慢病毒介导的重组TM9SF1蛋白通过诱导自噬和内质网应激抑制293T细胞生长

九次跨膜超家族蛋白成员1（transmembrane 9 superfamily protein member 1，TM9SF1）在进化过程中高度保守，在人体组织和多种细胞系广泛表达。目前，关于该蛋白质的功能研究十分有限和初步。本研究采用慢病毒介导的TM9SF1表达系统，研究了重组TM9SF1蛋白的生化特点及其对细胞生长的调控作用。慢病毒感染的293T全细胞裂解液的蛋白质免疫印迹结果揭示，TM9SF1蛋白具有表观分子质量约为70 kD的单体及寡聚体两种主要形式；在室温及加热37℃时蛋白质相对稳定，随变性温度升高（56 ℃以上）逐渐失去其稳定性。CCK8法显示，与慢病毒空载体感染的293T细胞比较，TM9SF1慢病毒表达载体感染的293T细胞在感染2 d后增殖明显减缓（P<0.001）。Western印迹结果证明，过表达TM9SF1引起LC3Ⅱ表达明显上调，LC3Ⅱ/LC3Ⅰ比例升高，说明TM9SF1可引起293T细胞发生自噬。荧光实时定量PCR结果显示，过表达TM9SF1的293T细胞内质网应激标志分子CHOP、GADD34和XBP1（S）表达水平是对照细胞的3～4倍，提示发生了内质网应激反应。以上结果提示，TM9SF1具有抑制293T细胞生长的功能，该功能可能与其引起的内质网应激和自噬有关。这一结论将进一步加深对TM9SF1在细胞生长调控中的功能的认识。

Lentivirus-Mediated Recombinant TM9SF1 Protein Inhibits 293T Cell Growth by Inducing Autophagy and Endoplasmic Reticulum Stress

The transmembrane 9 superfamily protein member 1（TM9SF1）is highly conserved throughout evolution and is widely expressed in human tissues and in a variety of cell lines. At present, the functional research on this protein is very limited and preliminary. In this study, some of the biochemical characteristics of recombinant TM9SF1 protein and its functional role in the regulation of cell growth have been studied, using a lentivirus-mediated TM9SF1 expression system. Western blotting for the proteins in the whole cell lysate from 293T cells infected by lentivirus-enveloped pLVX-IRES-TM9SF1 expression vector revealed that the TM9SF1 protein had the forms of the monomer with 70 kD and the oligomers. Moreover, the protein was relatively stable under the room temperature and 37 ℃, but lost its stability over 56 ℃. The CCK8 assays showed that the growth of 293T cells infected by lentivirus-enveloped TM9SF1 expression vector was markedly retarded after infection for 2 days, compared with lentivirus empty vector infected (control) cells (P<0.001). Western blotting demonstrated that over-expression of TM9SF1 up-regulated LC3Ⅱ and increased the ratio of LC3Ⅱto LC3Ⅰ, indicating that TM9SF1 may induce autophagy in infected 293T cells. Fluorescence real-time quantitative PCR (RT-qPCR)showed that overexpression of TM9SF1 increased the levels of endoplasmic reticulum stress markers such as CHOP, GADD34 and XBP1 (S) by 3-4folds, compared with that of control cells, indicating occurrence of endoplasmic reticulum stress. These data suggest that TM9SF1 may block 293T cell growth, which is presumably attributed to the induction of endoplasmic reticulum stress and autophagy by TM9SF1. These observations may provide aid in our better understanding of the roles of TM9SF1 in cell growth control.