长链非编码RNA NONHSAT101069在吉西他滨诱导胰腺癌细胞发生上皮间质转化中的机制研究

Metastasis and chemoresistance are the main causes for the poor prognosis of pancreatic cancer (PC) patients. Continuous exposure under gemcitabine treatment could result in the acquisition of epithelial mesenchymal transition (EMT) and enhance the migration and invasion in PC cells; however, the role of long non-coding RNA (lncRNA) within chemotherapy induced EMT in PC cells remains unclear. To identify the novel lncRNA concerning chemoresistance and EMT, Aligent lncRNA high-flux microarray was employed in parental and gemcitabine-resistant PC cells. NONHSAT101069, which was significantly upregulated in PC gemcitabine-resistant cells, cell lines and specimens, was selected in this study. Results of our preliminary studies revealed that knockdown of NONHSAT101069 significantly suppressed gemcitabine resistance, EMT, migration, invasion and metastasis of PC cells. And the NONHSAT101069/miR-129-5p/Twist1 axis might be responsible for NONHSAT101069-induced PC carcinogenesis. Taken together on the basis of high-flux microarray, we will elucidate the role and underlying mechanism of NONHSAT101069 on PC chemoresistance, EMT and metastasis by a series of assays, including cells in vitro, animals in vivo and PC specimens in clinic. Our study will provide a therapeutic target and theoretical basis for the prevention and treatment of PC chemoresistance and metastasis.

肿瘤转移和化疗耐药是胰腺癌预后不良的主要原因。吉西他滨化疗可以诱导胰腺癌细胞上皮间质转化（EMT）且增强其迁移侵袭能力，但具体机制尚不明确，长链非编码RNA（lncRNA）可能参与其中。基于Aligent lncRNA芯片测序亲代及耐药细胞中差异lncRNA，我们选择了差异表达较大且在胰腺癌耐药细胞、细胞系及组织中均高表达的NONHSAT101069。预实验提示：沉默NONHSAT101069可抑制胰腺癌细胞化疗耐药、EMT和侵袭转移；推测NONHSAT101069/miR-129-5p/Twist1轴是具体的调控机制。本项目在测序筛选差异lncRNA的基础上，将通过细胞水平、体外动物水平及临床标本水平探讨NONHSAT101069在胰腺癌化疗耐药、EMT和侵袭转移中的作用及具体机制。本项目的开展有助于为胰腺癌化疗及复发转移的防治提供新的靶点及理论基础。

胰腺癌是恶性程度极高的消化系统恶性肿瘤之一，预后极差，5年生存率不足8%，肿瘤转移和化疗耐药是其主要原因。吉西他滨化疗可以诱导胰腺癌细胞上皮间质转化并增强其迁移和侵袭能力，但其具体调控机制尚不明确，长链非编码RNA（LncRNA）可能参与其中。基于胰腺癌亲代细胞系SW1990，本项目首先通过吉西他滨持续化疗诱导建立了吉西他滨耐药细胞系SW1990/GZ。相比于亲代细胞系SW1990，SW1990/GZ迁移侵袭能力增强，且有明显的间质化改变。通过高通量测序及聚类分析SW1990和SW1990/GZ中差异表达的LncRNA，发现SW1990/GZ中有268个表达上调的LncRNA和49个表达下降的LncRNA。其中，位于第5号染色体的长链非编码RNA NONHSAT101069被选择用于本项目的研究。研究发现：NONHSAT101069在胰腺癌组织、胰腺癌细胞系和胰腺癌耐药细胞系中显著高表达。FISH定位及胞浆胞核RNA分离提取验证NONHSAT101069主要定位于胰腺癌细胞浆中。一系列体内外功能实验证实：过表达NONHSAT101069可显著促进胰腺癌细胞迁移、侵袭、上皮间质转化和吉西他滨耐药；反之，沉默NONHSAT101069可抑制胰腺癌细胞恶性生物学进展。结合NONHSAT101069亚细胞定位、生物信息学预测和LncRNA相关机制报道，进一步通过双荧光素酶报告基因实验、Ago2 RNA免疫共沉淀、Biotin pull-down和一系列功能回复实验验证：NONHSAT101069可作为ceRNA竞争性的结合miR-129-5p从而调控EMT的关键转录因子Twist1介导胰腺癌细胞迁移、侵袭和吉西他滨耐药等恶性生物学行为。综上所述，长链非编码RNA NONHSAT101069可通过NONHSAT101069/miR-129-5p/Twist1轴促进胰腺癌细胞吉西他滨耐药以及侵袭-迁移进展；NONHSAT101069在胰腺癌的发生发展中发挥着重要作用，可作为胰腺癌化疗和复发转移防治新靶点。