Rbm10 And Mutant-type C761Y Differentially Regulate ASPM Alternative Splicing To Control Cholangiocarcinoma Proliferation And Migration

Purpose: This study aims to explore the role and mechanism of RNA binding motif protein 10(RBM10)and its mutant-type C761 Y in cholangiocarcinoma,which may provide a theoretical basis for potential therapeutic targets for cholangiocarcinoma.Methods: Whole exome sequencing was performed on cholangiocarcinoma samples,which found RBM10 C761 Y mutation.RT-PCR and tissue chip immunohistochemical staining were used to analyze the expression of RBM10 in cholangiocarcinoma.Transfect cell lines with lentiviral vectors,overexpress wild-type RBM10 and C761 Y mutant in the cholangiocarcinoma cell lines QBC939 and RBE.To study the effects of RBM10 and its mutation C761 Y on the proliferation and migration of cholangiocarcinoma cells through plate cloning,EDU experiment,CCK8 experiment,cell cycle experiment,scratch experiment and transwell migration experiment.Transcriptome sequencing and r MATs software were used to analyze the exon skipping events affected by RBM10 and its mutation C761 Y.Verification of the binding of RBM10 and its mutation C761 Y to the target ASPM m RNA was conducted by RNA immunoprecipitation experiment.The correlation analysis was performed between the expression of RBM10 and the changes in ASPM transcripts in tissues.The changes in cell lines were verified at protein and RNA levels.Results: Whole-exome sequencing results of KEGG enrichment showed that posttranscriptional regulation-related genes were significantly enriched.Among them,RBM10 had 5 mutations in 67 samples from our center.It is predicted that C761 Y may have high pathogenicity.RT-PCR and tissue chip immunohistochemical staining analysis showed that down-regulation of RBM10 in cholangiocarcinoma is associated with poor prognosis.Functional experiments on the cell line constructed by lentiviral transfection showed that wild-type RBM10 can inhibit the proliferation and migration of cholangiocarcinoma cells,while the C761 Y mutant-type losed this function.Transcriptome sequencing and r MATs software analysis predicted that the skipping of exon 18 of abnormal spindle-like microcephaly-associated protein(ASPM)might be related to RBM10.The RNA immunoprecipitation experiment confirmed that RBM10 binds to ASPM m RNA,and the expression of RBM10 in tissues is positively correlated with the skipping frequency of ASPM18 exon.In QBC939 cell line,we verified that wild-type RBM10 can reduce ASPM203 containing exon 18 at the RNA and protein levels,while the C761 Y mutant has lost this function.Western blot results showed that the increase of ASPM203 can increase the level of β-catenin,CCND1,PCNA,and decrease the level of E-cadherin.Conclusion: Compared with RBM10 wild type,RBM10 C761 Y mutant type in cholangiocarcinoma cell lines can reduce the skipping of exon 18 of pre-ASPM messenger RNA after transcription,increase the content of ASPM203,and thus increase the level of β-catenin,which may promote the proliferation and migration of cholangiocarcinoma through Wnt/β-catenin.The loss-of-function mutation of RBM10 may provide a potential target for the treatment of cholangiocarcinoma.