1. RNA结合蛋白（RNA binding proteins, RBPs）是真核生物转录后调控的关键分子之一。RBPs与单链或双链RNA结合，决定它们从合成到降解的命运。RBPs通常具有一个或多个结构域，并以序列特异性的方式识别RNA，因此结合RNA的亲和力和特异性不同。除了这些可以直接与RNA结合的结构域之外，RBPs还包含辅助结构域，介导与其他蛋白质的相互作用。当RBPs与mRNA结合时，它们控制着mRNA生命的主要步骤，包括剪接、运输、定位、翻译和降解。RBPs表达和功能的改变都可能造成有害的影响，从而导致许多复杂的疾病，包括癌症和免疫紊乱。胰岛素样生长因子2 mRNA结合蛋白家族（Insulin-like growth factor 2 mRNA-binding proteins, IGF2BPs）是一类高度保守的RNA结合蛋白家族，包括IGF2BP1、IGF2BP2和IGF2BP3，三个成员在结构上均含有2个RNA识别基序（RRMs）和4个hnRNP K同源结构域（KH），能够特异性地识别和结合RNA。其中IGF2BP3又称为IMP3，与癌症联系最为密切，但其在不同恶性肿瘤进展中的报道主要通过免疫组化的方法进行分析，机制研究也多作为下游效应分子。近年来发现IGF2BP3可以通过与靶向mRNA结合发挥作用，更重要的是，IGF2BP3与家族其他两个成员被证实是N6-腺苷酸甲基化（m6A）的新的阅读器，并能够促进靶向mRNA的稳定和储存。作为癌胚胎蛋白，IGF2BP3在多种癌症中的重新合成或异常表达都影响着恶性肿瘤的发生发展，然而，如何影响仍不明确，尤其是在食管鳞癌中的报道几乎为空白。本研究通过构建稳定细胞系明确过表达IGF2BP3后显著抑制了食管癌细胞的增殖、克隆形成、迁移、侵袭以及裸鼠的体内成瘤；相反，敲降IGF2BP3则起到促进作用。IGF2BP3属于RNA结合蛋白，调控着mRNA的定位、翻译和稳定，且靶向mRNA的识别对于阐明其生物学作用至关重要。因此，我们利用RNA免疫共沉淀联合高通量测序（RIP-Seq）在IGF2BP3的数百个甚至上千个与之结合的RNA中筛选鉴定得到4个与食管癌细胞密切相关的候选靶向mRNA即PPP2CA、SMG7、SMAD2和SP3。进一步通过放线菌素D处理细胞，发现过表达IGF2BP3大大增加了候选靶向mRNA的半衰期。为了探讨是否有其他蛋白辅助IGF2BP3发挥功能，我们通过免疫共沉淀（Co-IP）联合质谱分析确定PABPC1和ELAVL1这两个已知的稳定mRNA的RBPs与IGF2BP3存在外源及内源性的结合。接着RIP-qPCR的结果表明PABPC1和ELAVL1都在这4个候选靶向mRNA的3’UTR有明显的的富集，且PABPC1或ELAVL1的缺失明显损害了IGF2BP3对候选靶向mRNA半衰期的延长，揭示了PABPC1与ELAVL1对IGF2BP3在食管癌细胞中发挥作用的重要性。值得注意的是，IGF2BP3在结构上包含2个RRMs和4个KH区域，我们的结果显示IGF2BP3的KH结构域（KH1-4）对于IGF2BP3发挥作用最为关键。最后通过GEO数据库分析临床数据发现PPP2CA在食管癌组织中的表达低于癌旁组织，初步确定其为IGF2BP3在食管癌中发挥作用的关键靶标。再利用RNA pull-down反向验证了PPP2CA与IGF2BP3的特异性结合，之后通过功能实验表明PPP2CA同样具有抑制食管癌细胞增殖的作用，且在过表达IGF2BP3后敲降PPP2CA明显抵消了IGF2BP3的抑制作用。综上所述，IGF2BP3具有抑制食管癌细胞增殖、迁移和侵袭的作用，发挥作用主要是通过与PABPC1和ELAVL1形成复合物来稳定具有潜在抑癌作用的靶向mRNA PPP2CA。本研究为临床上治疗食管癌寻找新靶点提供了一定的理论依据，但还需要更深入的机制探讨。2.近年来的研究表明，多个RNA结合蛋白（RBPs）在结直肠癌中异常表达，其中包括IGF2BPs。IGF2BP家族的三个成员IGF2BP1，IGF2BP2和IGF2BP3参与了结肠的发育和结直肠癌的进展，有报道称过表达IGF2BP1显著促进了结肠肿瘤细胞的生长。三个成员中，IGF2BP3与IGF2BP1的相似性较高，且IGF2BP3在大多数侵袭性结直肠癌中表达水平高于癌旁组织。为了探究IGF2BP3是否也参与了结直肠癌的进展过程，我们首先利用组织芯片检测了IGF2BP3的表达水平，发现其在结直肠癌组织中的表达显著高于癌旁组织。接下来通过功能实验明确了IGF2BP3具有促进结肠癌细胞增殖和克隆形成的能力，且IGF2BP3的缺失抑制了裸鼠的体内成瘤。为了探究作用机制，我们利用免疫共沉淀联合质谱分析，发现IGF2BP3与ELAVL1相互作用。通路富集分析表明，二者共同调控细胞增殖和周期相关的基因。进一步通过数据库预测IGF2BP3和ELAVL1共同调控的靶基因，并经RIP-qPCR验证后发现IGF2BP3和ELAVL1共同识别并结合多个mRNA，包括 MAP2K1，TPR，KRAS和CCNH。基于IGF2BP3与ELAVL1具有稳定mRNA的功能，我们利用放线菌素D处理结直肠癌细胞，发现过表达IGF2BP3后显著增加了MAP2K1，TPR，KRAS和CCNH的半衰期，进而促进mRNA的表达，这可能是IGF2BP3促进结直肠癌细胞增殖的原因之一。重要的是，敲降ELAVL1削弱IGF2BP3与候选靶向mRNA的结合及对其稳定性的调控作用，表明IGF2BP3依赖于ELAVL1发挥其稳定下游mRNA的功能。而作为 IGF2BP3在结直肠癌中发挥作用的关键靶点，MAP2K1和TPR的缺失显著抑制了结直肠癌细胞的增殖。回复实验表明在过表达IGF2BP3的结直肠癌细胞中敲降MAP2K1和TPR后，显著削弱了IGF2BP3的促增殖作用。反过来，在敲降IGF2BP3的细胞中过表达MAP2K1或TPR，细胞的生长得到一定程度的回复。此外，IGF2BP3的KH结构域对其稳定下游mRNA是不可或缺的，KH结构域（KH1-4）的缺失突变使IGF2BP3的促增殖和稳定mRNA的功能大大减弱。综上，我们的研究结果揭示了结直肠癌中IGF2BP3通过与ELAVL1相互作用来调控致癌转录本的稳定性从而促进癌细胞增殖的分子机制。进一步阐明RBPs与肿瘤相关mRNA之间的调控关系，有助于深化对RBPs作用机理的认识。

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. 2. 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.