Targeting Endoplasmic reticulum Stress-Induced CLGN Resensitizes Hepatocellular Carcinoma to Apoptosis: Paeonol Synergistically Enhances Efficacy by Dual Inhibition of CLGN and NF-κB

Sitong Yan^1,2Anqi Wang^2,3Xiang Chen²Weijia Jiang²Xiao Du²Yuhan Huang²Xiangyu Zu²Yue Zhu⁴Jiatao Liu⁵Lulu Fan²Lingling Zhang⁶Sun Guoping^2*

• ¹Department of Integrated Traditional Chinese and Western Medicine, The First Affiliated Hospital of Anhui Medical University, Hefei, China
• ²Anhui Medical University First Affiliated Hospital Department of Medical Oncology, Hefei, China
• ³Department of Medical Oncology,, The First Affiliated Hospital of Nanchang University, Nanchang, China
• ⁴Department of Oncology, The First Affiliated Hospital of Bengbu Medical University, Bengbu, China
• ⁵Department of Pharmacy, The First Affiliated Hospital of Anhui Medical University, Hefei, China
• ⁶Institute of Clinical Pharmacology,, Hefei Stomatological Hospital, Hefei, China

Purpose:Endoplasmic reticulum stress (ERS) drives hepatocellular carcinoma (HCC) progression and therapy resistance. This study identifies Calmegin (CLGN) as a novel ERS-induced pro-survival factor and explores shCLGN combined with Paeonol (Pae) to overcome apoptosis resistance via NF-κB suppression. Materials and Methods:CLGN was discovered by transcriptome sequencing of tunicamycin (TM)-induced ERS in HepG2 cells and validated via Western blot. Clinical significance was assessed using 93 paired HCC/adjacent tissues (IHC/WB) and TCGA data. Functional roles of CLGN (proliferation: CCK-8/EdU; migration/invasion: Transwell; apoptosis: flow cytometry) . shCLGN efficacy alone or with Pae was tested in vitro and in vivo (xenografts). Transcriptome sequencing combined with Western blot was performed after sh-CLGN to confirm that CLGN regulates apoptosis-related proteins (such as Bcl-2, Bax, and Caspase-3) through the NF-κB pathway, as well as the mechanism of sh-CLGN combined with paeonol on the pathway. Results: ERS significantly upregulated CLGN in HCC, correlating with advanced tumor stage and poor prognosis. CLGN promoted proliferation/migration and suppressed apoptosis. Crucially, sh-CLGN sensitized HCC cells to Pae, synergistically enhancing apoptosis and tumor suppression. Mechanistically, CLGN sustained survival via NF-κB activation; the combination (sh-CLGN + Pae) dual-blocked CLGN/NF-κB, reversing pro-survival signaling in vitro and in vivo. Conclusion: CLGN is a pivotal ERS effector mediating HCC apoptosis resistance through NF-κB. Sh-CLGN combined with Pae restores apoptotic sensitivity via dual CLGN/NF-κB inhibition, providing a potent strategy against ERS-adapted HCC.