BMI1和KLF4双信号通路阻滞在骨肉瘤干细胞的自我更新和肺转移中的作用及机制研究

The recurrence and metastasis of osteosarcoma (OS) is a major problem in clinical treatment. Recently studies show the existence of a small sub-population of osteosarcoma stem cells (OSCs) with infinite self-renewal capacity is the main cause of this clinical problem. Targeting the key regulators that selectively act on OSCs self-renewal may be an effective approach for its recurrence and metastasis. Both Bmi1 and KLF4 are involved in the initiation of OS and play a critical role in maintaining the self-renewal of the OSCs. Our preliminary study showed that Bmi1 and KLF4 were overexpressed in osteosarcoma and OSCs, while silencing Bmi1 or KLF4 reduced OSCs self-renewal and invasiveness, especially at the same time silencing the two genes，suggesting that Bmi1 and KLF4 play an important role in osteosarcoma OSCs self-renewing signaling pathways and metastases, but how they are regulated remains unclear. We also found that low expression of miR-135 in osteosarcoma and OSCs, but overexpression of miR-135 inhibited the expression of Bmi1 and KLF4, indicating that this new miR-135 / KLF4 / Bmi1 signaling pathway may play an important regulatory role in OSCs self-renewal. We will construct lentiviral plasmids expressing miR-135 and silencing both KLF4 and Bmi1 as well as tumor-specific delivery system (NanoKor). The roles and the mechanisms of miR-135 and KLF4 / Bmi1 in the OSCs self-renewal and metastasis of OS will be investigated in vitro and in vivo. This study will provide a new strategy for the treatment of relapsed and metastatic osteosarcoma.

骨肉瘤的转移是临床治疗的主要难题,研究表明骨肉瘤干细胞(OSCs)的存在是骨肉瘤转移的主要原因。我们前期研究发现: Bmi1和KLF4在骨肉瘤和OSCs中高表达,而沉默Bmi1或KLF4使OSCs自我更新和侵袭力下降，尤以同时沉默这两个基因的作用明显。提示：Bmi1和KLF4在骨肉瘤OSCs自我更新信号通路和转移中发挥重要作用，但其如何被调控仍不清楚。我们还发现，骨肉瘤中miR-135低表达,而过表达miR-135可抑制Bmi1和KLF4表达。表明这个新的miR-135/KLF4/ Bmi1信号通路对骨肉瘤OSCs自我更新能力有重要的调控作用。本课题组将构建高表达miR-135和沉默KLF4/ Bmi1的慢病毒质粒和肿瘤特异靶向递送系统（NanoKor）,探讨miR-135在体内外通过调控KLF4/ Bmi1及其信号通路,从而阻断骨肉瘤生长转移的机制。该研究将为治疗转移性骨肉瘤提供新思路。

骨肉瘤的转移是临床治疗的主要难题,研究表明骨肉瘤干细胞(OSCs)的存在是骨肉瘤转移的主要原因。我们前期研究发现: Bmi1和KLF4在骨肉瘤和OSCs中高表达,而沉默Bmi1或KLF4使OSCs自我更新和侵袭力下降，尤以同时沉默这两个基因的作用明显。提示：Bmi1和KLF4在骨肉瘤OSCs自我更新信号通路和转移中发挥重要作用，但其如何被调控仍不清楚。我们还发现，骨肉瘤中miR-135低表达,而过表达miR-135可抑制Bmi1和KLF4表达。表明这个新的miR-135/KLF4/Bmi1信号通路对骨肉瘤OSCs自我更新能力有重要的调控作用。本课题组在本研究中，我们发现miR-135a能够通过3‘-UTR与BMI1和KLF4结合，抑制BMI1/KLF4在OS中的翻译或降低BMI1/KLF4 mRNA的表达,miR-135在体内外通过调控KLF4/ Bmi1及其信号通路,从而阻断骨肉瘤生长转移。此外我们还发现，miR-135a还抑制了MMP2和MMP9的表达，抑制了MMP的活化，表现为FMT中MMPSense强度的降低，这在促进肿瘤的侵袭和转移中起着重要的作用。miR-135a的恢复通过靶向BMI1和KLF4在体内外抑制肿瘤的侵袭和肺转移，该研究将为治疗转移性骨肉瘤提供新思路。