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In vivo study revealed pro-tumorigenic effect of CMTM3 in hepatocellular carcinoma involving the regulation of peroxisome proliferator-activated receptor gamma (PPARγ)

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Abstract

Purpose

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.

Conclusion

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.

Graphical abstract

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. RNA-seq dataset is available at Sequence Read Archive (accession no. PRJNA850897).

Abbreviations

BP:

Biological process

CC:

Cellular component

CKLFSF:

Chemokine-like factor superfamily

CMTM3:

CKLF-like MARVEL transmembrane domain-containing member 3

CRISPR-Cas9:

Clustered regularly interspaced short palindromic repeats-associated protein 9

CYP2E1:

Cytochrome P450 2E1

DEN:

N-nitrosodiethylamine

GO:

Gene ontology

HCC:

Hepatocellular carcinoma

HE:

Hematoxylin-eosin

IHC:

Immunohistochemistry

KEGG:

Kyoto Encyclopedia of Genes and Genomes

KO:

Knock out

KOG:

EuKaryotic Ortholog Groups

MF:

Molecular function

PCR:

Polymerase chain reaction

PPAR:

Peroxisome proliferator-activated receptor

PPI:

Protein-protein interaction

RNA-seq:

Whole transcriptome sequencing

SD:

Standard deviation

TUNEL:

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling

VEGF:

Vascular endothelial growth-factor

WT:

Wild type

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Acknowledgements

We thank Prof. Wenling Han from Peking University Center for Human Disease Genomics, China for generously providing resources and expertise, Prof. Jian Chen from the Medical Oncology Department, Yantai Yuhuangding Hospital for providing human tissue samples, and Mr. Jun Li of the Central Laboratory, Yantai Yuhuangding Hospital for animal maintenance.

Funding

This work was supported by grants from the Shandong Medicine and Health Science and Technology Development Plan, China (Grant no.: 202102080625, 202102080643), National Natural Science Foundation of China (Grant no.: 81971438), and Natural Science Foundation of Shandong Province (Grant no.: ZR2020MH075). The funding body was not involved in any form in the design of the study and collection, analysis, interpretation of data or in writing the manuscript.

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Authors and Affiliations

  1. Central Laboratory, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, 20 Yuhuangding East Road, Yantai Yuhuangding Hospital, Zhifu, Yantai, 264000, Shandong, China

    Jiahui Wang, Hongjin Chu, Zhixin Wang, Xuexia Liu, Zhan Song & Fujun Liu

  2. Department of Clinical Laboratory, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, 264000, Shandong, China

    Xuebo Wang

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Contributions

F.L. and conceived of this study, supervised the project and critically revised the manuscript. J.W. primarily performed the investigations, analyzed data, and wrote the first draft manuscript. H.C., Z.W., X.L., Z.S., and X.W. participated in material preparation, data collection and analysis. All authors commented on previous versions of the manuscript, and have read and approved the final manuscript.

Corresponding author

Correspondence to Fujun Liu.

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Research involving human participants and/or animals

This study was approved by the Ethics Committee of Yantai Yuhuangding Hospital (IRB: 2019–398). All procedures for animal care and use were carried out following the guidelines for the care and use of laboratory animals in accordance with the US NIH Guide for the Care and Use of Laboratory Animals (no. 8023, revised in 1996). All efforts were made to reduce the number of animals used and bodily suffering during the experiment.

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Written informed consent was obtained from all participants for the use of human samples and clinical information for research and publication.

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Wang, J., Chu, H., Wang, Z. et al. In vivo study revealed pro-tumorigenic effect of CMTM3 in hepatocellular carcinoma involving the regulation of peroxisome proliferator-activated receptor gamma (PPARγ). Cell Oncol. 46, 49–64 (2023). https://doi.org/10.1007/s13402-022-00733-1

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