Abstract:

WD repeat domain， phosphoinositide interacting 2 （WIPI2） is overexpressed in colorectal cancer （CRC）， suggesting that WIPI2 may be closely related to CRC progression， but the precise mechanism remain unclear. Therefore， the construction of a CRC cell model with stable overexpression of WIPI2 can provide a powerful experimental tool for in-depth study of the effect mechanism of WIPI2 in CRC and discovery of potential therapeutic targets for CRC. In this study， lentiviral vectors overexpressing WIPI2 were designed according to the human WIPI2 gene sequence and infected with CRC cells to evaluate the effect of the recombinant lentiviral vectors in enhancing the expression of WIPI2 protein. The sequence of the recombinant lentiviral vector was confirmed to be consistent with the target sequence of WIPI2 by gene sequencing， and the viral titer of the recombinant lentivirus was measured after viral packaging using 293T， and the titer of GL190-WIPI2 was 2.16×10⁸ TU·mL^-1； the expression level of WIPI2 protein in the overexpression of WIPI2 stably-transfected cell line was obviously up-regulated， and the lentiviral vector for overexpressing WIPI2 was successfully constructed and established. A lentiviral vector for WIPI2 overexpression was successfully constructed， and a stable transient CRC cell line overexpressing WIPI2 gene was established， which provides a cell model for studying the role of WIPI2 in CRC.

Key words: colorectal cancer, WIPI2, lentiviral vector

摘要：

WD重复结构域磷酸肌醇相互作用蛋白2（WD repeat domain，phosphoinositide interacting 2，WIPI2）在结直肠癌（colorectal cancer，CRC）中过度表达，提示WIPI2可能与CRC进展密切相关，但确切作用机制尚不清楚，因此，构建稳定过表达WIPI2的CRC细胞模型，可为深入研究WIPI2在CRC中的效应机制及发现CRC潜在治疗靶点提供有力的实验工具。根据人类WIPI2基因序列设计，构建过表达WIPI2基因的慢病毒载体，并感染CRC细胞，评价重组慢病毒载体提高WIPI2蛋白表达的效果。经基因测序证实重组慢病毒载体的序列与WIPI2目标序列一致，利用293T进行病毒包装后测定重组慢病毒的病毒滴度，GL190-WIPI2的滴度为2.16×10⁸ TU·mL^-1；过表达WIPI2稳转细胞株中WIPI2蛋白的表达水平明显上调，成功构建了WIPI2过表达慢病毒载体，并建立了过表达WIPI2基因的稳转CRC细胞株，为研究WIPI2在CRC中的作用提供了细胞模型。

关键词: 结直肠癌, WIPI2, 慢病毒载体