HUESCC-LncRNA5调控人食管鳞状细胞癌侵袭及迁移的功能及机制研究

Long non-coding RNA (LncRNA) has been reported to be associated with the development and progression of various human cancers. Our microarray analysis and large-sample qRT-PCR validation confirmed that HUESCC-LncRNA5 was up-regulated in ESCC and its expression was close associated with the clinical stage and lymph node metastasis of ESCC patients. Meanwhile, we tentatively confirmed that its over-expression promoted migration and invasion of ESCC in vitro and in vivo.LncRNA has been reported as ceRNA to silent miRNA, then up-regulated the expression of the target gene of miRNA. HUESCC-LncRNA5 comprises a plurality of binding sites of miR-200 family via bioinformatics predicts, and our preliminary experiments confirmed that HUESCC-LncRNA5 was combined with miR-200a/b/429,up-regulated target genes ZEB1 and Kindlin-2, and then promote ESCC matestasis.Recent studies confirmed that PI3K/AKT pathway was excessively activated in ESCC,and its activation promoted the ZEB1 transcription. It has been also reported that PI3K inhibitors reduce the HUESCC-LncRNA5 expression. Bioinformatics analysis should that the promoter region of HUESCC-LncRNA5 contained the ZEB1 binding sites. Our preliminary experiments found that ZEB1 may has effect on the expression of HUESCC-LncRNA5. Our project intends to research the regulatory mechanism of matestasis and the expression of HUESCC-LncRNA5 among clinical samples, in vitro and in vivo in ESCC.

长链非编码RNA（LncRNA）被证实与多种肿瘤发生发展密切相关。课题组经芯片及大样本验证确认HUESCC-LncRNA5在ESCC中高表达，且其表达与患者临床分期及淋巴结转移相关，并在细胞及动物层面证实该指标可促进ESCC细胞迁移及侵袭。文献报道LncRNA可作为ceRNA沉默miRNA，上调靶基因表达。生物信息学预测该LncRNA含多个miR-200家族结合位点，预实验证实其可与miR-200a/b/429结合，上调靶基因ZEB1、Kindlin-2，促进ESCC转移。文献报道PI3K/AKT通路在ESCC中被过度激活，该通路激活可促进ZEB1的转录，而PI3K抑制剂可下调该LncRNA表达，生物信息学预测该LncRNA启动子区域含ZEB1结合位点，预实验证实ZEB1可影响该LncRNA表达。本项目拟在组织、细胞、动物等多层面研究该LncRNA下游促进ESCC转移及其自身上游调控机制。

编码RNA被证实与多种肿瘤包括ESCC的发生发展密切相关。课题组使用来自10名ESCC患者的血浆样本（包括5例T3N0M0和其他5例T3N1-3M0以及5名正常对照进行高通量人circRNA微阵列评估差异ECCC进展中circRNA表达谱的变化。基于测序结果，课题组经过大样本验证证实了血清circ-SLC7A5可作为ESCC检测的新型诊断生物标志物。我们扩增了样本并检测了87名ESCC患者血浆中circ-SLC7A5的表达，选择了53名健康人作为对照。结果显示，与正常病例相比，ESCC血浆中circ-SLC7A5表达明显上调，ROC曲线下面积（AUC）总计为0.7717。临床病理数据分析显示，circ-SLC7A5水平的上调与TNM分期呈正相关。 Kaplan-Meier生存分析和对数秩检验结果显示，circ-SLC7A5表达水平较高的ESCC患者的circ-SLC7A5表达水平较低的患者的生存时间明显较短。单因素和多因素分析表明相对circ-SLC7A5表达水平和TNM分期均被确定为ESCC患者总生存率的独立预后指标。因此，我们将circ-SLC7A5确定为潜在的ESCC循环生物标记。在机制方面，课题组证实了circ-ZDHHC5在体外ESCC细胞中具有促癌作用，circ-ZDHHC5的下调抑制了细胞的侵袭。克隆形成实验和CCK-8分析表明，敲除circ-ZDHHC5后，TE-10和TE的增殖能力显着减少。伤口愈合实验表明，circ-ZDHHC5沉默显着降低了细胞迁移率。通过生物信息学分析发现circ-ZDHHC5与miR-217种子区具有互补序列。circ-ZDHHC5直接与miR-217结合并抑制miR-217活性，miR-217靶向ZEB1并抑制ESCC细胞的增殖和侵袭。在本项目的赞助下，课题组发表SCI 9篇，其中大于5分2篇，申报发明专利10项，已获批发明专利2项。培养硕士4名，博士2名。该项目的开展经临床大样本验证后可用于ESCC早期诊断试剂盒的制备以及提供可能的ESCC治疗分子靶点，可更好的区分癌变患者中病情进展迅速的亚型，及时进行微创治疗，阻断其癌变进程，既可避免漏诊又可减少过度治疗，从而有利于实现癌前病变患者的个体化治疗。