PTBP3 Splicing Factor Promotes Hepatocellular Carcinoma By Destroying The Splicing Balance Of Non-coding RNAs

?Background?The incidence of human hepatocellular carcinoma?HCC?has been rising worldwide.Although the survival has improved due to advances in surgical techniques,malignant growth and metastasis are major causes of cancer-related death.RNA binding proteins?RBPs?are proteins that directly bind to RNA.RBPs interact with RNA through one or multiple RNA binding domains.RBPs critically regulate RNA expression by affecting all aspects of gene metabolism.They play key roles in post-transcriptional processes,such as alternative splicing?AS?regulation,maturation,transport,translation,RNA decay,storage and turnover.The regulatory function of many RBPs,especially in the occurrence of tumor,depends on AS,which is a significant checkpoint to tune gene expression and diversity at the RNA level.Polypyrimidine tract-binding proteins?PTBPs?are RBPs that are mainly enriched in the nucleus.The AS event is controlled by the PTBP family members PTBP1?also known as hnRNP I?,PTBP2,and PTBP3.PTBP1 is upregulated in pancreatic cancer and breast cancer,and its increased recruitment to PKM pre-mRNA promotes PKM2 splicing.PTBP2 is a PTBP1homolog,and is mostly localized in neurons.In the nervous system,PTBP2 is an essential AS factor.By comparison,the physiological roles or molecular functions of PTBP3remain unclear.Recent studies have reported disease associated variations,functional variations and trait mapping to non-coding RNAs?ncRNAs?,including long non-coding RNAs?lncRNAs?,microRNAs?miRNAs?,small nuclear RNAs and small nucleolar RNAs.LncRNAs?longer than 200 nt?and miRNAs?20-24 nt?are increasingly being identified.For example,HOTAIR acts as a scaffold for chromatin modifying complexes,XIST silences the inactive X chromosome,and miRNAs?miR-151,-143?can promote or suppress cancer cell metastasis?miR-200a,-503,-338,-125?.In this study,we confirmed that nuclear-enriched abundant transcript 1?NEAT1?was recruited by PTBP3.In many types of cancer,high NEAT1 expression levels play an important role in malignant growth,metastasis and chemosensitivity.NEAT1 gives rise to two transcripts,NEAT1₁?3.7 kb?and NEAT1₂?23 kb?,which are produced by alternative 3?-end processing and are transcribed from the same promoter.The effect of NEAT1₂ on the formation of RNA-RBP complexes is stronger than that of NEAT1₁.Remarkably,miR-612 is transcribed from chromosome 11,and its DNA fragments are part of the NEAT1₂ DNA sequences.In addition,down-regulation of miR-612 inhibits the invasive-metastatic cascade in HCC.These findings suggest that the unbalanced expression levels of NEAT1₁,NEAT1₂ and miR-612 are modulated by some pre-RNAs splicing factors.However,the extent to which AS processing and splicing variants contribute to the specialized functions of HCC is poorly understood.?Objective?Nuclear-enriched RBPs are mainly involved in transcriptional regulation,which is a critical checkpoint to tune gene diversity and expression levels.Therefore,we investigated nuclear-enriched RBPs and their physiological roles and molecular functions in HCC.?Methods?We analyzed several nuclear RBPs in HCC tissues and matched normal control tissues.HCC cell lines were transfected with siRNAs or lentiviral vectors of the candidate RBPs.We analyzed the invasion,migration and proliferation of these cells in vitro.We established three in vivo models.To identify lncRNAs that were directly recruited by the candidate RBPs,RNA immunoprecipitation?RIP?sequencing was performed.Fluorescence in situ hybridization?FISH?and qRT-PCR assays verified the sequencing results.The splicing variants were evaluated by RIP,FISH and qRT-PCR.?Results?Based on the gene expression levels,PTBP3 was identified as top-ranked in the nuclei of HCC cells.PTBP3 promoted HCC cell proliferation and metastasis both in vitro and in vivo.RIP,FISH and qRT-PCR assays verified that PTBP3 protein recruited abundant lnc-NEAT1 splicing variants?NEAT1₁ and NEAT1₂?and pre-miR-612?precursor of miR-612?in the nucleus.NEAT1₁,NEAT1₂ and miR-612 expression levels were determined by PTBP3.Correlational analyses revealed that PTBP3 was positively correlated with NEAT1,but they were all inversely correlated with miR-612 in HCC.The P53/CCND1 and AKT2/EMT pathways were determined by NEAT1 and miR-612respectively in HCC.The PTBP3^high and NEAT1^high/miR-612^lowow patients had a shorter overall survival.?Conclusion?In this study,we identified a novel interaction among PTBP3,NEAT1 and miR-612that regulates HCC cell activity.The high-expression of PTBP3 promoted HCC cell metastasis and malignant growth.PTBP3 served as a nuclear protein that modulated NEAT1 and miR-612 balance at the transcriptional level.To the best of our knowledge,this is the first study to identify a nuclear protein that modulates AS between lncRNAs and miRNAs in HCC.