Silencing AREG Enhances Sensitivity to Irradiation by Suppressing the PI3K/AKT Signaling Pathway in Colorectal Cancer Cells

Wenbing Zhang,1,* Wenjuan Zhang,2,* Chenling Tang,3,4,* Yan Hu,5 Ke Yi,5 Xiaohui Xu,3– 5 Zhihua Chen3,4 1Department of Gastrointestinal Surgery, Anqing First People’s Hospital Affiliated to Anhui Medical University, Anqing, Anhui, 246000, People’s Republic of China; 2Department of Anesthesiology, QingPu Branch of Zhongshan Hospital Affiliated to Fudan University, Shanghai, People’s Republic of China; 3The First People’s Hospital of Taicang City, Taicang Affiliated Hospital of Soochow University, Suzhou, Jiangsu, People’s Republic of China; 4Suzhou Medical College of Soochow University, Suzhou, Jiangsu, People’s Republic of China; 5Central Laboratory, The First People’s Hospital of Taicang, Taicang Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215400, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhihua Chen; Xiaohui Xu, The First People’s Hospital of Taicang, Taicang Affiliated Hospital of Soochow University, No. 58 Changsheng South Road, Taicang, 215400, Jiangsu, People’s Republic of China, Email [email protected]; [email protected]: It has been established that Spalt-Like Transcription Factor 4 (SALL4) promotes Colorectal Cancer (CRC) cell proliferation. Furthermore, Amphiregulin (AREG) is crucially involved in cancer cell proliferation and therapeutic resistance regulation. In this regard, this study aimed to establish whether SALL4 affects the radiosensitization of CRC cells via AREG expression regulation.Methods: Transcriptome sequencing and the Human Transcription Factor Database (HumanTFDB) were used to identify the potential SALL4 targets. The dual-luciferase reporter analysis was used to confirm the SALL4-induced AREG activation. Western Blot (WB) and Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays were used to examine the effect of X-ray irradiation on SALL4 and AREG expression. The AREG-KD (Knockdown) stable cell lines were created through lentiviral infection. Cell proliferation was tracked using Cell Counting Kit 8 (CCK-8) and 5-Ethynyl-2′-deoxyuridine (EdU)-incorporation assays. Cell cycle and apoptosis were examined through flow cytometry. The cells were exposed to a controlled X-ray radiation dose (6 Gy) for imaging purposes.Results: SALL4 could bound to the AREG promoter, enhancing AREG expression. Furthermore, irradiation upregulated SALL4 and AREG in CRC cells. Additionally, AREG knockdown in CRC cells led to reduced DNA replication efficiency, suppressed cell proliferation, increased DNA damage, and enhanced G1 phase arrest and apoptosis following irradiation. On the other hand, AREG overexpression reversed the inhibitory effects of SALL4 downregulation on AREG expression.Conclusion: In CRC cells, SALL4 downregulation suppressed AREG expression, regulating CRC cell radiosensitivity via the PI3K-AKT pathway, thus presenting a potential therapeutic pathway for CRC treatment using Radiotherapy (RT).Keywords: colorectal cancer, SALL4, AREG, PI3K-AKT, radiosensitivity