The effect of CRISPR/Cas9-based gene knockout of SQSTM1 on the cell growth and albumin nanodrug efficacy in human colorectal cancer cells

• Main Authors: Tseng, Tzu-Yen, 曾子硯
• Other Authors: Chao, Jui-I
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/9b74y7

碩士 === 國立交通大學 === 生物科技學系 === 108 === Colorectal cancer (CRC) has become one of the major cause and cancer death in Taiwan and worldwide. Autophagy is an important pathway that displays the biological functions for degrading foreign pathogens and intracellular organelles to keep cellular homeostasis. Autophagy is not a simple non-specific degradation process. Various cargos can be recognized by specific selective autophagy receptors that can mediate the selective autophagosomal degradation. SQSTM1 is a vital selective autophagy receptor which can recruit LC3 and ubiquitinated substrates for degradation. However, the roles of SQSTM1 in regulating cancer cells and whether can be used as a drug target for cancer therapy are still unclear. In this study, we used the CRISPR/Cas9 gene editing technique to generate the SQSTM1 gene knockout in human CRC cells. The protein levels of SQSTM1 in the SQSTM1 knockout cells were identified by western blot and immunofluorescence staining. The gene knockout fragments of SQSTM1 were further verified by DNA sequencing. CRISPR/Cas9 gene editing technique specifically deleted SQSTM1 gene but not affected other autophagy receptors in human CRC cells. Moreover, we found that the knockout of SQSTM1 inhibited the cell growth ability and increased the fractions of G0/G1 phase and cyclin D1 proteins. Albumin is one of the serum proteins that contains high biocompatibility and binding affinity for drug delivery and cancer therapy. The knockout of SQSTM1 reduced the nanodrug efficacy of albumin-bound Paclitaxel (Abraxane) and albumin-conjugated Rapamycin. Taken together, this study demonstrated that the knockout of SQSTM1 affected the cell growth ability and decreased the efficacy of albumin-bound nanodrugs in the CRC cells. We suggest that SQSTM1 is a potential selective autophagy receptor that can be developed as target for CRC therapy.