The Study Of The Roles And Molecular Mechianisms Of MBNL3 And Induced Alternative Splicing Transcript POLDIP3-β In HCC

Alternative splicing plays critical roles in many pathophysiological processes and abnormal regulations of splicing occur in many cancers.It is well known that different subtypes of gene can have distinct effects on cancer.Therefore,comprehending the roles of splicing factors and splicing events during human tumorigenesis would make a contribution to new targeted therapeutics for cancer.Here we find a splicing factor MBNL3,which has oncofetal feature.Our research indicates that MBNL3 can strongly drive hepatocellular carcinoma tumorigenesis.First,we found that the expression of MBNL3 is higher in human fetal liver tissues than that in adult liver tissues.Furthermore,MBNL3 expression was also detected in hepatocellular carcinoma(HCC)tissues and noncancerous hepatic tissues.The results showed that the expression of MBNL3 is higher in HCC tissues and higher MBNL3 expression was associated with poorer prognosis.Stem cell markers OCT4,SOX2,and NANOG upregulate the expression of MBNL3.Cell Counting Kit-8 assays(CCK-8),EdU immunofluorescence staining assays and colony formation assays showed that MBNL3 promotes cell proliferation of HCC cells and nontransformed liver cells.TUNEL and apoptotic markers detection showed that MBNL3 suppresses cell apoptosis.Also,overexpressing of MBNL3 promotes tumor growth in vivo.The silencing of MBNL3 produced opposite results in HCC cells.In addition,we further verified the effects of MBNL3 in HCC tumorigenesis by depleting MBNL3 in many cell lines.These results indicated that the growth of HCC cells was strongly suppressed in vitro and in vivo by MBNL3 depletion.In view of the strong biological function of MBNL3,we analyzed its downstream target.High-throughput sequencing results indicated that MBNL3 could regulate the alternative splicing of POLDIP3.Therefore,we fixed attention on the research of different transcripts of POLDIP3.POLDIP3 is a target of ribosomal protein S6 kinase 1,which regulates DNA replication and mRNA translation.In this study,we identified two transcripts of POLDIP3.One containing exon 3 was named as POLDIP3-α,the other lacking exon 3 and 29 amine acids was named as POLDIP3-β.The effects of POLDIP3-α and POLDIP3-β on the proliferation of HCC cells were detected by Glo cell viability assays,EdU assays and clone formation assays.The roles of POLDIP3-α and POLDIP3-β on apoptosis of HCC cells were detected by TUNEL assays,annexin V-propidium iodide staining and flow cytometry.The effects of POLDIP3-α and POLDIP3-β on the migration of HCC cells were examined by transwell assays and wound healing assays.In vitro functional experiments results showed that POLDIP3-β could significantly increase the proliferation of HCC cells,inhibit the apoptosis of HCC cells and accelerate the migration of HCC cells.However,POLDIP3-α had relatively weak effects.Finally,we found that POLDIP3-β significantly promotes tumor growth in vivo.In conclusion,our study showed that POLDIP3-β is upregulated in HCC,could be regarded as an oncofetal gene,and playes an important role in HCC.Selectively silencing POLDIP3-β would be a new strategy for the treatment of HCC.The above data reveals the relationship between alernative splicing factors,splicing events and HCC tumorigenesis.At the meanwhile,we determined that alternative splicing factors and events can serve as potential therapeutic targets.