基于MALAT1介导的SFPQ/PTBP-2复合物解离途径研究健脾解毒方抗结肠癌侵袭转移机理

As traditional Chinese medicine compound---JianPi JieDu Recipe (JPJDR) is known to inhibit invasion and metastasis of colon cancer, preliminary data suggested it did so through a previously unknown mechanism involving the inhibition of MALAT1 expression, and mechanism study demostrated that, MALAT1 could mediate the dissociation of SFPQ/PTBP-2 complex and release free PTBP-2 which had ability of promoting migration. Therefore using colon cancer derived cell line LoVo (high expression of MALAT1 and high metastasis ability) and HCT116 (comparative low expression of MALAT1 and low metastasis ability), control cells versus MALAT1 knockdown and over-expression cells by lentiviral construct, we propose to study the effect of JPJDR on tumor cell invasion and metastasis, how it affects the down-regulation of MALAT1 expression, inhibits the dissociation of SFPQ/PTBP-2 complex mediated by MALAT1, and decreases the formation of free PTBP-2. We further propose to establish orthotopic xenograft mouse model of human invasive colon cancer, thus enabling us to investigate JPJDR's effect on tumor invasion and metastasis as well as conducting expression and correlation analysis of MALAT1, SFPQ and PTBP-2 in JPJDR treated tumors. The results for these studies will uncover the inhibitive mechanism of JPJDR on invasion and metastasis of colon cancer basing on the dissociation pathway of SFPQ/PTBP-2 complex mediated by MALAT1. This discovery will surely provide important pre-clinical evidence supporting largely use of JPJDR in prevention and treatment of colon cancer，and provide a reference for the prevention and treatment of other gastrointestinal tumors.

中药复方健脾解毒方具有抗结肠癌侵袭转移功效，前期研究发现其通过抑制MALAT1表达以达到抗侵袭转移的新作用机理，而机制研究提示MALAT1可以介导SFPQ/PTBP-2复合物解离以释放具有促转移能力的PTBP-2。本课题拟选择结肠癌LoVo细胞（高转移，MALAT1高表达），HCT116细胞（低转移，MALAT1低表达），及相应的MALAT1基因慢病毒干扰和过表达细胞株，研究健脾解毒方是否通过下调MALAT1，进而抑制MALAT1介导的SFPQ/PTBP-2复合物解离以减少自由PTBP-2的生成，最终达到抗侵袭转移作用；制作裸小鼠结肠癌原位移植瘤模型，观察健脾解毒方对组织浸润和转移的影响，并分析肿瘤组织中MALAT1、SFPQ、PTBP-2的表达水平和相关性，从而揭示健脾解毒方抗结肠癌侵袭转移的新作用机理，为健脾解毒方广泛应用于临床结肠癌防治奠定基础，也为其他消化道肿瘤的防治研究提供借鉴。

长期的临床应用表明，中药复方健脾解毒方在临床治疗中具有非常好的抗肿瘤效果。前期研究结果提示，健脾解毒方可能通过降低长链非编码RNA-MALAT1的表达来抑制结肠癌细胞的侵袭转移能力，而MALAT1的分子机制可能跟PTBP-2和SFPQ有重要联系，但是具体的作用机理并不清楚。本项目主要通过体外细胞实验和体内动物实验，探索MALAT1调控结肠癌侵袭转移的分子机制，揭示健脾解毒方抑制结肠癌侵袭转移的具体作用机理。研究结果显示，MALAT1在人结肠癌转移灶和癌组织中的表达水平明显高于癌旁组织，并且与结肠癌的浸润转移显著相关。进一步的分子机制研究提示，MALAT1可以竞争性地与SFPQ蛋白结合，促使SFPQ/PTBP-2复合物的解离，释放自由形式PTBP-2，从而促进结肠癌细胞的生长和转移。另一方面，健脾解毒方可以通过抑制MALAT1介导的SFPQ/PTBP-2复合物解离以达到抗结肠癌侵袭转移的作用机理。本研究提示，MALAT1等长链非编码RNA在结肠癌的发生发展中有着非常关键的调节作用，同时健脾解毒方的抗肿瘤作用跟调节非编码RNA的表达有着重要的联系。本研究成果为健脾解毒方广泛应用于临床结肠癌的防治奠定了非常扎实的实验基础。