从表观遗传调控角度探讨miRNA-140对骨肉瘤恶性表型的抑制作用

Disruptions of stem cell osteogenic differentiation can lead to the development of osteosarcoma(OS). Previous researches have indicated that up-regulated expression of osteogenic regulator Runx2 can promote OS cells to differentiate osteogenically, and can inhibit OS cells to proliferate. However, the function of Runx2 can be epigenetically inhibited by HDAC4. Our researches found that 1) OS tissue exhibited high endogenous expression level of HDAC4, whereas low endogenous expression level of miRNA-140. 2) HDAC4 is the target gene of miRNA-140. 3) exogenous expression of miRNA-140 restrains the migration of OS cells and downregulates HDAC4. On this basis, we postulate that miRNA-140 inhibit OS cells proliferation and promote OS cells osteogenic differentiation through down-regulating HDAC4 to epigenetically reactivate the function of Runx2. In this study, we expect to investigate the effect and mechanism of miRNA-140 inhibiting the malignant phenotypes of osteosarcoma from the role of epigenetic regulation through the following experiments: miRNA-140 down-regulation and up-regulation experiments in OS cells, OS cells osteogenic differentiation experiment regulated by miRNA-140, the osteaosarcoma-burdened experiment in nude mice, as well as the following methods: malignant phenotype detection, gene cloning and silencing, protein expression and etc. The purposes of this study are to find miRNAs that could effectively inhibit the development of OS, and to establish a theoretical base of bio-therapy for OS. Meanwhile, this research will further expound the mechanism of epigenetic regulation in the genesis, development and lapse of tumors.

骨肉瘤（OS）是干细胞成骨分化障碍形成的恶性骨肿瘤。既往研究发现:上调成骨转录基因Runx2可诱导OS细胞成骨分化、并抑制其增殖，而Runx2受HDAC4的表观负调控。我们研究发现:1）OS组织高表达HDAC4，但低表达miR140；2）HDAC4是miR140的靶基因；3）转染miR140抑制了OS细胞的迁移并下调了HDAC4的表达。据此，我们的推测:miR140通过HDAC4表观调控Runx2，从而抑制OS细胞增殖，并促其成骨分化。本研究拟通过OS细胞miR140抑制实验，过表达实验，miR140调控OS细胞成骨分化实验和OS荷瘤实验，以及通过OS恶性表型检测、基因克隆和沉默、蛋白质表达等技术方法，从表观遗传角度探讨miR140抑制OS恶性表型的效果和机制。研究旨在探寻有效调控OS的miRNA，为OS的生物治疗奠定基础。同时，将进一步阐释表观遗传调控在肿瘤发生、发展及转归中的作用机制。

众多研究证实miRNAs在肿瘤的发生发展扮演着重要的角色。本研究首先检测了40例骨肉瘤(OS)患者肿瘤组织与癌旁组织中miR-140 的表达，结果显示miR-140在OS组织中表达下调.然后我们鉴定了HDAC4是miR-140的直接靶基因，同时我们在OS细胞系HOS、U2中过表达miR-140和敲低HDAC4(si-HDAC4)，观察其对细胞增殖、周期、凋亡、迁移及侵袭的影响，结果显示miR-140以及si-HDAC4可明显的抑制细胞增殖、迁移及侵袭，同时早期凋亡的细胞明显增多。miR-140通过直接靶向HDAC4在OS发展中发挥作用。最后，我们利用裸鼠进行荷瘤实验，多点注射miR-140 mimics 观察其对瘤块生长的影响，进一步证明了过表达miR-140可明显地抑制肿瘤的生长。我们研究证实miR-140能够明显抑制OS细胞的增殖，促进肿瘤细胞的凋亡，抑制肿瘤细胞的周期进程，上述作用可通过抑制HDAC4的表达实现的。我们的实验为miR-140用于OS的诊断和治疗奠定了生物学基础。