miR-378a-5p/Sp1/IGF1R调控网络促进结直肠癌发生的分子机制

The expression and biological function of miR-378a-3p and 5p in colorectal cancer (CRC) have not been fully clarified. In our previous study, we found that miR-378a-3p and 5p were down-regulated in CRC compared with matched normal colorectal tissues. Ectopic miR-378a-3p and 5p expression inhibited proliferation and colony formation, induced apoptosis and G1-phase cell cycle arrest in CRC cells; up-regulation of both decreased the level of IGF1R protein. We further verified that IGF1R was a direct target of miR-378a-3p but not of 5p. Bioinformatic analysis showed that Sp1 was the target of miR-378a-5p and Sp1 could bind to the promoter of IGF1R gene directly inducing its transcription. Does miR-378a-5p regulate IGF1R expression indirectly promoting colorectal carcinogenesis by targeting Sp1? It has not been reported in the literature. Taken together, we make a hypothesis that (1) miR-378a-5p can regulate Sp1 expression by targeting Sp1 mRNA 3’UTR directly;(2) transcription factor Sp1 can bind to the promoter of IGF1R gene activating its transcription;(3) miR-378a-5p regulates IGF1R signaling pathway indirectly by targeting Sp1. In this project, we first explore expression of miR-378a-5p, Sp1, and IGF1R in different stages of CRC and their association with clinicopathological features of CRC. Then, we verify whether miR-378a-5p targets Sp1 in CRC cells by Dual Luciferase reporter. Finally, we study the molecular mechanism of miR-378a-5p/Sp1/IGF1R signaling pathway in promoting carcinogenesis of CRC. Our object is to provide new insight into the mechanism of carcinogenesis in CRC and find a novel target for CRC treatment.

申请者前期研究发现miR-378a-3p和5p在结直肠癌中低表达。分别上调两者表达可抑制结肠癌细胞增殖和平板克隆形成并诱导细胞凋亡和细胞周期G1期阻滞，两者高表达可降低IGF1R蛋白水平，进一步研究发现IGF1R是miR-378a-3p而非5p的直接靶标。生物信息学分析Sp1是miR-378a-5p的直接靶标。有报道Sp1能结合IGF1R启动子促进其转录。miR-378a-5p是否通过Sp1间接调控IGF1R参与结直肠癌发生发展未见报道。本课题将在不同发展阶段结直肠癌组织中检测miR-378a-5p、Sp1和IGF1R表达及临床病理关系基础上，采用双荧光素酶报告基因系统验证结肠癌细胞中Sp1是否为miR-378a-5p的直接靶基因，进一步探讨miR-378a-5p/Sp1/IGF1R通路促进结直肠癌发生的分子机制。为阐明结直肠癌发生发展机制提供理论和实验依据，为其靶向治疗提供新的靶点。

申请者前期研究发现miR-378a-5p在结直肠癌中低表达。上调其表达可抑制结肠癌细胞增殖和平板克隆形成并诱导细胞凋亡和细胞周期G1期阻滞，其高表达可降低IGF1R蛋白水平；有报道Sp1能结合IGF1R启动子促进其转录，并且生物信息学分析miR-378a-5p可结合到SP1的3’UTR；采用WB和免疫组化方法证实SP1和IGF1R在结直肠癌新鲜组织和石蜡组织中高表达，并且分析其表达量与临床病理特征之间的关系；SP1和IGF1R的表达量呈正相关；进一步采用双荧光素酶报告基因实验证实SP1能够结合到IGF1R的启动子并促进其表达，而SP1却并不是miR-378a-5p的直接靶标。.随后采用生物信息学预测Sp1是miR-133a-3p的直接靶标；miR-133a-3p是否通过Sp1间接调控IGF1R参与结直肠癌发生发展未见报道。本课题检测了不同发展阶段的结直肠癌组织中miR-133a-3p的表达及与临床病理特征之间的关系，结果表明miR-133a-3p在结直肠癌组织中低表达，起抑癌基因的作用；双荧光素酶实验证实miR-133a-3p能够结合到SP1的3’UTR，抑制其表达；结肠癌细胞株SW480中转入miR-133a-3p mimic后，SP1和IGF1R蛋白表达降低。该课题初步探讨了miR-378a-5p/Sp1/IGF1R通路能否在结直肠癌发生发展中起作用，并且发现了miR-133a-3p/Sp1/IGF1R通路能够促进结直肠癌发生，为阐明结直肠癌发生发展机制提供理论和实验依据，为其靶向治疗提供新的靶点。