背景： 基因转录后调控是影响真核生物基因表达的一个重要机制。RNA结合蛋白(RBPs)和非编码RNA都是基因转录后调节者，二者之间存在交互作用，它们与靶RNA结合从而影响后者的稳定性和翻译过程。IGF2BP3是一种重要的RNA结合蛋白，以往的研究发现IGF2BP3在多种肿瘤细胞中高表达，促进肿瘤的生长和转移，但其在肿瘤发生发展中的作用机制还有待于进一步研究。因此，阐明IGF2BP3在肿瘤中的分子调控机制，将为肿瘤诊断和治疗提供新的靶标。 本课题研究发现：IGF2BP3在乳腺癌和口腔鳞状细胞癌(口腔鳞癌)中表达显著高于对照组，干涉其表达有效地抑制肿瘤细胞增殖。进一步研究发现：在乳腺癌， IGF2BP3通过抑制miR-3614成熟而减弱后者对靶基因TRIM25表达的抑制作用，从而保护TRIM25 RNA，促进乳腺癌细胞生长；而在口腔鳞癌，IGF2BP3通过稳定E2F5表达从而促进肿瘤细胞生长。本研究揭示了IGF2BP3与靶RNA相互作用调控肿瘤细胞生长的一种新机制，对了解乳腺癌和口腔鳞癌细胞增殖失控提供了新视角。 方法： 1.通过TCGA数据库和免疫组化分析IGF2BP3在乳腺癌和口腔鳞癌组织中的表达情况；采用qRT-PCR和Western Blot方法检测IGF2BP3在乳腺癌细胞系MCF-7和MDA-MB-231以及口腔鳞癌细胞系SCC-25中的表达情况。 2.利用siRNA干涉IGF2BP3在乳腺癌和口腔鳞癌细胞中表达，qRT-PCR和Western Blot验证干涉效果，通过MTT、流式细胞仪等方法检测干涉IGF2BP3后对细胞增殖和周期的影响，并分析周期调控蛋白CDK4和Cyclin D1的表达变化。 3.通过生物信息学预测IGF2BP3的靶基因。在乳腺癌中，雌激素反应基因TRIM25的3'UTR区存在IGF2BP3的结合位点，同时，在这些结合位点附近有一个pri-miR-3614的转录区，显示TRIM25既是miR-3614的靶基因，同时又是它的宿主基因。我们推测这三者之间可能存在紧密的相互调控作用。RIP测序分析IGF2BP3结合的靶基因，显示E2F5，一种具有促进细胞增殖作用的转录因子，是IGF2BP3的潜在靶基因。 4.分别在乳腺癌细胞和口腔鳞癌细胞中下调IGF2BP3表达，通过qRT-PCR和Western Blot分析TRIM25(在乳腺癌)和E2F5(在口腔鳞癌)的表达变化；并通过qRT-PCR检测乳腺癌中miR-3614成熟体的含量变化，明确IGF2BP3-TRIM25-miR-3614的调控关系。 5.通过RIP-PCR和Pull Down实验明确IGF2BP3与TRIM25和E2F5的靶向结合位点。 6.在乳腺癌细胞中，分别利用pre-miR-3614真核表达载体和miR-3614-3p抑制剂改变miR-3614-3p的表达水平，通过qRT-PCR和Western Blot分析TRIM25的表达变化。根据miRscan网站预测miR-3614-3p结合TRIM25的3'UTR区靶向序列，构建野生型和突变型双荧光素酶报告基因载体，采用双荧光素酶报告系统验证TRIM25与miR-3614-3p的靶向关系，以及miR-3614是否存在自我靶向性。 7.通过MTT、细胞周期和Western Blot检测干涉TRIM25或过表达miR-3614-3p后对乳腺癌细胞增殖和周期的影响，并分析周期调控蛋白CDK4和Cyclin D1的表达变化。同样的方法检测下调E2F5后对口腔鳞癌细胞增殖的影响。 8.在裸鼠皮下荷瘤模型中，分别验证干涉IGF2BP3对乳腺癌和口腔鳞癌生长的抑制作用。 结果： 第一部分：IGF2BP3在乳腺癌细胞通过抑制mLR-3614的成熟保护TRIM25 RNA从而促进肿瘤生长 1.IGF2BP3在乳腺癌组织和细胞系MCF-7和MDA-MB-231中呈高表达，体内外实验均证实下调IGF2BP3表达明显抑制乳腺癌细胞的增殖。 2.通过生物信息学分析发现IGF2BP3与TRIM25呈正相关，TRIM25的3'UTR区存在6个IGF2BP3的结合位点，并且存在pfi-miR-3614的基序，显示TRIM25既是miR-3614-3p的靶基因，同时又是它的宿主基因。qRT-PCR和Western Blot等实验结果显示干涉IGF2BP3表达能够抑制TRIM25的转录和翻译，并促进miR-3614-3p的生成；而过表达miR-3614能够抑制TRIM25表达水平，同时双荧光素酶报告实验证实miR-3614-3p作用于TRIM25 RNA的3'UTR区抑制其翻译。最后，通过RIP、Pull Down实验证实IGF2BP3通过作用于TRIM25的3'UTR区，抑制miR-3614成熟体的剪切，进而减弱miR-3614-3p对TRIM25表达的抑制作用，最终稳定TRIM25的RNA。 3.在乳腺癌细胞中通过干扰RNA下调TRIM25的表达后明显抑制细胞的增殖活性，并阻滞细胞周期于G1期，相应周期蛋白CDK4和Cyclin D1的表达也有所下调，提示TRIM25在乳腺癌中发挥促癌基因作用。 4.过表达miR-3614-3p，导致乳腺癌细胞发生G1期阻滞现象，并抑制周期调控蛋白CDK4和Cyclin D1的表达，结果表明miR-3614-3p在乳腺癌中发挥抑癌基因作用。 第二部分：IGF2BP3在口腔鳞癌细胞通过增强E2F5的表达促进肿瘤生长 1.IGF2BP3在口腔鳞癌组织和细胞系中呈高表达，且体内外实验均证实下调IGF2BP3能够明显抑制口腔鳞癌细胞的增殖。 2.IGF2BP3与E2F5在口腔鳞癌中呈正相关；干涉IGF2BP3表达抑制E2F5的转录和翻译。并通过RIP、Pull Down实验证实IGF2BP3结合于E2F5的3'UTR区加强其mRNA的稳定性。 3.干涉E2F5能够明显抑制口腔鳞癌细胞增殖活性并且阻滞细胞周期于G1期，抑制相应周期蛋白CDK4和Cyclin D1的表达，提示E2F5在口腔鳞癌中发挥促癌基因的作用。 结论： 1.在乳腺癌，IGF2BP3通过抑制miR-3614成熟而减弱后者对靶基因TRIM25表达的抑制作用，从而保护TRIM25 RNA，促进乳腺癌细胞生长。 2.在口腔鳞癌，IGF2BP3通过稳定E2F5促进口腔鳞癌细胞生长。 关键词：IGF2BP3；TRIM25；E2F5；乳腺癌；口腔鳞状细胞癌 论文类型：应用基础

 Backgrounds: Post-transcriptional regulation of genes is an important mechanism that affects eukaryotes gene expression. RNA binding protein (RBPs) and non-coding RNA are both post-transcriptional regulators of genes, and there are interactions between them. Their binding with target RNA affects the stability and translation process of the latter. IGF2BP3 is an important RNA-binding protein. Previous studies have found that IGF2BP3 is highly expressed in a variety of tumor cells, promoting tumor growth and metastasis. However, its