LncRNAH19/miR-675促进胰腺癌细胞上皮间质转化及对肿瘤干细胞的调控机制研究

The long non-coding RNA H19 regulates various signaling pathways via miR-675 in embryonic development. Studies have shown that H19/miR-675 was critical in regulating tumorigenesis and affecting the malignant behaviors in many kinds of human malignancies. However, the effects and mechanisms of H19/miR-675 on the cancer stem cells in human pancreatic cancer has not been elucidated. Our previous studies showed that H19 was highly expressed in human pancreatic cancer cells, and it is positively correlated with miR-675 expression level. The expression levels of H19 in pancreatic cancer cell lines were correlated with the proliferation ability, the proportion of CD24+CD44+ESA+ cancer stem cells, and the chemosensitivity to gemcitabine. Furthermore, H19/miR-675 was found to be involved in the regulation of Epithelial-Mesenchymal Transition (EMT) process as well as the cancer stem cell transformation in pancreatic cancer cells. In this study, we will explore the possible mechanisms of H19/miR-675 on modulating the EMT process and maintenance of cancer stem-like cells in human pancreatic cancer cells through silencing or overexpressing the key gene of H19/miR-675 signaling in vitro and in vivo. The expression levels of H19 and miR-675 in both resected cancer tissues and the serum from pancreatic cancer patients would be detected, and their relationship of H19/miR-675 and the clinical data will be analyzed to figure out their clinical significance. The aim of this study is to elucidate the mechanisms of H19/miR-675 on regulating the stemness of pancreatic cancer cells via inducing EMT process and modulating the pluripotency transcription factors expression, and to assess the value of H19/miR-675 as a therapeutic target of pancreatic cancer.

长链非编码RNA H19可能通过直接转录miR-675表达调控多种人类肿瘤的发生发展，影响肿瘤细胞的生物学行为，但其与胰腺肿瘤干细胞的相关性研究未见报道。本研究前期工作表明：H19在胰腺癌组织中高表达且与miR-675的表达水平正相关；调控H19的表达水平可影响胰腺癌细胞增殖能力及CD24+CD44+ESA+肿瘤干细胞比例；进一步研究发现H19/miR-675参与调控胰腺癌Panc-1细胞EMT过程及体外成球能力。本研究拟在此基础上，通过沉默或过表达H19/miR-675关键基因的水平，探讨H19/miR-675对胰腺癌细胞EMT和干细胞化过程的调控机制；评价其与临床病理特征的相关性，探讨其在预测患者化疗敏感性、肿瘤复发、远处转移、预后等方面的作用；阐明H19/miR-675通过介导EMT和多能性转录因子的表达水平，调控肿瘤干细胞化的机制，为其作为靶点的临床应用提供理论依据及实践基础。

LncRNA H19是源自H19基因的具有调控功能的非编码RNA，可影响下游靶蛋白或RNA的表达，通常通过招募转录因子或吸附microRNAs等参与肿瘤发生、发展进程。课题组前期发现Hedgehog信号通路异常激活参与调控胰腺癌上皮间质转化、干细胞生成和化疗耐药；抑制胰腺癌细胞Hedgehog通路后，lncRNA H19表达水平显著下调；lncRNA H19在胰腺癌干细胞（CD24+CD44+ESA+细胞）群、吉西他滨耐药胰腺癌细胞株中表达水平均显著上调，提示lncRNA H19可能参与调控Hedgehog通路介导的胰腺癌细胞干性转化及化疗耐药，但具体分子机制不明。.编码LncRNA H19来的H19的第1个外显子区域能够转录生成高度保守的miR-675，本项目通过分析胰腺癌组织中lncRNA H19与miR-675表达情况，发现二者表达水平正相关；在胰腺癌细胞中过表达或沉默lncRNA H19，miR-675的表达水平随之上调或下调，肿瘤细胞干性转化能力也相应增强或减弱，同时肿瘤细胞增殖、迁移、侵袭能力也增强或减弱。通过生物信息学分析，课题组发现miR-675可能靶向STAT3通路的负性调控因子SOCS5和转录因子E2F-1。由此，课题组将研究内容分为两个方向继续研究，一方面确证了lncRNA H19/miR-675/E2F-1对胰腺癌细胞增殖的调控作用，并通过裸鼠成瘤进行体内验证；另一方面，课题组通过双荧光素报告酶实验和相应的功能试验，明确miR-675-3p对STAT3通路调控因子SOCS5的直接靶向和调控作用，经后续营救实验确证lncRNA H19/miR-675-3p/SOCS5/STAT3通路诱导胰腺癌细胞干性转化及由此导致细胞增强迁移、侵袭、化疗耐药、上皮间质转化等恶性表型，并亦通过体内验证此部分研究结果。这两部分研究内容协同证实了lncRNA H19/miR-675-3p对胰腺癌增殖、迁移、侵袭表型及上皮间质转化、肿瘤细胞干性转化的调控机制。 .综上，本项目现已按照计划完成研究内容并取得重要进展，发表SCI论文8篇、中文核心期刊论文11篇，并培养5名博士研究生毕业，另有3名博士研究生和1名硕士研究生仍在进行后续研究。该项目所提供材料详实，数据真实可靠，研究成果有助于解析胰腺癌发生发展和化疗耐药的分子机制，有望为靶向胰腺癌化疗耐药和肿瘤干细胞的肿瘤分