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.
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,