mechanism of action in the development of tumors is still unclear. The elucidation of its molecular regulatory mechanisms in tumors will provide new targets for tumor diagnosis and treatment. In this study, it was found that IGF2BP3 expression was significantly higher in breast cancer and oral squamous cell carcinoma (OSCC) than that in the control group, and interference in its expression effectively inhibited tumor cell proliferation. Further studies found that in breast cancer, IGF2BP3 attenuated the inhibitory effect of miR-3614 on TRIM25 expression of target gene by inhibiting the maturation of miR-3614, thus protecting TRIM25 RNA to promote the growth of breast cancer cells. Besides, IGF2BP3 promotes tumor cell growth by stabilizing E2F5 in OSCC. This study revealed a new mechanism of interaction between IGF2BP3 and target RNAs to regulate tumor cell growth, providing a new perspective for understanding the uncontrolled proliferation of breast cancer and OSCC. Methods: 1.The expression of IGF2BP3 in breast cancer and OSCC tissues was analyzed by TCGA database and immunohistochemistry; qRT-PCR and Western Blot were used to detect IGF2BP3 expression in breast cancer cell lines MCF-7 and MDA-MB-231 and OSCC cell line SCC-25. 2.Inhibition of IGF2BP3 expression in breast cancer and OSCC cells by siRNA and verified by qRT-PCR and Western Blot. The effect of si-IGF2BP3 on cell proliferation and cell cycle were detected by MTT and flow cytometry, and the expression changes of cyclin-regulated proteins CDK4 and Cyclin D1 were analyzed. 3.The target genes of IGF2BP3 were predicted by bioinformatics. In breast cancer, the binding sites of IGF2BP3 exists in the 3'UTR region of the estrogen response gene TRIM25. There is also a transcription region of pri-miR-3614 near these binding sites, indicating that TRIM25 is not only a target gene but also a host gene of miR-3614. Thus, we speculate that there may be close interaction between them. Next, RIP sequencing analysis of IGF2BP3-bound target genes revealed that E2F5, a transcription factor that promotes cell proliferation, is a potential target gene for IGF2BP3. 4.Down-regulated IGF2BP3 expression in breast cancer cells and OSCC cell and analyzed the expression changes of TRIM25 (in breast cancer) and E2F5 (in OSCC) by qRT-PCR and Western Blot. Using qRT-PCR detected the expression of miR-3614-3p in breast cancer to define the regulatory relationships of between IGF2BP3-TRIM25-miR-3614. 5.Targeted binding sites of IGF2BP3 with TRIM25 and E2F5 were determined by RIP-PCR and Pull Down experiments. 6.In breast cancer cells, the expression level of miR-3614-3p was altered by pre-miR-3614 overexpression vector and miR-3614-3p inhibitor to detect the expression of TRIM25 by qRT-PCR and Western Blot. Based on miRscan website prediction of miR-3614-3p binding sites to TRIM25 3'UTR region, we constructed wild-type and mutant type of pmiRGLO dual-luciferase reporter vector. Using dual-luciferase reporter system to verify the targeting relationships between TRIM25 and miR-3614-3p, and miR-3614 self-targeting. 