Abstract: Objective To investigate the impact of Egl⁃9 family hypoxia⁃inducible factor 1 (EGLN1) deficiency on the proliferation, migration, invasion, and autophagic pathway of colorectal cancer (CRC) cells, and to provide potential targets for precision therapy in CRC. Methods EGLN1⁃knockout LoVo and RKO cell lines were generated using CRISPR⁃Cas9 technology. Cell proliferation, invasion, and migration were assessed through CCK⁃8 assay, colony formation assay, Transwell assay, and scratch wound⁃healing assay, respectively. Western blot analysis was performed to determine the LC3B⁃Ⅱ/LC3B⁃Ⅰratio and p62 protein expression levels, thereby assessing the activation of the autophagy pathway. A subcutaneous xenograft model in nude mice was developed to evaluate the effects of the autophagy activator Rapamycin on tumor growth in EGLN1 knockout xenografts. The cell proliferation of xenograft tissues was detected by hematoxylin and eosin (HE) staining and Ki⁃67 immunohistochemical staining. Results Stable EGLN1 knockout cell lines were successfully developed. The deletion of EGLN1 significantly enhanced proliferation, migration, and invasion in both LoVo and RKO cell lines (all P<0.05). The analysis results of autophagy⁃related proteins revealed a decreased LC3B⁃Ⅱ/LC3B⁃Ⅰratio and significant accumulation of p62 following EGLN1 knockout.  Treatment with the late⁃stage autophagy inhibitor chloroquine or the early⁃stage inhibitor 3⁃methyladenine  further exacerbated the inhibition of autophagy. In vivo experiments demonstrated that the EGLN1 knockout group developed significantly larger tumor volumes compared to the EGLN1 wild⁃type group (P<0.05), whlie treatment with Rapamycin effectively suppressed tumor growth. HE staining and Ki⁃67 immunohistochemistry confirmed that EGLN1 knockout promoted cell proliferation in xenografts, an effect that was reversible with rapamycin treatment. Conclusions EGLN1 appears to suppress the malignant phenotype of CRC cells by activating the autophagic pathway. Its deficiency may lead to  autophagy inhibition, thereby promoting tumor cell proliferation, migration, and invasion. Targeting the EGLN1/autophagy axis may represent an effective therapeutic strategy for CRC.

Key words: Colorectal cancer, Egl-9 family hypoxia inducible factor 1,  Proliferation, Migration,  Invasion, Autophagy