MALAT1调控ATG9B基因介导细胞自噬在结直肠癌转移中的作用和机制

The role and regulative machenism of long noncoding RNA in tumour has been the focus of research recently. In the previous study, it has been found that long noncoding RNA MALAT1 plays a very important role in Colorectal Carcinoma Metastasis, wherein ATG9B is the key target gene regulated by MALAT1 and it is autophagy-related gene involving in autophagy through interaction with actin and tubulin. Some reseraches have already proved that MALAT1 may regulate the alternative splicing of relevant gene through SR protein in the cells. Moreover, preliminary experiments found that ATG9B is related to Colorectal Carcinoma Metastasis. This project intends to analyze the regulative function of MALAT1 on SR protein and ATG9B gene through RNA-FISH and RNA-IP; to analyze ATG9B interactive protein through immunoprecipitation in ATG9B up-regulated/silenced cells; to detect autophagy, mTOR siganaling, EMT and so on. At last, we will discuss the possible mechanism of ATG9B's mediating autophagy and promoting Colorectal Carcinoma Metastasis. This research will not only reveal the unknown molecular mechanism of MALAT1 in regulating ATG9B-mediated autophagy, but also is expected to to offer new clues for developing new drugs for resisting tumor metastasis, and thus is of great theocratic significance and potential application value.

长非编码RNA在肿瘤中的作用和调控机制是当前研究热点。前期研究发现长非编码RNA MALAT1在结直肠癌转移中起重要作用，其中ATG9B是受MALAT1调控的重要靶基因，ATG9B是自噬相关基因，可与Actin和tubulin相互作用参与细胞自噬；预实验发现ATG9B与结直肠癌转移相关。已有研究表明MALAT1可通过细胞内SR蛋白而调控相关基因的可变剪接，本项目拟在前期研究基础上，通过RNA-FISH和RNA-IP等分析MALAT1对SR蛋白和ATG9B基因的调控作用；上调或沉默ATG9B基因，通过免疫共沉淀分析ATG9B相互作用蛋白，在体内外检测自噬﹑mTOR通路﹑EMT等指标，探讨ATG9B是否通过介导自噬促进结直肠癌转移及可能机制。本研究不仅将揭示前所未知的MALAT1调控ATG9B介导自噬在转移中的分子机制，且有望为开发新型抗肿瘤转移药物提供新线索，具有重大理论意义和潜在应用价值。

我们前期研究结果证实非编码RNA，MALAT1能促进结直肠癌细胞体内外增殖，侵袭和转移。ATG9B是MALAT1调控的相关靶基因。然而，MALAT1调控ATG9B的分子机制以及ATG9B是否在结直肠癌的发展和转移中起着重要的作用，这些我们都尚未清楚。在本项研究中，我们研究了在结直肠细胞SW480中MALAT1调控ATG9B基因的分子机制以及ATG9B在结直肠癌发展和转移中的作用。我们发现MALAT1可与SRPK1和SRSF1蛋白相互作用，MALAT1通过调控SRPK1所介导的SRSF1蛋白的磷酸化作用，从而调控ATG9B的表达。ATG9B在SW620、LOVO等转移结直肠癌细胞株的表达水平明显高于SW480、DLD1等原发直肠癌细胞株，过表达ATG9B可促进结直肠癌细胞体内外的增殖、侵袭、转移，而敲低ATG9B的表达则与上述结果相反。另外，ATG9B在结直肠癌组织中高表达与病人的低生存率密切相关。有趣的是，我们将结直肠癌细胞经过EBSS饥饿处理，证实过表达ATG9B的结直肠癌细胞经过EBSS诱导后促进自噬发生。通过免疫共沉淀，我们发现了ATG9B的相互作用蛋白myosin9，过表达ATG9B可增加myosin-9的表达，但敲低myosin-9的基因MYH9，过表达ATG9B结直肠癌细胞的侵袭、转移的能力降低，且ATG9B诱导结直肠癌细胞骨架重组及上皮间质转化的能力降低。综上所述，这些结果证明了MALAT1通过正向调节SRPK1所介导的SRSF1蛋白的磷酸化作用，从而调控ATG9B的表达；ATG9B通过调控myosin9介导的细胞骨架重组及EMT调控结直肠癌的发展和转移。