Ascl2调控miR-200家族在结肠癌细胞EMT的作用及其分子机制研究

Ascl2 is an important transcription factor related to intestinal crypt stem cell, which controls the intestinal crypt stem cell fate. Ascl2 knockout mice exerted an influence on the disappearance of the intestinal crypt stem cells. Our previous findings firstly demonstrated that Ascl2 is involved in regulation of "stemness" of colon cancer stem-like cells (colon cancer progenitor cells), Ascl2 knockdown in cultured HT-29 and LS174T cells reduced their cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133+ HT-29 cells was significantly higher than in CD133- HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells resulted in 26.2% of cells staining CD133+ compared with 54.7% in control. The levels of 'stemness' associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were signi?cantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in vivo. The shRNA-Ascl2/HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells and 178 two-fold up-regulated miRNAs and 172 two-fold down-regulated miRNAs in shRNA-Ascl2/LS174T cells. Expression levels of let-7b, miRNA-124, miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of 'stemness', were quantified with qPCR, which confirmed their identities. Restoration of miRNA-302b, via its mimic, led to the restoration of shRNA-Ascl2/HT-29 'stemness' characteristics, including tumorsphere formation and 'stemness' associated genes levels. Elevated levels of miR-200 family: miR-200a, miR-200b, miRNA-200c, miR-141, and miR-429, involved in the regulation of 'epithelial mesenchymal transition ( EMT )', were quantified with qPCR, which confirmed their identities in shRNA-Ascl2/LS174T cells. We found that Ascl2 is involved in regulation of epithelial mesenchymal transition ( EMT ) of colon cancer cells of LS174T cells (0.45% of CD133 positive staining), but the exact mechanism was unclear. This study intends to validate direct relationship between Ascl2 and miR-200 family promoter though chromatin immunoprecipitation microarray analysis, dual luciferase report gene experiments and EMSA, then confirm the functional outcome of the transcriptional regulation through observing changes of precursor and mature miRNA of miR-200 family after Ascl2-RNAi though q-PCR, at last, predict and verify EMT-related target genes of the miR-200 family, and study EMT-related cell biological behavior changes. The study is to clarify the molecular mechanism among Ascl2, miRNA-200 family members and targets of EMT in colon cancer cells, and seek for the novel therapeutic targets and theoretical basis for intestinal tumor.

Ascl2作为WNT信号的一个靶基因，是控制肠隐窝干细胞命运的一个重要转录因子。我们前期研究发现抑制结肠癌细胞Ascl2的表达致其体内外生长的抑制是通过miR-302b抑制结肠癌前体细胞的干性；同时发现Ascl2的抑制导致结肠癌细胞上皮间质转变（EMT）的逆转，但其调控机制不清。本研究拟通过染色质免疫沉淀芯片分析Ascl2与miR-200家族的启动子是否存在直接的连接，并通过双萤光素酶报告基因实验和EMSA实验揭示Ascl2对miR-200家族的转录作用，再通过q-PCR明确Ascl2-RNAi后miR-200家族的前体和成熟miRNA的变化以证实其转录调控的功能结局，最后探讨miR-200家族调节EMT相关靶基因的作用，并研究其相关细胞生物学行为的变化。通过上述研究，明确Ascl2通过调控miRNA-200家族调节结肠癌细胞EMT-MET转变的分子机制，为结肠癌治疗提供新靶点和理论依据。

Ascl2已经证明其控制 CBC 细胞的“干性”。CBC 细胞向终末分化细胞的分化是上皮向间充质转变（EMT)的生物学基础。研究表明，EMT 及其逆转过程 MET 在胚胎发育、肿瘤的进展和转移起重要的调节作用。miRNA-200家族作为肿瘤的抑制基因，在侵袭性肿瘤中表达下调，可抑制肿瘤的EMT进展。本课题组早期研究表明ascl2影响结肠癌细胞株EMT标记E-cadherin、Vimentin和N-cadherin的表达，且ascl2干扰的结肠癌细胞株其miR-200家族的表达发生显著上调，因此推测Ascl2 通过转录调控 miRNA-200s，进而调节结肠癌细胞 EMT 的作用。本课题构建了Ascl2干扰HT-29和LS174T细胞株，检测其EMT标记基因及miR-200家族的表达变化；观察Ascl2干扰后LS174T体外增值、迁移、侵袭情况；运用Ascl2特异性抗体的染色质免疫沉淀证实Ascl2与miR-200b/a/429和miR-200c/141簇的启动子存在直接连接；通过双荧光素酶报告基因系统及点突变证明Ascl2通过E-box元件调控miR-200家族启动子的活性；通过Ascl2干扰及其对照细胞株中miR-200家族的表达抑制，检测EMT标志基因的变化；通过细胞划痕实验，观察干扰Ascl2的细胞株中，miR-200a和miR-429的表达抑制能否阻碍细胞的增殖和迁移；通过Q-PCR分析结直肠癌组织样本中Ascl2与miR-200家族的表达趋势。重要结论：干扰Ascl2导致结肠癌细胞株EMT的逆转及miR-200家族表达的上调；结Ascl2的干扰抑制ZEB1/ZEB2的表达，导致其与miR-200家族启动子上的E-BOX元件的结合减少从而激活miR-200家族启动子的活性；定点突变E-BOX元件的保守位点导致miR-200b/a/429启动子的活性增加；干扰Ascl2的结肠癌细胞中，miR-200家族的表达抑制导致EMT的发生及部分MET的逆转，且增强细胞株的增殖及迁移能力；检测Ascl2与miR-200家族在结肠癌临床标本中的表达， 发现两者的表达呈现负相关，且Ascl2高表达的患者大部分伴随着淋巴结转移。研究意义：通过对Ascl2转录调控miRNA-200家族进而调控结肠癌细胞EMT的分子机制的研究，揭示Ascl2在结肠癌细胞EMT-MET的平衡维持的分