As shown in Figure?3D,E, silencing of PLAGL2 in SK\Hep\1 and SMMC\7721 cells induced the arrest of C\MET and STAT3, whereas the co\occurrence of PLAGL2, p\C\MET (Y1349), and p\STAT3 (Y705) overexpression was found in constructed Bel\7402 and Bel\7404 cells compared to that in vector\transfected cells (Figure?3K,L). significant anti\proliferative and apoptosis\inducing effects on HCC cells in a PLAGL2\dependent manner. Mechanistically, SeS2 suppressed C\MET/STAT3, AKT/mTOR, and MAPK signaling and triggered Bcl\2/Cyto C/Caspase\mediated intrinsic mitochondrial apoptosis both in vitro and in vivo. Conclusions Our data reveal an important role of PLAGL2 in apoptosis resistance in HCC and highlight the potential of using SeS2 as a PLAGL2 inhibitor in patients with HCC. gene encodes a protein comprising 496 amino acids. PLAGL2 contains six zinc fingers and is usually located in the nucleus.5 It has been reported that PLAGL2 contributes to tumorigenesis and the development of a wide variety of different tumors. AZ32 For instance, PLAGL2 overexpression is associated with lung cancer progression, where advanced stages of lung cancer are associated with a higher PLAGL2 expression.6 In addition, PLAGL2 is positively correlated with the degree of tumor invasion in gastrointestinal cancer and colorectal cancer.7, 8 In HCC, PLAGL2 can regulate the EMT\related Wnt/\catenin and EGFR/AKT signaling pathways.9 Furthermore, PLAGL2 and Pirh2 dimers can negatively regulate the levels and stability of p53.10 In neuroblastoma, PLAGL2 induces cell cycle regulation and apoptosis by activating the Nip3 promoter independent of HIF\1;11 however, the mechanism underlying PLAGL2\mediated apoptosis regulation in HCC is not yet fully understood. In addition, no clinically approved drugs targeting PLAGL2 are available to date. Therefore, it would be valuable to study the role of PLAGL2 in HCC and explore potential drugs that can target it. The expression of mesenchymal\epithelial transition factor (C\MET) is commonly upregulated in various cancers.12 The abnormal activation of C\MET plays crucial roles in AZ32 cancer cell proliferation and the resistance of programmed apoptosis.13 The binding of intracellular adapter proteins to C\MET leads to the activation of specific cascades, such as STAT3, AKT/mTOR, and MAPK signaling cascades.14 However, numerous C\MET inhibitors, including the well\known drugs cabozantinib and capmatinib, have failed in clinical trials involving HCC patients.15 These data highlight the significance of clarifying the role of the intracellular regulator of C\MET in HCC. Drug repositioning offers a relatively shorter approval period and a simpler path to clinical translation than traditional drug structural design and high\throughput screening.16 Selenium sulfide (SeS2) is a clinical agent used for the treatment of DFNA56 seborrheic dermatitis and tinea versicolor.17, 18 Several studies have shown that Se exerts anti\cancer effects in addition to supplying substrates for selenoprotein synthesis, such as sodium selenite.18, 19 However, the potential mechanism of action of SeS2 in HCC growth suppression has not been reported to date. Therefore, based on the principle of drug repositioning, we aimed to discover new functions of SeS2 and further investigated the anti\HCC effect and the underlying mechanism of action of SeS2. AZ32 Here, we found that PLAGL2 expression was upregulated in HCC tumor tissues and that PLAGL2 AZ32 overexpression substantially promoted apoptosis resistance in HCC cells. Moreover, we found that the C\MET/STAT3 signaling axis acted as a novel downstream target of PLAGL2 and contributed to the inhibition of PLAGL2\mediated proliferation and apoptosis induction of SeS2 in HCC in vitro and in vivo. Our findings suggest that PLAGL2 plays a vital role in HCC apoptosis resistance and supports the use of SeS2 as a promising PLAGL2 inhibitor for HCC therapy. 2.?MATERIALS AND METHODS 2.1. Chemicals and reagents The details of the reagents used in this study are listed in Table S1. For western blotting analysis, all primary antibodies except GAPDH were used at a dilution of 1 1:1000, and GAPDH and goat anti\rabbit IgG secondary antibodies were used at a dilution of 1 1:10000. 2.2. Cell AZ32 lines and cell culture The human normal hepatocyte cell lines L\02 and the HCC cell lines Hep3B and Huh\7 were purchased from the Shanghai Institute of Cell Biology at the Chinese Academy of Sciences (Shanghai, China). The HCC cell lines SMMC\7721, Bel\7402, Bel\7404, SK\Hep\1, and HepG2 were obtained from Genechem Co., Ltd. (Shanghai, China). The HCC cell line MHCC\97L was obtained as a gift from the First Affiliated Hospital of Xi’an Jiaotong University. MHCC\97L, Huh\7, and HepG2 cells were cultured in DMEM containing 10% FBS. L\02, SMMC\7721, Bel\7402, and Bel\7404 cells were cultured in RPMI\1640 medium, while Hep3B and SK\Hep\1 cells were.