内源性钙调神经磷酸酶调节剂RCAN1在肺癌骨转移中的作用及机制研究

Cancers of lung and bronchus is the third most commonly diagnosed types of cancer and the leading cause of cancer death. Development of skeletal metastasis occurs in over 30% to 40% of lung cancer cases. It may decrease the quality of life and increase the rate of death for cancer patients. No curative therapy exists for bone metastasis and this clinical management is generally palliative. Little progress has been made in treating skeletal metastasis. New molecules and mechanisms related to osteotropism metastasis are needed to be studied. . Previously we found that calcineurin overexpresses in SBC-5 which has special priority in metastasis to bone in a multiple-organ metastasis NOD-SCID mice model，then we testified that CnAα is closely related to osteotropism metastasis of SCLC and a candidate tumor promotor gene for developing bone metastases. We found groundbreakingly that CaN may play a role in identification, attachment and lodgement in bone of SCLC by increasing integrin expression though CaN-NFAT- NF-κB-αvβ3 pathway. In vivo down or up regulation of CnAα gene expression can only decrease or increase the bone metastasis rate, but not affect the metastasis rate to the visceral organs. .RCANs (Regulators of Calcineurin) are endogenous proteins that interact with calcineurin and alter its function by interfering with calcineurin/nuclear factor in activated T cells (NFAT) binding. This interference reduces the nuclear import of NFAT and its subsequent function as a transcription regulator. Previously we found that RCAN1 expression levels between SBC-5 and SBC-3 are different and overexpression of RCAN1 was associated with reduced calcineurin activity. Some studies show that RCNA1 also play a role in cancer and present a motility suppressor gene in vitro. However the roles of RCAN1 in SCLC and osteotropism of SCLC remain to be elucidated. .The present study is to research the function of RCAN1 on SCLC in vitro and vivo and to prove that the RCAN1 is a functional osteotropism metastasis suppressor gene. It reduces attachment and lodgement of SCLC to bone by down-regulation integrin αvβ3 expression though direct binding and inhibiting of Calcineurin.Understanding the interactions between RCAN1-calcineurin-NFAT-NF-κB-αvβ3 pathway and bone metastasis may help identify these potential targets for chemotherapeutic intervention to halt tumor growth and bone metastasis.

肺癌是发病率和死亡率都非常高的恶性肿瘤，约30%-40%的肺癌患者会发生骨转移，是降低患者生活质量、增加癌症相关死亡的重要原因。对肺癌骨转移机理的研究是研发新的治疗方法的前提，具有重要的意义。我们前期研究发现，在发生骨转移的小细胞肺癌中过表达的钙调神经磷酸酶CaN通过NFAT上调αvβ3表达、在癌细胞识别、粘附、定居于骨发挥作用，以siRNA下调CaN、体内可特异性减少骨转移的发生，首次证实CaN是小细胞肺癌嗜骨转移促进因子；RCAN1做为CaN内源性抑制剂，在不同骨转移潜能的小细胞肺癌中差异表达并与CaN活性呈负相关；已知其体外可下调部分癌细胞转移能力，但RCAN1对小细胞肺癌及其骨转移的作用未见报道。我们推测：RCAN1是小细胞肺癌骨转移抑制因子;拟应用分子生物学、细胞、动物实验验证以上推测，探讨RCAN1通过CaN抑制肺癌骨转移的作用及机制，为将来肺癌骨转移的防治奠定基础。

我们通过real-time PCR检测RCAN1 mRNA在SBC-5中含量为SBC-3的60%；WB检测RCAN1的蛋白在SBC-5中的表达比SBC-3中低，p<0.05；应用比色法检测CaN活性在SBC-5中比SBC-3高，p<0.05。构建了RCAN1表达载体GV146-RCAN1，筛选获得了RCAN1稳定高表达的细胞系GV146-RCAN1-SBC-5，其RCAN1mRNA较GV146-NC-SBC-5增高21.95788倍，p=4.25379E-13；RCAN1蛋白相比增加3.11倍，p=2.684E-8。构建RCAN1-RNAi慢病毒载体GV248并获得GV248-RCAN1-RNAi-SBC-3细胞。其RCAN1mRNA较GV248-NC-SBC-3及SBC-3下降了94%，p=7.4425E-11；RCAN1蛋白相比下降了53%，p=1.1313E-9。通过实验观察上调RCAN1抑制了SBC-5的增殖和克隆形成能力（PcolonSBC-5 6.07515 E-6）；阻滞了其G1向G2期的转化；PG1=0.50071 E-4、PS=0.40301、PG2=1.09819 E-4；减弱了迁移和侵袭能力，P分别为1.61995E-10,9.98117 E-8；减弱了骨片粘附能力，P=0.00504；而敲减RCAN1后SBC-3的增殖和克隆形成能力显著升高（PcolonSBC-3为7.33517 E-7）；促进了G1向G2期的转化，PG1=0.21647、PS=0.0174、PG2=6.59563E-5；提高了迁移和侵袭能力，P值分别为：3.68044E-8,1.00032E-9；提高了其骨片粘附能力，P=0.01924。但对两组细胞的凋亡没有产生有意义的影响，P分别为0.22335、0.06019、0.51723、0.6286。.随后拟通过骨陷窝形成实验、肺癌细胞与骨片共培养系统观察RCAN1对破骨细胞分化成熟的影响；利用肺癌骨转移动物模型，研究RCAN1对肺癌细胞骨转移能力的影响；进一步通过电泳迁移率分析法（EMSA）、明胶酶谱法等方法分析RCAN1通过CaN-NFAT通路对基质金属蛋白酶和整合素αⅤβ3表达的作用。.由于目前文章发表周期比较长，研究内容对应的SCI论文正在撰写中，尚未发表，也有待今后研究结果的补充。