The Functional Role Of IGF2BP3 In Esophageal Squamous Cell Carcinoma And Colorectal Cancer

RNA binding proteins(RBPs)were one of the key molecules in post-transcriptional regulation in eukaryotes.RBPs bound to single-stranded or double-stranded RNA and determined their fate from synthesis to degradation.RBPs usually had one or more domains and recognized RNA in a sequence-specific way,leading to the different affinity and specificity to bind RNA.In addition to these domains that can directly bind to RNA,RBPs also contained auxiliary domains that mediated interactions with other proteins.When RBPs bound to mRNA,they modulated the key steps of mRNA fate regulation,including splicing,transportation,localization,translation and degradation.Dysregulation of RBPs can result in many complex diseases,including cancers.Insulin-like growth factor 2 mRNA binding protein family(IGF2BPs)was a highly conserved RBP family,including IGF2BP1,IGF2BP2 and IGF2BP3,which contained two RNA recognition motifs(RRMs)and four hnRNP K homologous domains(KH),can specifically recognize and bind to RNA.Among them,IGF2BP3,also known as IMP3,was often dysregulated in cancer development with limited evidence focused as downstream mRNA targets.More significantly,IGF2BP1-3 have been proved to be a new reader of N6-adenylate methylation(m6A)to maintain the stability of mRNA targets.As a carcinoembryonic protein,the de novo synthesis or abnormal expression of IGF2BP3 played critical roles in the occurrence and development of malignant tumors.However,the underlying mechanism was still unclear.In this study,we focused on its role in esophageal squamous cell carcinoma(ESCC).We found that overexpression of IGF2BP3 significantly inhibited the proliferation,clone formation,migration and invasion of ESCC cells.On the contrary,knockdown of IGF2BP3 exerted an opppsite function.Mechanistically,we performed RNA immunoprecipitation combined with high-throughput sequencing(RIP-Seq)to identify potential mRNA targets interacted with IGF2BP3.Through pathway analysis and functional characterization,we selected and verified that four mRNA,namely PPP2CA,SMG7,SMAD2 and SP3 can be recognized by IGF2BP3.Furthermore,after the cells were treated with actinomycin D,overexpression of IGF2BP3 greatly prolonged the half-life of the candidate mRNAs.To explore whether other proteins facilitated IGF2BP3-mediated mRNA recognition,we identified PABPC1 and ELAVL1,two known mRNA-binding proteins interacted with IGF2BP3 by co-immunoprecipitation(Co-IP)combined with mass spectrometry.RIP-qPCR analysis showed that both PABPC1 and ELAVL1 were significantly enriched in the regions closed to 3’UTR of the four candidate mRNAs,and the depletion of PABPC1 or ELAVL1 significantly impaired the IGF2BP3-enhanced mRNA half-life,indicating IGF2BP3 regulation of mRNA half was dependent on PABPC1 or ELAVL1.It was worth noting that IGF2BP3 was composed of 2 RRMs and 4 KH domains.We constructed several IGF2BP3 deletion mutants’ to examine the specific domains responsible for mRNA recognition and stability.We found that KH domain played essential role in IGF2BP3-mediated mRNA binding.Finally,by analyzing the clinical database,we found that the expression of PPP2CA in ESCC was much lower than that of adjacent tissues,which was consistent with tumor suppressive role of IGF2BP3 in ESCC.Furthermore,we performed RNA pull-down assay to verify the interaction between PPP2CA and IGF2BP3 reciprocally.Moreover,ectopical expression of PPP2CA inhibited the proliferation of ESCC cells.More importantly,knockdown of PPP2CA in IGF2BP3-overexpressing ESCC cells compromised inhibitory effects of IGF2BP3 on ESCC progression.In summary,overexpression of IGF2BP3 significantly inhibits the proliferation,migration and invasion of ESCC cells.IGF2BP3 interacts with PABPC1 and ELAVL1 to stabilize the downstream mRNA targets.Further studies are also needed to understand the molecular mechanisms in IGF2BP3-mediated regulation of mRNA recognition and stability.In recent years,it has been found that multiple RNA binding proteins are abnormally expressed in colorectal cancer,including IGF2BPs.The three members of the IGF2BP family,IGF2BP1,IGF2BP2 and IGF2BP3,were involved in the development of the colon and the progression of colorectal cancer.IGF2BP1 was reported that its overexpression significantly promoted the growth of colon tumor cells.Because of the high similarity between IGF2BP3 and IGF2BP1,and the expression level of IGF2BP3 in most aggressive colorectal cancer was higher than that in adjacent tissues,we speculated that IGF2BP3 may be also involved in the development of colorectal cancer.We first examined the expression of IGF2BP3 in CRC tissue microarray and found that its expression in CRC was significantly higher than that in adjacent tissues.And then we confirmed that overexpression of IGF2BP3 significantly increased the proliferation,clone formation of CRC cells.By immunoprecipitation combined with mass spectrometry analysis,we found that IGF2BP3 interacted with ELAVL1.More importantly,RIP-qPCR analysis found that IGF2BP3/ELAVL1 jointly recognized and bound to multiple mRNAs,including MAP2K1,TPR,KRAS and CCNH.Given that IGF2BP3 and ELAVL1 were reported to stabilize mRNA,we treated CRC cells with actinomycin D,and found that overexpression of IGF2BP3 significantly prolonged the half-life of MAP2K1,TPR,KRAS and CCNH,thereby increased mRNA expression.Importantly,knockdown of ELAVL1 inhibited the binding of IGF2BP3 to candidate mRNA and impaired IGF2BP3-enhanced mRNA stability,indicating that IGF2BP3 regulation of mRNA stability was dependent on ELAVL1.Knockdown of MAP2K1 and TPR significantly inhibited the proliferation of CRC cells.Furthermore,knockdown of MAP2K1 and TPR in IGF2BP3-overexpressing CRC cells impaired the growth-promoting effect of IGF2BP3.Reciprocally,ectopical expression of MAP2K1 or TPR in IGF2BP3-depleted CRC cells restored their proliferative capabilities.Importanly,the KH domains of IGF2BP3 were indispensable for mRNA stabilization.The deletion mutant of KH domain(KH1-4)greatly inhibited IGF2BP3-mediated mRNA stabilization,leading to CRC cell growth inhibition.In summary,our findings reveal the molecular mechanism of IGF2BP3 in stabilizing mRNA in CRC.By interacting with ELAVL1,IGF2BP3 stabilizes carcinogenic mRNA transcripts to promote proliferation of CRC cells.Further studies will be needed to focus on how IGF2BP3 recognize the downstream mRNAs and other potential collaborating protein partners involved in this process to deepen the understanding of function of RBPs in cancer cells.