Ets-2/UCA1调控CYP4F12促进结直肠癌进展及耐药的研究

Our previous study demonstrated that high expression of LncRNA-UCA1(UCA1) maybe associated with the progression of carcinogenesis and 5-FU resistance of CRC(colorectal cancer), However, the underlying mechanism remains poor understood. We recently found that the transcription factor Ets-2 can directly activate the expression of UCA1. Moreover, UCA1 could regulate CYP4F12 in promoting tumor progression and drug resistance of CRC. Therefore, we hypothesize that Ets-2/UCA1 could promote tumor progression and drug resistance by regulated CYP4F12 in CRC. In order to confirm the hypothesize, first, we will examine UCA1 and CYP4F12 expression in normal mucosa, adenoma, non-metastatic and metastatic colorectal cancer tissues, and investigate the clinical significance and the correlation between them. Methods such as EMSA, CHIP, truncated mutant, and so on, will be performed to prove the interaction between Ets-2/UCA1 and CYP4F12, to identify the key domains that mediate the interaction. Finally, we will then investigate the biological function and molecular mechanism of UCA1 in CRC by the lentiviral expression system, RNAi and nude mice model. This study will not only reveal the molecular mechanism about how Ets-2/UCA1 regulated CYP4F12 in promoting tumor progression, and to provide new evidences for the mechanism of 5-FU resistance in CRC.

我们前期研究发现LncRNA-UCA1在结直肠癌中高表达，并可能与肿瘤的进展及5-FU耐药相关，但其相关机制仍不清楚。初步探索发现转录因子Ets-2可直接激活UCA1的表达，进一步发现UCA1可以下调CYP4F12表达促进肿瘤增殖转移和5-Fu耐药。据此我们推测：Ets-2转录激活UCA1，后者下调CYP4F12的表达，最终导致肠癌的发生发展和化疗耐药。本课题拟首先在临床样本中验证UCA1和CYP4F12与肠癌进展和5-FU耐药的相关性；然后通过EMSA、CHIP、截短突变体等实验证实Ets-2对UCA1的调控作用，以及UCA1与CYP4F12的相互作用；最后，通过RNAi、慢病毒感染、裸鼠成瘤模型等实验进一步揭示UCA1促进肠癌发生发展和5-FU耐药的分子机制。本项目有望阐明Ets-2/UCA1调控CYP4F12在结直肠癌进展中的关键作用和调控机制，为5-FU耐药逆转机制研究提供新思路。

结直肠癌（Colorectal cancer，CRC）是常见的恶性肿瘤之一，近年来虽其发病率和死亡率呈缓慢下降趋势，但在中国癌症致死率仍排名第五位。癌症的发生发展是复杂的多因素调控过程，探讨其发生发展的机制将有助于寻找有效的治疗靶标，实现基础研究到临床应用的转化。多项研究表明长链非编码RNA可参与调控CRC的进展及化疗耐药。本课题组前期采用高通量LncRNA表达芯片及qPCR实验发现UCA1在结直肠腺瘤及肠癌中高表达，在结直肠正常组织中低表达，其表达水平与淋巴结转移、远处转移及肿瘤分期相关，高表达UCA1可预测不良的预后。课题研究发现在CRC细胞中沉默表达UCA1可以抑制癌细胞的增殖、侵袭转移，促进细胞凋亡，降低5-fluorouracil(5-FU)化疗敏感性，促进5-FU诱导的细胞凋亡；而过表达UCA1则相反。进一步，我们结合生物信息学并通过荧光素酶实验、细胞实验和凝胶迁移实验（EMSA）等实验验证了转录因子Ets-2对UCA1的调控作用；然后通过Fluorescence in situ hybridization（FISH）、qPCR、免疫组化、RNAi、慢病毒感染和RNA pull down实验验证了UCA1与CYP4F12的相互作用。最后裸鼠移植瘤实验结果表明，敲低UCA1组小鼠较对照组小鼠能显著抑制裸鼠成瘤，在移植瘤小鼠注射5-FU后能减小移植瘤大小，而敲低UCA1组小鼠对5-FU更敏感。UCA1作为潜在的结直肠癌的分子标志物和治疗靶点，有望为解决结直肠癌诊疗及化疗耐药这一难题提供依据。