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Ubiquitin-specific protease 3 facilitates cell proliferation by deubiquitinating pyruvate kinase L/R in gallbladder cancer

Abstract

Ubiquitin-specific protease 3 (USP3), a kind of cysteine protease, is a crucial family member of deubiquitinating enzymes. USP3 is aberrantly expressed in several tumors, which may contribute to cancer progression. However, the role of USP3 in gallbladder cancer (GBC) is still unknown. In the current study, we detected the expression of USP3 in GBC tissues, measured its contribution to the cell proliferation in GBC progression, and further studied the underlying mechanism of USP3 in GBC through pyruvate kinase L/R (PKLR; a kind of glycolytic enzyme). We found that the expression of USP3 in GBC tissues were higher than that of adjacent tissues, and the protein levels of USP3 and PKLR were positively correlated. Additionally, overexpressed USP3 significantly promoted cell proliferation in vitro and tumor growth in vivo, while the silencing of USP3 inhibited proliferation and tumor growth. Glycolysis in GBC cells ws promoted by the USP3 overexpression and inhibited bye USP3 downregulation. Moreover, the loss of USP3 promoted the ubiquitination and weakened the stability of PKLR. Results of the rescue assay confirmed that PKLR knockdown suppressed USP3-induced oncogenic activity in USP3 overexpressed GBC cells. These findings imply that USP3 is an essential positive regulator in GBC progression, and USP3-PKLR plays a vital role in the progression and metabolism of GBC.

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Fig. 1: USP3 is aberrantly expressed in gallbladder carcinoma (GBC) samples.
Fig. 2: USP3 promotes GBC cell proliferation.
Fig. 3: USP3 ectopic expression regulates tumor growth in vivo.
Fig. 4: USP3 regulates the ubiquitination and stability of PKLR in GBC cells.
Fig. 5: USP3 regulates glycolysis in GBC-SD and NOZ cells.
Fig. 6: USP3 affects the malignant phenotypes and glycolysis of GBC cells by the regulation of PKLR.

Data availability

The datasets generated or analyzed during the study are available from the corresponding author upon reasonable request.

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Acknowledgements

This research was supported by grants from the Co-operation Research Plan of Medical Science and Technology in Henan Province (Grant no. LHGJ20190149), the Key Scientific Research Projects of Universities in Henan Province (Grant no. 21A320052), and the National Natural Science Foundation of China (Grant no. 82172944).

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Yuling Sun and Ruopeng Liang conceived and founding the experiments; Jie Zhao, Rongtao Zhu, Weijie Wang and Qinwei Lu contributed sample collection/reagents/materials/analysis tools, performed all experiments. Xiaoxue Zhang analyzed the data and wrote the paper. All authors reviewed the manuscript for publication.

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Correspondence to Yu-Ling Sun.

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The study was conducted according to the guidelines of the declaration of Helsinki, and approved by the Medical Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Approval No. 7, 2019).

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Liang, RP., Zhang, XX., Zhao, J. et al. Ubiquitin-specific protease 3 facilitates cell proliferation by deubiquitinating pyruvate kinase L/R in gallbladder cancer. Lab Invest 102, 1367–1376 (2022). https://doi.org/10.1038/s41374-022-00836-1

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