According to the World Health Organization, it is estimated that more than 1 million patients will die of hepatocellular carcinoma (HCC) by 2030, and the 5-year survival rate is only 18 % (Feng et al., 2019, Villanueva, 2019). Aflatoxin, alcohol-induced liver disease, and obesity-related fatty liver all have the potential to develop into HCC (Eugen, 2020, Mantovani et al., 2022, Péneau et al., 2022). HCC has strong resistance to traditional chemotherapy and radiotherapy, and is prone to recurrence and metastasis after surgical treatment. Although the level of diagnosis and treatment of HCC has been greatly improved in recent years, the research on the improvement of prognosis of HCC still needs to be further strengthened. Therefore, the elucidation of the mechanism of the occurrence and development of HCC has become particularly important and needs to be further studied.

Autophagy is an evolutionarily conserved catabolic process that maintains homeostasis in cells. Beclin1 and LC3I/II, which are critical for autophagosome formation and maturation (Li et al., 2021, Nakamura et al., 2021). Beclin-1, a core component of the Class III PI3K complex, initiates autophagy by recruiting downstream effectors, while the conversion of LC3I to LC3II serves as a hallmark of autophagic flux. Notably, autophagy was found to promote tumor cell survival during cancer progression (Debnath et al., 2023, Li et al., 2020). There is growing evidence that autophagy inhibitors can enhance the therapeutic efficacy of different types of cancer. Lys05 is a potent autophagy inhibitor with single-agent anti-tumor activity and can effectively inhibit autophagy in multiple cancers, such as lung cancer, clear cell ovarian cancer and HCC (Amaravadi and Winkler, 2012, Cechakova et al., 2019, DeVorkin et al., 2017, Gade et al., 2017, Ma et al., 2020). The autophagy inhibitor hydroxychloroquine, an FAD-approved antimalarial drug in the United States, has been reported to improve the medium-term survival of patients with brain tumors (Eldredge et al., 2013, Sotelo et al., 2006). Recently, a large number of evidences have revealed that the autophagy of tumor cells is closely related to EMT. EMT is driven by transcription factors such as Snail, which directly represses E-cadherin (a key epithelial marker) and upregulates Vimentin (a mesenchymal marker), thereby enhancing tumor cell motility and invasivity. Autophagy activation contributes to the occurrence of EMT and promotes tumor cell migration and invasion, which leads to tumor metastasis (Han et al., 2022). Inhibition of autophagy impairs EMT of tumor cells (Gundamaraju et al., 2022). Impaired Beclin1 expression may drive oncogenic transformation and EMT progression, ultimately leading to diminished clinical prognosis (Cicchini et al., 2014). Targeting autophagy-related molecules (mTOR inhibitor AZD2014) can significantly reduce the migration ability of HCC cells by inhibiting the autophagy EMT axis (Liao et al., 2015). Therefore, the underlying mechanisms regulating the autophagy-dependent EMT process in HCC need to be further understood to develop effective therapeutic strategies.

TSPAN15, a member of the tetraspanin family of proteins, has increasingly become a focal point of interest in cancer research due to its potential role in tumor progression and metastasis. TSPAN15 has been implicated in the modulation of various cellular processes, including cell adhesion, migration, proliferation, and signal transduction, which are often deregulated in cancer (Zhang and Huang, 2012). Within the tumor microenvironment, TSPAN15's expression pattern and function have been observed to significantly differ from those in normal tissues (Huang et al., 2022). Aberrant expression of TSPAN15 can contribute to the malignant phenotype of various cancers, including but not limited to, oral squamous cell carcinoma, ovarian cancer, and HCC (Hiroshima et al., 2019, Sidahmed-Adrar et al., 2019, Tavares et al., 2020). Notably, TSPAN15 has been shown to interact with E3 ubiquitin ligases like BTRC, facilitating the oesophageal squamous cell carcinoma metastasis (Zhang et al., 2018). It is posited that TSPAN15 may facilitate tumor growth and metastasis by enhancing cell motility, promoting EMT, and altering the tumor cell's interaction with the extracellular matrix. The precise molecular mechanisms by which TSPAN15 exerts its effects in HCC are still under investigation.

The programmed cell death 4 (PDCD4), known for its pro-apoptotic functions, has been shown to negatively regulate the initiation and progression of EMT and autophagy. Knockdown of PDCD4 can lead to a decrease in the expression of epithelial markers (such as E-cadherin) and a significant upregulation of stromal markers (such as N-cadherin, Snail/Slug), thereby inducing the EMT process (Wang et al., 2013, Wang and Yang, 2018). In HCC, PDCD4 knockdown HCC cells exhibit stronger migration ability and promote liver and lymph node metastasis in nude mice. Low expression of PDCD4 has also been confirmed to be associated with poor prognosis in HCC patients in clinical samples (Ding et al., 2016, Wang et al., 2013). In addition, the inhibitory effect of PDCD4 on autophagy is one of its important mechanisms for inhibiting tumor progression. PDCD4 binds to the mRNA of autophagy key gene ATG5 through the MA3 domain, inhibiting its protein translation and thereby blocking autophagosome formation (Song et al., 2013). Inhibiting ATG5 dependent autophagy can reduce the expression of EMT transcription factors such as Snail/Slug (Hu et al., 2018). It is worth noting that the dysregulation of PDCD4 ubiquitination process is highly correlated with abnormal tumor metastasis, especially BTRC mediated PDCD4 ubiquitination (Blees et al., 2012, Cui et al., 2019, Dorrello et al., 2006, Li et al., 2019). These observations suggest that PDCD4 may be a key target for the regulation of autophagy EMT network in HCC, but the molecular mechanism regulating the expression of PDCD4 in HCC is not fully understood.

In this study, we aimed to systematically investigate the role of TSPAN15 in HCC progression and metastasis, with a focus on its regulatory mechanisms in autophagy and EMT. Specifically, we used bioinformatics methods combined with relevant online databases to mine and analyze the related data of liver cancer, and analyze the expression and clinical value of TSPAN15. Subsequently, relevant molecular biology related experiments were used to explore the mechanism of TSPAN15 in autophagy, EMT and metastasis of HCC cells, particularly its interaction with BTRC to mediate PDCD4 ubiquitination and degradation. Our findings may provide a theoretical basis for TSPAN15 as a target molecule for clinical treatment.