MicroRNA-19a/b通过正反馈环路促进胃癌细胞转移的机制研究

It has not been elucidated why malignant cancer cells are prior to metastasize. From what we have studied prior to others, the highly expression of miR-19a/b had positive correlation with the metastasis of gastric cancer. From functional assays, we found that miR-19a/b promoted the in vitro and in vivo metastatic ability of gastric cancer cells. Furthermore, report gene assay and functional assays revealed that MXD1 is a direct target of miR-19a/b. Moreover, the highly expression of MXD1 suppressed the expression of c-Myc and miR-19a/b and inhibited the malignant phenotypes of gastric cancer cells. From what has been reported, the proto-oncogene c-Myc directly trans-activates the expression of miR-19a/b and MXD1 could directly or indirectly antagonize the function of c-Myc. Furthermore, the above molecules were reported to be associated with cancer metastasis. Therefore, we rank the first in the world to speculate that the feedback loop constituted by c-Myc, miR-19a/b and MXD1 might be involved in the process of gastric cancer metastasize. In future studies, we will first explore the expression pattern of miR-19a/b, MXD1 and c-Myc in metastasized gastric cancer tissues and cells. Secondly, using gene transfection assay and functional assays, we will change the expression levels of the above molecules and explore their regulatory ability in the metastasis of gastric cancer cells. Furthermore, report gene assay, CoIP, western blot and real-time PCR will be used to explore the existence of the potential positive feedback loop (c-Myc－miR-19a/b－MXD1) and the mechanisms in regulating the metastasis of gastric cancer. The present study would provide theoretical evidence and would be helpful for the understanding of cancer metastasis, the finding of new cancer metastatic markers, and the designation of new anti-metastatic therapeutic strategies.

恶性肿瘤转移的调控机制尚未完全阐明。我们在国际上率先发现：miR-19a/b的高表达与胃癌转移正相关；miR-19a/b能促进胃癌细胞的体内外转移；MXD1是miR-19a/b的靶基因；MXD1的高表达能抑制c-Myc和miR-19a/b的表达并阻遏胃癌恶性表型的发生。文献回顾提示：c-Myc能直接转录激活miR-19a/b；MXD1能直接或间接拮抗c-Myc的功能；上述分子均与肿瘤转移相关。我们推测，c-Myc-miR-19a/b-MXD1组成的环路可能参与了胃癌的转移。本项目拟先明确上述分子在胃癌转移细胞和组织中的表达规律，再利用基因转染分别调变它们的表达，通过体内外转移实验阐明它们对胃癌转移的调控，并借助报告基因实验、免疫共沉淀、western blot、PCR等明确上述正反馈环路的存在及其对胃癌转移的调控机制，为明确miRNA对胃癌转移的调控作用及机制提供理论依据。

肿瘤转移是恶性肿瘤患者死亡的主要原因，但是其调控机制仍未完全阐明。MicroRNA是一类在转录后水平调控靶基因蛋白表达的非编码小RNA分子，它们在恶性肿瘤转移的发生过程中发挥了重要的调控作用。我们前期对miR-17-92与胃癌恶性表型进行研究时发现miR-17-92成员miR-19a/b的表达与胃癌患者淋巴结和远处转移密切相关，miR-19a/b能够促进胃癌细胞的体内外侵袭和迁移能力，并且通过靶基因MXD1发挥功能；进一步研究发现MXD1能够抑制胃癌细胞恶性表型的发生，影响miR-19a/b的体外侵袭能力，并能够抑制miR-19a/b及miR-19a/b的上游转录因子c-Myc的表达。此外，利用本实验室前期从胃癌亲本细胞SGC7901和MKN28中筛选出的两株转移能力不同的细胞（SGC7901高转移潜能细胞和低转移潜能细胞、MKN28高转移潜能细胞和低转移潜能细胞）研究胃癌肿瘤微环境的过程中，我们发现SGC7901和MKN28高低转移胃癌细胞均能够分泌外泌体，高转移胃癌细胞分泌的外泌体能够促进低转移胃癌细胞的转移、侵袭、发生上皮间质转化(EMT)，这一功能可能通过激活低转细胞的NF-κB通路实现；进一步利用miRNA芯片筛选高转移胃癌细胞和低转移胃癌细胞的miRNA表达差异，发现在高转移胃癌细胞内表达上升两倍的miRNA中，有三个miRNA（miR-17-5p，miR-20a-5p，miR-19b-3p）属于miR-17-92基因簇。进一步研究发现，高转移胃癌细胞分泌的外泌体携带miR-17-92基因簇能够通过靶向PTEN，激活AKT，从而激活NF-κB通路来发挥促进低转移胃癌细胞转移的功能。本研究表明miR-17-92基因簇能够通过c-Myc－miR-19a/b－MXD1—c-Myc环路促进胃癌细胞的转移；并且miR-17-92基因簇能够通过激活NF-κB通路在恶性程度较强的胃癌细胞分泌的外泌体内发挥促进恶性程度较弱的胃癌细胞的转移能力；说明miR-17-92基因簇有可能作为预测胃癌转移的标志物，甚至可能作为防治胃癌转移的靶标。本项目的研究结果有助于进一步揭示胃癌转移发生的分子机理，明确miRNA在胃癌转移中的相关功能及机制，对于理解胃癌转移发生和研究新的预防治疗策略具有重要意义。