miR-361-3p调控SH2B1在非小细胞肺癌侵袭转移中的作用及机制研究

Preliminary data confirmed SH2B1 play an important role in the invasion and metastasis of non-small cell lung cancer (NSCLC) and its mechanism of action is unclear. JAK2 and IGF-1R signaling pathway mediated SH2B1 phosphorylation was associated with cell migration; we found that miR-361-3p expression was significantly downregulated in NSCLC tissues. Furthermore, the SH2B1 is predicted to be the direct target of miR-361-3p by bioinformatics methods. Moreover, our studies showed that forced expression of miR -361-3p significantly downregulated the SH2B1 protein expression and suppressed cellular migration and invasion in NSCLC cells. The study aims to confirmed low expression of miR-361-3p in NSCLC maybe decreased SH2B1 expression, enhanced JAK2 and IGF -1R cell signaling pathways and increased the expression of the target genes which result in cell migration, cell extracellular matrixc degradation and angiogenesis involved in invasion and metastasis through gene transfection, quantitative reverse transcription polymerase chain reaction, luciferase reporter gene assay, angiogenesis in the chick chorioallantoic membrane , animal models, tissue microarray et al. The project will not only help to elucidate the mechanisms SH2B1promoting the cells invasion and metastasis in NSCLC, and provide a novel biomarker for predicting metastasis and recurrence of NSCLC, and provide enough scientific evidence to establish a new target for antimetastatic therapy of HSCLC.

前期工作证实SH2B1在非小细胞肺癌(NSCLC)侵袭转移中发挥重要作用，其作用机制不清楚。JAK2及IGF-1R两信号通路介导SH2B1磷酸化与细胞迁移相关；我们发现miR-361-3p 在NSCLC中表达明显下调，生物信息学预测SH2B1是miR-361-3p 直接靶基因，过表达miR-361-3p 下调SH2B1 蛋白表达，同时细胞的迁移侵袭能力明显下降。本项目拟采用基因转染、定量PCR、报告基因、鸡胚绒毛尿囊膜血管形成、动物模型、组织芯片等方法分析证实NSCLC中miR-361-3p表达下调导致SH2B1表达增加，上调JAK2及IGF-1R两信号传导通路及相应靶基因表达及活性促细胞迁移、细胞外基质降解及新生血管生成，参与侵袭转移。项目不仅有助于阐明SH2B1表达上调促进NSCLC 侵袭转移分子机制，且为预测肺癌转移复发提供新的生物标志物和确立肺癌转移治疗新靶点提供充分的科学依据。

研究背景.microRNAs是一类长度约19~24个核苷酸的非编码的单链RNA分子，大量的microRNA被发现和证实在非小细胞肺癌发生发展过程中起着重要作用。近年来的研究发现miR-361在宫颈癌、前列腺癌、胃癌、结直肠癌、肝癌中参与肿瘤的发生发展。但miR-361-3p在非小细胞肺癌中的具体调剂机制国内外尚未见报道。.目的.研究miR-361-3p在非小细胞肺癌组织中的表达水平及其临床病理特征的关系，证实miR-361-3p对非小细胞肺癌细胞的作用和具体作用机制。.方法.1. PCR技术检测miR-361-3p在非小细胞肺癌组织及细胞系中的表达情况，分析其与临床病理特征的相关性；.2. 干预非小细胞肺癌细胞中miR-361-3p的表达，观察其对非小细胞肺癌细胞增殖及侵袭迁移能力的影响；.3. 采用荧光素酶报告基因及Western blot、免疫组化方法对miR-361-3p在非小细胞肺癌细胞中预测的靶基因进行验证，并分析靶蛋白与miR-361-3p的关系； .4. 通过恢复靶基因表达，证实下游靶点及具体作用通路；.结果.1. miR-361-3p的在非小细胞肺癌患者中呈现普遍低表达，其表达水平与非小细胞肺癌患者的TNM分期和淋巴结转移相关。.2. miR-361-3p抑制体内和体外非小细胞肺癌细胞的生长增殖及侵袭和迁移。.3. SH2B1是miR-361-3p的直接作用靶基因，且SH2B1在非小细胞肺癌中呈高表达，其高表达与淋巴结转移和TNM分期相关，并与miR-361-3p的表达呈负相关。.4. miR-361-3p上调对体外非小细胞肺癌细胞的抑制生长增殖及侵袭和迁移作用可以被上调靶基因SH2B1所修复。.5. 低表达miR-361-3p失去对靶基因SH2B1的抑制作用，激活JAK2/Rac1信号通路.结论.低表达miR-361-3p与非小细胞肺癌患者高淋巴转移及TNM分期有关，低表达miR-361-3p失去对SH2B1的抑制作用介导了JAK2/Rac1通路的激活促进非小细胞肺癌的细胞增殖及转移能力。