LUNAR1作为ceRNA与miR-630相互作用抑制Notch信号调控结直肠侵袭转移的作用和机制

Notch signal plays important role in cancer. We have previously proved that Notch1 could promote colorectal cancer invasion and metastasis and found that this effect might be through its regulation on miR-630 and LUNAR1. However, in clinical colorectal specimens, we found a significantly negative correlation between the expression of miR-630 and LUNAR1. For that, we found there was specific binding sequence position of miR-630 on LUNAR1 with relatively low free energy through bioinformatics analysis. Based on these results, we deduced that LUNAR1 might inhibit miR-630 by ceRNA effect, which together play a suppressive effect in the oncogenic effect of Notch pathway. In the present study, we are going to prove the ceRNA effect of LUNAR1 on miR-630; use reporter gene and RIP to clarify the mechanism by which LUNAR1 regulate miR-630; verify this ceRNA effect on the oncogenic role of Notch signal on colorectal cancer in cell lines and animal models; use array and iTRAQ to screen effector of this pathway and verify those molecules.

Notch信号在肿瘤中的作用复杂多样，申请人率先发现Notch1在结直肠癌与侵袭转移正相关，并发现Notch1可通过诱导下游的miR-630和长链非编码LUNAR1促进肿瘤侵袭转移。但是在临床大样本中却发现LUNAR1与miR-630的表达负相关，为此申请人通过生物信息学分析发现LUNAR1序列上存在miR-630的特异性结合位点，并且自由能较低。根据上述研究，申请人提出了LUNAR1通过ceRNA效应与miR-630相互作用抑制Notch信号通过非编码RNA途径促进结直肠癌侵袭转移的推论。在本研究中，申请人将利用多种方法证明LUNAR1与miR-630的作用方式；利用报告基因、R-IP等方法阐明LUNAR1对miR-630的作用机制；在细胞系和动物模型中验证LUNAR1与miR-630的相互作用对Notch信号促癌作用的影响；并利用芯片和iTRAQ等技术筛选通路下游的效应分子并验证。

非编码RNA在结直肠癌侵袭转移中发挥十分重要的作用。miR630是Notch1信号通路下游十分重要的分子，发挥促癌作用。本项目聚焦长链非编码LUNAR1与miR-630在结直肠癌侵袭转移中的作用及机制开展一系列研究。首先，我们证实LUNAR1在结肠癌中的表达与miR-630的表达呈负相关。LUNAR1在结直肠癌组织中低表达，miR-630在结肠癌组织中高表达，且在临床III期癌组织中表达更高。其次，我们证实miR-630过表达能够促进结肠癌细胞侵袭迁移，而miR-630 inhibitor能够逆转miR-630对结肠癌细胞侵袭迁移的促进作用。过表达miR-630时，EMT的上皮标记E-钙粘蛋白表达减少，间质标记波形蛋白表达增加，表明miR-630可能通过对EMT的促进作用促进了结肠癌细胞的侵袭迁移。第三，LUNAR1可能通过调控miR-630及其效应靶分子发挥促进结肠癌侵袭转移的作用。第四，KLF6是miR-630的下游靶基因。miR-630过表达显着降低KLF6 mRNA和蛋白的表达，而转染miR-630 inhibitor则增加了KLF6 mRNA和蛋白的表达。双萤光素酶报告基因检测发现miR-630 mimic降低了HEK 293T细胞中包含KLF6基因的报告载体的荧光强度，表明miR-630直接靶向KLF6。下调KLF6的表达，产生了与过表达miR-630相似的促进结肠癌侵袭迁移的表型。KLF6 siRNA和miR-630 mimic的共转染显着增强SW480和SW620细胞的迁移和侵袭，而KLF6 siRNA和miR-630 inhibitor的共转染抵消了对迁移侵袭的增强作用，表明miR-630靶向KLF6以促进结肠癌细胞的迁移和侵袭。最后，进一步研究miR630及其靶基因KLF6对EMT相关转录因子Snail的调控作用发现，KLF6的下调导致Snail表达显着增加。将miRNA-630 mimic或miR-630 inhibitor与KLF6 siRNA共转染，结果发现KLF6的下调和miR-630的上调增加了snail的表达，而与KLF6 siRNA和miR-630 inhibitor共转染可逆转这种作用。本项目研究揭示了miR-630通过其靶基因KLF6促进结直肠癌侵袭转移的相关分子机制，为提出新的分子干预靶点提供了理论依据。