Abstract：
OBJECTIVE: To clarify the ambiguity of the function of CMTM3 in the development of hepatocellular carcinoma (HCC) and explore its molecular mechanism.
METHODS: The Cmtm3-KO C57BL/6 mouse strain was established using CRISPR-Cas9. Acute liver damage and HCC models were induced by peritoneal injection of 100 or 25 mg/kg.BW N-Nitrosodiethylamine (DEN) to male mice. Liver function and histology were evaluated by blood serum levels of AST and ALT, and HE staining. Gene and protein expression in liver tissues was investigated by RNA-seq, RT-qPCR, Western blotting, immunohistochemistry, and immunofluorescence. Protein-protein interactions were studied by STRING and topological measures. The mRNA expression of CMTM3 and PPARs and patient survival were analyzed using the UALCAN database.
RESULTS: Global knockout of Cmtm3 in KO mice was successfully confirmed. Cmtm3 knockout alleviated DEN-induced acute damage to liver histological integrity and liver function, reduced DNA damage and apoptosis, and also caused a significantly reduced number (WT: 8.7 ± 5.5 vs. KO: 2.7 ± 3.1, P = 0.0394) and total size of tumors (WT: 130.9 ± 181.8 mm2 vs. KO: 9.3 ± 11.5 mm2, P = 0.026) in the liver. Mechanistically, Cmtm3 knockout resulted in reduced expression and inactivation of Pparγ and its downstream lipid metabolism genes (e.g. Adipoq) upon DEN intoxication. CMTM3 and PPARγ were both overexpressed in HCC, and higher levels of both genes were associated with worse overall survival of HCC patients.
CONCLUSIONS: This study clarified the pro-tumorigenesis role of CMTM3 in HCC in vivo, possibly through the upregulation of PPARγ and activation of the PPAR pathway.
METHODS: The Cmtm3-KO C57BL/6 mouse strain was established using CRISPR-Cas9. Acute liver damage and HCC models were induced by peritoneal injection of 100 or 25 mg/kg.BW N-Nitrosodiethylamine (DEN) to male mice. Liver function and histology were evaluated by blood serum levels of AST and ALT, and HE staining. Gene and protein expression in liver tissues was investigated by RNA-seq, RT-qPCR, Western blotting, immunohistochemistry, and immunofluorescence. Protein-protein interactions were studied by STRING and topological measures. The mRNA expression of CMTM3 and PPARs and patient survival were analyzed using the UALCAN database.
RESULTS: Global knockout of Cmtm3 in KO mice was successfully confirmed. Cmtm3 knockout alleviated DEN-induced acute damage to liver histological integrity and liver function, reduced DNA damage and apoptosis, and also caused a significantly reduced number (WT: 8.7 ± 5.5 vs. KO: 2.7 ± 3.1, P = 0.0394) and total size of tumors (WT: 130.9 ± 181.8 mm2 vs. KO: 9.3 ± 11.5 mm2, P = 0.026) in the liver. Mechanistically, Cmtm3 knockout resulted in reduced expression and inactivation of Pparγ and its downstream lipid metabolism genes (e.g. Adipoq) upon DEN intoxication. CMTM3 and PPARγ were both overexpressed in HCC, and higher levels of both genes were associated with worse overall survival of HCC patients.
CONCLUSIONS: This study clarified the pro-tumorigenesis role of CMTM3 in HCC in vivo, possibly through the upregulation of PPARγ and activation of the PPAR pathway.
摘要：
目的：阐明CMTM3在肝细胞癌（HCC）发生发展中的作用，并探讨其分子机制。
方法：使用CRISPR-Cas9建立Cmtm3-KOC57BL/6小鼠品系。通过腹膜注射100或25mg/kg诱导急性肝损伤和HCC模型。BWN-亚硝基二乙胺(DEN)给雄性小鼠。通过血清AST和ALT水平评估肝功能和组织学，HE染色。通过RNA-seq研究肝组织中的基因和蛋白质表达，RT-qPCR,西方印迹,免疫组织化学，和免疫荧光。通过STRING和拓扑测量研究了蛋白质-蛋白质相互作用。使用UALCAN数据库分析CMTM3和PPARs的mRNA表达和患者生存。
结果：成功证实了KO小鼠中Cmtm3的整体敲除。Cmtm3基因敲除减轻DEN诱导的肝脏组织学完整性和肝功能的急性损伤，减少DNA损伤和细胞凋亡，并导致数量显着减少（WT：8.7±5.5与KO：2.7±3.1，P=0.0394）和肿瘤总大小（WT：130.9±181.8mm2vs.肝脏KO:9.3±11.5mm2,P=0.026)。机械上,Cmtm3敲除导致DEN中毒后Pparγ及其下游脂质代谢基因（例如Adipoq）的表达降低和失活。CMTM3和PPARγ在HCC中均过表达，两种基因水平较高与HCC患者总体生存率较差相关。
结论：本研究阐明了CMTM3在肝癌体内的促肿瘤发生作用，可能通过PPARγ的上调和PPAR途径的激活。
方法：使用CRISPR-Cas9建立Cmtm3-KOC57BL/6小鼠品系。
结果：成功证实了KO小鼠中Cmtm3的整体敲除。
结论：本研究阐明了CMTM3在肝癌体内的促肿瘤发生作用，
