表观重编程矫正MMPs/TIMPs失衡改变胃癌细胞生物学行为及其机制研究

Our previous study had confirmed that gastric cancer cells could be reprogrammed with induced pluripotent Stem cells (iPSCs) technology. After reprogramming and nondirectional differentiation, the migration, invasion and tumorigenicity were apparently reduced. Our further study showed that, (i)the imbalance between MMPs and TIMPs was corrected after reprogramming,(ii)the gastric cancer cells before reprogramming presented abnormal DNA methylation and histone modification,(iii)miRNA chip and bioinformatic analysis revealed that miR-202 and miR-30b might target MMP1 and TIMP3, respectively. Based on these, for the first time we hypothesized that DNA methylation and histone modification together with miRNAs played a vital role in correcting the imbalance between MMPs and TIMPs, which would elucidate the changed biological behavior of reprogrammed gastric cancer cells. In order to gain reliable evidence that iPSCs reprogramming could change the biological behavior of gastric cancer cells, we would further investigate the mechanisms of DNA methylation and histone modification in the regulation of MMPs and TIMPs using the methods of MSP, BSP and ChIP. More over, we would explore the role of miRNA in regulating the MMPs and TIMPs using lentiviral expression and luciferase test. Hopefully, it would provide some experimental evidence for iPSCs in treating the gastric cancer.

我们已经证明诱导多能干细胞（iPSCs）技术能重编程胃癌细胞，并明显抑制其迁移侵袭及致瘤等能力。进一步实验显示：1、胃癌细胞MMPs/TIMPs表达失衡得到纠正，尤以MMP1及TIMP3明显；2、重编程前胃癌细胞MMP1及TIMP3的DNA存在异常甲基化和组蛋白修饰；3、miR-202及miR-30b可能分别参与调控MMP1及TIMP3。由此，我们首次提出胃癌细胞iPSCs重编程改变胃癌细胞生物学行为的可能机制是DNA甲基化及组蛋白修饰与miRNA共同调控矫正MMPs/TIMPs失衡。本项目拟进一步采用MSP、BSP及ChIP等方法探讨DNA甲基化及组蛋白修饰对MMPs/TIMPs的调控机制；同时采用慢病毒表达及荧光素酶实验等技术探讨miRNA对MMPs/TIMPs的调控作用，旨在获得iPSCs重编程改变胃癌细胞生物学行为的可靠依据，为iPSCs技术应用于胃癌治疗提供理论依据。

胃癌仍然是全球最常见的恶性肿瘤之一，死亡率高居第二位，我国尤甚。胃癌严重威胁人类的健康和生活，对社会经济造成巨大负担，是当前亟待解决的重要问题。尽管胃癌治疗手段日益更新，然而肿瘤复发转移却经常发生，严重影响患者的预后。积极寻找新的治疗手段已成为目前研究的重点和难点。本研究进一步证实，使用iPS技术重编程可改变胃癌细胞的生物学行为，即降低胃癌细胞迁移侵袭能力，降低胃癌细胞的体内及体外成瘤能力，通过表观遗传学技术，我们发现⑴使用MSP和BSP法，我们发现5’CpG岛在分化后Post-iPS-AGS细胞呈现去甲基化状态，而诱导前AGS细胞则呈甲基化状态。这提示iPS重编程改变了TIMP3的DNA甲基化状态从而导致其表达上调。⑵使用CHIP法，我们发现MMP1和TIMP3的组蛋白H3-K4、H3-K9及H3-K27的启动子甲基化以及H3和H4的乙酰化发生改变，从而改变了MMP1及TIMP3 mRNA及蛋白质水平的表达，进而降低胃癌细胞的迁移侵袭能力及成瘤能力；⑶通过WB法，我们发现重编程胃癌细胞与诱导分化前胃癌细胞相比，p38-MKK-Hsp27磷酸化通路发生改变，推测该通路在胃癌细胞重编程过程降低其肿瘤性方面可能发挥重要作用；⑷通过miRNA芯片，我们发现多个miRNA在重编程过程中可能发挥重要作用，其中miR-223及miR-21在重编程后下降较为明显。通过生物信息学软件预测，结果发现它们均可靶向作用于黏膜屏障相关蛋白Claudin-8，而后者在胃癌组织及细胞表达明显下降。体外细胞荧光素酶实验证明miR-223及miR-21可靶向作用于Claudin-8，miR-223及miR-21可促进胃癌细胞侵袭迁移能力，而Claudin-8则相反，后者与胃癌分化程度、胃癌患者TNM分期呈负相关，与预后可呈正相关。上述研究结果为iPS技术应用于胃癌治疗提供理论依据及新的思路。