7.MTT, cell cycle and Western Blot were used to detect the effect of si-TRIM25 or overexpression of mir-3614-3p on the proliferation of breast cancer cells. The same methods were used to examine the effect of downregulating E2F5 on the proliferation of OSCC cells. 8.In the subcutaneous tumor-bearing model of nude mice, the inhibitory effects of IGF2BP3 intervention on the growth of breast cancer and OSCC. Results: Part I: IGF2BP3 protects TRIM25 RNA in breast cancer cells by inhibiting the maturation of miR-3614 to promote tumor growth 1.IGF2BP3 is highly expressed in breast cancer tissues and cell lines MCF-7 and MDA-MB-231. Both in vitro and in vivo experiments have confirmed that downregulation of IGF2BP3 significantly inhibits the proliferation of breast cancer cells. 2.Bioinformatics analysis found that IGF2BP3 was positively related to TRIM25. There were six IGF2BP3 binding sites in the 3'UTR region of TRIM25, and also a motif of pri-miR-3614. It showed that TRIM25 is the target gene and host gene of miR-3614-3p. The results of qRT-PCR and Western Blot experiments showed that si-IGF2BP3 inhibited the transcription and translation of TRIM25 and promoted the production of miR-3614-3p. However, overexpression of miR-3614 can inhibit the expression of TRIM25. Double fluorescence report confirmed that miR-3614- 3p acts on the 3' UTR region of TRIM25 RNA to inhibit translation. Finally, RIP and Pull Down experiments confirmed that IGF2BP3 inhibited the cleavage of miR-3614 matures by acting on the 3' UTR region of TRIM25, thereby weakening the inhibitory effect of miR-3614-3p on TRIM25 expression, and finally stabilizing TRIM25 RNA. 3.Downregulation of TRIM25 expression by RNA interference in breast cancer cells significantly inhibited cell proliferation and arrested the cell cycle in G1 phase. The corresponding cyclin CDK4 and Cyclin D1 expression was also down-regulated, suggesting that TRIM25 play the role of oncogene effect in breast cancer. 4.Overexpression of miR-3614-3p resulted in G1 arrest in breast cancer cells and inhibition of the expression of the cyclic regulatory proteins CDK4 and Cyclin Dl. The results indicated that miR-3614-3p plays a tumor suppressor gene role in breast cancer. Part II: IGF2BP3 promotes tumor growth in OSCC cells by enhancing the expression of E2F5 1.IGF2BP3 is highly expressed in OSCC tissues and cell line, and both in vivo and in vitro experiments have confirmed that downregulation of IGF2BP3 can significantly inhibit the proliferation of OSCC. 2.IGF2BP3 and E2F5 are positively related in OSCC. Downregulated of IGF2BP3 inhibited the transcription and translation of E2F5. The RIP and Pull Down experiments confirmed that the binding of IGF2BP3 to the 3' UTR region of E2F5 enhanced its mRNA stability. 3.Downregulation of E2F5 significantly inhibited the proliferation of OSCC cells and arrested cell cycle in G1 stage. Conclusions: 1.In breast cancer, IGF2BP3 attenuates the inhibitory effect of miR-3614 on the expression of target gene TRIM25 by inhibiting the maturation of miR-3614, thus protecting TRIM25 RNA to promote the growth of breast cancer cells. 2.IGF2BP3 promotes tumor cell growth in OSCC by stabilizing E2F5. KEY WORDS: IGF2BP3; TRIM25; E2F5; Breast cancer; OSCC TYPE OF DISSERTATION: Application Fundamentals