lncRNA LOC389641通过VCAM1/整合素α4近分泌激活效应促进胰腺癌神经浸润及进展的研究

Neural invasion is a distinctive and common biological behavior, which is strongly correlated with the invasion and metastasis, but the concrete mechanism remains unclear. In our previous research, we found that lncRNA LOC389641,which is closely related to the neural invasion, promotes the invasion and metastasis of pancreatic cancer. Further preliminary experiments showed that:1. LOC389641 significantly upregulated the expression of VCAM1 and subsequently facilitated neural invasion. 2. Mechanistically, LOC389641 bound KAT2A histone acetyltransferase, acted as a modular scaffold of KAT2A, coordinated its localization, specified the H3K9 histone modification pattern on the target genes, including VCAM1, and consequently transactivated the transcription of VCAM1 gene. VCAM1 is able to bind the integrin α4 and reversely activates the PI3K/AKT signal pathway, regulating the process of neural invasion and tumor progression. As mentioned above, we put forward the scientific hypothesis that lncRNA LOC389641 promotes neural invasion and tumor development via VCAM-1/integrin α4 juxtacrine activation. Several important experimental methods including RNA pull-down assay, dual-luciferase reporter assay and sequence truncated mutation test will be used to illuminate the exact molecular mechanism. Based on our previous randomized controlled trial of nerve radical dissection of pancreatic cancer, we aim to lay the foundation for the development of combination therapy model targeting on both nerve radical dissection of pancreatic cancer and postoperative nerve infiltration.

神经浸润是胰腺癌独特而普遍的生物学行为，与复发转移密切相关，但其具体机制远未阐明。我们前期已报道lncRNA LOC389641可促进胰腺癌细胞的侵袭转移，且与神经浸润显著相关。进一步预实验表明：①基因芯片提示LOC389641显著上调VCAM1表达而促进胰腺癌神经浸润；②LOC389641可能作为支架分子招募KAT2A，促进H3K9组蛋白乙酰化修饰，激活VCAM1转录，后者可结合神经细胞表面整合素α4反向激活胰腺癌细胞PI3K/AKT通路，调控神经浸润过程及演进。据此本项目提出“lncRNA LOC389641通过VCAM-1/整合素α4近分泌激活效应促进胰腺癌神经浸润及进展”的假说，通过RNA pull-down、荧光素酶报告和突变序列构建等方法阐明其具体分子机制，结合申请人团队前期胰腺癌神经清扫术的RCT临床研究，拟为“胰腺癌神经清扫+术后靶向神经浸润”组合治疗模式的应用奠定基础。

胰腺癌预后极差，能否根治性手术切除是影响胰腺癌患者预后的重要因素。重要原因之一是胰腺癌广泛存在神经侵犯特性，神经侵犯在胰腺癌高达90～100%。我们前期临床研究表明神经浸润与胰腺癌患者预后密切相关。本项目在前期筛选鉴定促癌长链非编码基因LOC389641基础上，进一步通过体内外实验及临床预后分析证实：.（1）胰腺癌细胞与神经细胞的细胞粘附实验模型、胰腺癌细胞与神经细胞的直接共培养模型及胰腺癌细胞与大鼠背根神经节的离体共培养模型均证实LOC389641显著促进胰腺癌细胞的神经浸润及进展；（2）利用上述三个体外模型同时证实VCAM1通过结合整合素α4发挥近分泌效应促进促进胰腺癌细胞的神经浸润；（3）通过核浆分离实验，我们证实LOC389641主要定位于细胞核，且LOC389641特异性结合KAT2A的位点主要位于1000bp-1500bp片段碱基，特异性募集KAT2A介导其乙酰化修饰进而发挥表观修饰作用；（4）体内动物模型证实LOC389641高表达后，小鼠的胰腺癌细胞的坐骨神经浸润程度显著增强，而添加整合素α4的中和抗体后，小鼠的胰腺癌细胞的坐骨神经浸润程度显著减弱，且LOC389641高表达后显著促进胰腺癌细胞肺转移；（5）通过在胰腺癌组织标本中对LOC389641的表达进行原位杂交实验检测，发现胰腺癌较癌旁中LOC389641的表达显著上调，进一步在60例神经浸润明显的胰腺癌组织及对应的癌旁组中检测LOC389641的表达，发现癌组织中LOC389641的表达较癌旁呈显著性上调，通过临床样本及TCGA分析，证实LOC389641高表达的胰腺癌患者预后不良。.结合申请人团队前期开展关于胰腺癌扩大神经清扫根治术的RCT临床研究，将为“胰腺癌扩大神经清扫根治术+术后靶向神经浸润关键调控分子”组合治疗模式的应用奠定基础。

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