CAFs源性exosomalmiR-206靶向调控LASP-1在胃癌侵袭转移中的作用及机制研究

Our previous research has identified LIM and SH3 protein 1 (LASP-1) as a novel gastric cancer (GC) metastasis-associated protein, which promotes GC invasion and metastasis and is closely related with poor prognosis of GC patients. The regulatory mechanisms of LASP-1 abnormal expression, however, are still unclear. Tumor-associated fibroblasts(CAFs) are main stromal cells of tumor microenvironment and regulate tumor cell invasion and metastasis through various ways. In pre-test, we found that cancer-associated fibroblasts (CAFs) secreted and released exosomes and tumor cells could uptake exosomes, which led to up-regulated expression of LASP-1 compared with normal fibroblasts (NFs) group in gastric cancer. MicroRNA expression chip was used to detect the differential expression of microRNA in the exosomes of CAFs and NFs. MiR-206 was found to be down-regulated in exosomes of CAFs, bioinformatics analysis and pre-test in vitro confirmed that miR-206 might interact with LASP-1 3'-UTR and regulate its expression. Based on that, we hypothesize that exosomal miR-206 released by CAFs can regulate the invasion and metastasis of gastric cancer by targeting LASP-1. These hypotheses will be verified by clinical specimens, cell culturing and in vivo research involving animal model. This study may elucidate the regulatory mechanism of exosomes and microRNA in the expression of LASP-1, which can influence the invasion and metastasis of gastric cancer. Based on the results of this study, we can illustrate the regulatory mechanism of LASP-1 in GC, providing new target and theoretical basis for clinical treatment of gastric cancer.

LASP-1是我们前期研究新发现的胃癌转移相关蛋白，首次报道LASP-1可促进胃癌的侵袭转移并导致患者预后不良，但其异常表达的调控机制尚不清楚。癌相关成纤维细胞（CAFs）是肿瘤微环境的主要基质细胞，通过多种方式调控肿瘤的侵袭转移。预实验我们发现CAFs可以分泌和释放外泌体，胃癌细胞摄取CAFs源性外泌体后LASP-1的表达高于正常成纤维细胞（NFs）组。我们对CAFs和NFs分泌的外泌体进行microRNA差异表达谱检测，生物信息学分析结合体外预实验初步证实CAFs源性外泌体内低表达miR-206可与LASP-1基因3'-UTR区结合并调控其表达。据此我们推测：CAFs源性外泌体内miR-206靶向调控LASP-1介导了胃癌的侵袭和转移。本项目拟从临床组织标本、细胞和动物体内三个层面验证我们提出的假设，揭示LASP-1促进胃癌侵袭转移的调控机制，为胃癌的临床治疗提供新的靶标和理论依据。

MicroRNAs（miRNAs）能够与其靶基因mRNA的3'非翻译区结合，抑制靶基因蛋白的合成，从而发挥相应的生物学调节作用。LASP-1是我们前期研究新发现的胃癌转移相关蛋白，首次报道LASP-1可促进胃癌的侵袭转移并导致患者预后不良，但其异常表达的调控机制尚不清楚。癌相关成纤维细胞（CAFs）合成和释放外泌体，外泌体中microRNA可传递给胃癌细胞。本研究对CAFs和正常成纤维细胞（NFs）分泌的外泌体进行microRNA差异表达谱检测，结果显示miR-206在CAFs分泌的外泌体中低表达，荧光实时定量PCR检测验证了miRNA高通量测序的结果。生物信息学分析结合双荧光素酶报告基因实验发现CAFs外泌体内低表达的miR-206可靶向调控LASP-1的表达，miR-206可直接与LASP-1 mRNA的3'-非翻译区结合调控LASP-1蛋白的表达。体外细胞学实验和动物实验结果表明miR-206可在体内外抑制胃癌细胞的增殖、侵袭和转移能力。CAFs外泌体来源miR-206可以被胃癌细胞直接摄取，过表达LASP-1能够减弱miR-206表达上调所引起的抑制胃癌进展的作用。机制研究发现miR-206可通过影响细胞的上皮-间质转化（EMT）、F-actin聚合及MMP-2和MMP-9的表达从而调控胃癌细胞的侵袭和转移。本研究初步探讨了CAFs通过外泌体调控胃癌进展的分子机制，阐明了CAFs释放的外泌体可作为胃癌早期诊断和判断预后的生物学标记及治疗的新靶点。由于CAFs释放的外泌体及外泌体内的miRNA具有可控性，通过人为上调外泌体内miR-206的合成和释放，最终达到抑制胃癌增殖、侵袭和转移，延长患者生存时间，提高患者生存质量的目的。