MiR-148b调控长链非编码RNA MCF2L-AS1参与胃癌细胞侵袭转移的机制研究

Gastric cancer is one of the most common cancers with a heavy mortality in China. Metastasis and diffusion are the leading causes of poor prognosis. In our previous work, we have already found significant down-regulation of miR-148b in gastric cancer tissues. Moreover, subsequent experiments confirmed that miR-148b inhibited cell proliferation in gastric cancer, depending on negative regulation of the expression of CCKBR. On the other hand, overexpression of miR-148b in gastric cancer could inhibit the invasion capacity significantly. Using microarray analysis and PCR, we found that the expression of growth arrest-specific 6(GAS6), which was related to aberrant proliferation and invasion in gastric cancer, was significantly reduced in cells which were overexpression of miR-148b. However, GAS6 is not the target gene of miR-148b. Moreover,using microarray analysis, we found the expression of long non-coding RNA MCF2L-AS1 was significantly increased in cells which were overexpression of miR-148b. In addition, bioinformatic analysis indicated that MCF2L-AS1 just located in the upstream of GAS6 gene. Furthermore, after interfering the expression of MCF2L-AS1 with siRNA, GAS6 mRNA expression was significantly increased. Taken together, we try to further explore the downstream molecular mechanisms of miR-148b, as well as to demonstrate that miR-148b plays an important role in the invasion and metastasis of gastric cancer through regulating the expression of MCF2L-AS1 and then affecting GAS6 expression.

胃癌是我国主要恶性肿瘤之一，转移扩散是其预后不良的首要原因。申请者前期工作率先发现miR-148b在胃癌组织中显著低表达，并通过负性调控CCKBR表达，影响细胞增殖；同时发现过表达miR-148b的胃癌细胞侵袭能力显著下降。经芯片筛查和PCR验证发现与胃癌异常增殖和侵袭相关的生长停滞特异性基因6(GAS6)的表达水平在过表达miR-148b的胃癌细胞中显著降低。然而，GAS6并不是miR-148b的靶基因。经芯片筛查miR-148b过表达前后胃癌细胞中长链非编码RNA的变化,发现MCF2L-AS1在过表达miR-148b胃癌细胞中显著高表达，而生物信息学分析提示它恰位于GAS6基因的上游,并且siRNA干扰其表达后，GAS6表达水平升高。基于此，我们拟深入探寻miR-148b下游调控的分子机制，试图证明miR-148b通过调控MCF2L-AS1表达进而影响GAS6的表达，参与胃癌侵袭转移。

胃癌是我国主要恶性肿瘤之一，转移扩散是其预后不良的首要原因。申请者前期工作率先发现 miR-148b 在胃癌组织中显著低表达，并能抑制胃癌细胞的增殖与侵袭能力。经芯片筛查和 PCR 验证发现与胃癌异常增殖和侵袭相关的生长停滞特异性基因 6(GAS6)的表达水平在过表达miR-148b 的胃癌细胞中显著降低，同时GAS6上游基因lncRNA-MCF2L-AS1在过表达miR-148b 的胃癌细胞中显著高表达。基于此，我们拟深入探寻 miR-148b 下游调控的分子机制，试图证明 miR-148b 通过调控 MCF2L-AS1 表达进而影响 GAS6 的表达，参与胃癌侵袭转移。.本项目在国自然基金委资助下按计划顺利进行并完成预期目标。我们首先在胃癌细胞中明确了miR-148b、MCF2L-AS1及GAS6表达的相关性：miR-148b可以促进MCL2L-AS1并抑制GAS6的表达，而MCL2L-AS1则可以抑制GAS6表达。通过构建稳定转染细胞系，应用划痕实验、Transwell 小室侵袭实验、CCK8、流式细胞术、 Annexinⅴ-FITC 染色凋亡等实验方法，我们证实了MCL2L-AS1可以抑制胃癌细胞的增殖与侵袭能力，影响细胞周期分布，并促进细胞凋亡。随后，通过在沉默MCF2L-AS1的细胞系中恢复miR-148b的表达，我们证明了miR-148b可以调控MCF2L-AS1对胃癌细胞的作用。紧接着，我们应用real-time PCR及western blotting在稳定过表达/沉默细胞系中证明了miR-148b与MCL2L-AS1均能调控GAS6及其下游通路蛋白的表达。同时，我们在GAS6过表达细胞中恢复MCF2L-AS1，证明了lncRNA-MCF2L-AS1通过抑制GAS6的表达进而抑制了胃癌细胞系的增殖、侵袭能力。最后，通过裸鼠移植瘤实验，我们在体内证明了MCF2L-AS1对胃癌侵袭、转移的抑制作用。.综上，我们构建了miR-148b通过MCF2L-AS1调控GAS6进而调控胃癌细胞增殖、侵袭的非编码RNA调控网络，为胃癌的早期诊断与精准治疗提供了新的靶点。同时我们针对CLDN4的非编码RNA调控网络及其他的lncRNA位点也行了深入的研究，试图找到新的诊断及联合治疗靶点。基于上述研究成果发表SCI论文12篇，培养博士生7人，硕士生2人，社会效益显著。