结直肠癌肝转移相关miR-199b通过RISC调控lnc-AC012313.1-1表达的作用机制

Tumor metastasis could be regulated by miRNAs and long non-coding RNAs (lncRNAs). Previously we have found that both miR-199b and lnc-AC012313.1-1 have a close relationship with liver metastasis of colorectal cancer (CRC); lnc-AC012313.1-1 could be negatively regulated by miR-199b; RNA-induced silencing complex (RISC) may be a key mechanism during this process. This study plans to elucidate the regulative mechanism between lnc-AC012313.1-1 and miR-199b during the the liver metastasis process of CRC; We will prove the sequence specific targeting between miR-199b and lnc-AC012313.1-1, and that the regulation of lnc-AC012313.1-1 by miR-199bis through the RISC, with Ago2 as its core protein. What’s more, phosphorylation of Ago2 during this process will be studied. Finally, we will verify that it is through the regulation of lnc-AC012313.1-1 that miR-199b participate in the liver metastasis of CRC. This study will provide us with the regulation mechanisms between miR-199b and lnc-AC012313.1-1, so as to providing new therapeutic targets for the treatment of colorectal cancer.

miRNA、长链非编码RNA(lncRNA)调控肿瘤侵袭转移。我们前期发现：miR-199b和lnc-AC012313.1-1与结直肠癌肝转移相关；miR-199b负性调控lnc-AC012313.1-1；形成RNA诱导沉默复合体（RISC）可能为重要机制。本课题将明确miR-199b通过调控lnc-AC012313.1-1影响结肠癌侵袭转移；验证miR-199b与lnc-AC012313.1-1序列特异性结合；明确miR-199b通过Ago2蛋白形成RISC参与lnc-AC012313.1-1调控；探索Ago2磷酸化在 miR-199b调控lnc-AC012313.1-1表达的作用；动物体内验证miR-199b通过调控lnc-AC012313.1-1参与结直肠癌肝转移。本课题拟阐明miR-199b通过lnc-AC012313.1-1调控结直肠癌肝转移的作用机制，希望为治疗提供新靶点。

肝转移是结直肠癌患者最常见的远处转移方式，与患者预后密切相关。miRNA和长链非编码RNA（lncRNA）分子都可参与恶性肿瘤的侵袭转移并相互调控。我们在前期研究中发现，miR-199b分子与结直肠癌肝转移密切相关，而lnc-AC012313.1-1（亦作linc-ZNF837）也是一个潜在参与了结直肠癌肝转移的分子，且两者间可能存在相互作用共同参与调控结直肠癌肝转移过程，但具体机制尚不完全清楚。本研究发现，lnc-AC012313.1-1在结直肠癌肿瘤组织中的表达水平显著高于正常组织（P＜0.05）。与患者临床病理因素分析发现，lnc-AC012313.1-1高表达与肿瘤脉管侵犯及肿瘤转移相关，且可能提示患者的不良预后。体外实验证实，干扰lnc-AC012313.1-1表达后，可以显著抑制结肠癌细胞系的增殖、迁移、克隆形成能力，并且可促进细胞凋亡发生。在体内实验方面，我们通过建立裸鼠体内肝转移模型证实，干扰lnc-AC012313.1-1后，可以显著抑制结肠癌细胞系RKO发生肝脏转移的能力。通过基因芯片，我们发现lnc-AC012313.1-1调控肿瘤转移可能是通过作用于COL4A2、FXYD5、MMP10等基因而实现的。双荧光素酶报告基因实验证实miR-199b可与lnc-AC012313.1-1直接结合。干扰Ago2后，miR-199b与lnc-AC012313.1-1的结合作用消失。这提示miR-199b是通过与Ago2蛋白结合形成RNA诱导沉默复合体（RISC）实现对lnc-AC012313.1-1的沉默作用。在SW480细胞中，lnc-AC012313.1-1与miR-199b的表达水平存在相互负向调控关系。功能逆转实验显示，lnc-AC012313.1-1的促进结肠癌细胞增殖和迁移的作用可被过表达miR-199b所逆转。有关lnc-AC012313.1-1/miR-199b相互调控关系的研究有助于进一步理解结直肠癌肝转移的分子机制，并为临床治疗提供潜在靶点。