SMS和自噬途径协同抑制PUMA表达促进结直肠癌细胞存活的机制研究

结直肠癌（CRC）的发生发展与多胺异常调节密切相关，我们前期研究发现一种多胺生物合成酶——精胺合成酶SMS在CRC中高表达，SMS负责催化亚精胺生成精胺。抑制SMS使CRC细胞生长受抑制，但不导致细胞凋亡。进一步研究显示，抑制SMS可使亚精胺生成增多，后者可升高CRC细胞自噬水平，在此基础上抑制自噬途径可致细胞凋亡，提示抑制SMS活性诱导的自噬可能帮助CRC细胞逃避凋亡。因此，联合抑制SMS和自噬途径可能是治疗CRC的一种新策略，但具体机制尚不清楚。我们的预实验结果发现，抑制自噬途径可使FOXO3a水平升高，而抑制SMS可增强FOXO3a的转录活性，二者协同促进促凋亡蛋白PUMA表达。本项目拟通过CRISPR/Cas9、激光共聚焦、荧光素酶报告实验、IP和ChIP等，阐明以下假说：SMS和自噬途径协同抑制PUMA表达促进CRC细胞存活。本项目将为CRC的治疗提供新的理论基础和实验依据。

Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC). Our previous research showed that spermine synthase (SMS), a polyamine biosynthetic enzyme, is overexpressed in CRC. SMS is an enzyme which converts spermidine into spermine. Inhibition of SMS restrains CRC cell growth but have no effect on cell apoptosis. Further studies show that inhibition of SMS function results in an increase in the spermidine level, which induces a higher level of autophagy in CRC cells. What's more, combined inhibition of SMS and autophagy pathway dramatically induces apoptosis in CRC cells. These data suggest that autophagy induced by SMS inhibition may help CRC cells escape apoptosis. Thus, combined inhibition of SMS and autophagy pathway may be a new strategy for the treatment of CRC, but the underlying mechanism remains unclear. Our preliminary results show that inhibition of autophagy pathway upregulates the expression of FOXO3a, while inhibition of SMS enhances the transcriptional activity of FOXO3a. And concurrent inhibition of SMS and autophagy pathway synergistically induces the expression of proapoptotic protein PUMA. In this study, CRISPR/Cas9, confocal laser, luciferase reporter assay, IP and ChIP assay will be performed to clarify the hypothesis that SMS and autophagy pathway cooperate to promote CRC cell survival by repressing PUMA expression. This study will provide the new theoretical foundation and experimental evidence for the treatment of CRC.