasion and metastasis of hepatocellular carcinoma cells.Results: 1.The expression of SRA1-L was significantly increased in HCCLM3 cells,while the expression of SRA1-S was significantly decreased in HCCLM3 cells;2.Compared with normal tissues,the expression level of SRA1 in liver cancer samples increased,and the expression in the four stages of liver cancer development was higher than that in normal tissues,and the expression level of SRA1 was positively correlated with the degree of liver cancer deterioration;3.SRA1-L can encode proteins,SRA1-S does not have the ability to encode proteins;4.SRA1 isoforms have no significant effect on cell proliferation;SRA1-L can promote cell migration and invasion,SRA1-S can inhibit cell migration and invasion;SRA1-L can increase the expression of phosphorylated ERK and phosphorylated AKT,SRA1-S has no significant effect on the expression of phosphorylated ERK and phosphorylated AKT;SRA1-L can bind to PPARγ;5.Compared with Hep G2 and LO2 cells,the expression of SRSF1 was increased in HCCLM3,a highly transformed liver cancer cell;compared with normal samples,the expression of SRSF1 was increased in liver cancer samples;6.In liver cancer cells,SRA1 is one of the downstream target genes of SRSF1.SRSF1 can promote the alternative splicing process of SRA1 Exon3;overexpression of SRSF1 can promote the generation of endogenous and exogenous SRA1-L;7.MS2-GFP-IP,CLRIP and RNA-pulldown experiments show that SRSF1 can bind to the "GGAACAGGCATTGGAAGA" sequence in exon 3 of SRA1;8.SRSF1 can promote the migration and invasion of liver cancer cells;SRSF1 can increase the expression of phosphorylated ERK and phosphorylated AKT;9.SRSF1 promotes the invasion and metastasis of liver cancer cells in part depending on the expression level of SRA1-L.Conclusions: 1.Alternative splicing of SRA1 in liver cancer cells;2.There are functional differences between SRA1 isoforms in the process of proliferation,invasion and metastasis of liver cancer cells.The SRA1-L isoform promotes the invasion and metastasis of liver cancer cells,while the SRA1-S isoform inhibits the invasion and metastasis of liver cancer cells.SRA1-L has a stronger binding ability with the receptor PPARγ;3.SRA1 is one of the major downstream target genes of SRSF1 in hepatocellular carcinoma cells;SRSF1 can promote the alternative splicing process of SRA1 Exon3,causing the expression of SRA1-L isoform in hepatocellular carcinoma cells to increase;4.The expression level of SRSF1 is positively correlated with the degree of liver cancer progression.SRSF1 can promote the migration and invasion of liver cancer cells;5.Finally,the effect of SRSF1 on the migration and invasion of liver cancer cells depends in part on the alternative splicing of SRA1;...