Targeting TRMT5 suppresses hepatocellular carcinoma progression via inhibiting the HIF-1α pathways

Targeting TRMT5 suppresses hepatocellular carcinoma progression via inhibiting the HIF-1α pathways 靶向TRMT5抑制HIF-1α信号通路调控肝癌进程 1 2 first-author 1 2 1 2 1 2 1 2 3 4 5 corresp corresp Ye CHEN, yechency@zju.edu.cn Ye CHEN, yechency@zju.edu.cn Ruolang PAN, panrl@zju.edu.cn Ruolang PAN, panrl@zju.edu.cn panrl@zju.edu.cn 1 2 corresp Ye CHEN, yechency@zju.edu.cn Ye CHEN, yechency@zju.edu.cn yechency@zju.edu.cn https://orcid.org/0000-0003-3671-2504 浙江大学医学院附属儿童医院遗传代谢科，国家儿童健康临床医学研究中心，中国杭州，310052 浙江大学医学院附属儿童医院遗传代谢科，国家儿童健康临床医学研究中心，中国杭州，310052 Department of Genetics, and Department of Genetic and Metabolic Disease, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou 310052, China Department of Genetics, and Department of Genetic and Metabolic Disease, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou 310052, China 浙江省遗传缺陷与发育障碍研究重点实验室，浙江大学遗传研究所，中国杭州，310058 浙江省遗传缺陷与发育障碍研究重点实验室，浙江大学遗传研究所，中国杭州，310058 Zhejiang Provincial Key Laboratory of Genetic and Developmental Disorders, Institute of Genetics, Zhejiang University, Hangzhou 310058, China Zhejiang Provincial Key Laboratory of Genetic and Developmental Disorders, Institute of Genetics, Zhejiang University, Hangzhou 310058, China 传染病诊治国家重点实验室，国家感染性疾病临床医学研究中心，浙江大学医学院附属第一医院，中国杭州，310003 传染病诊治国家重点实验室，国家感染性疾病临床医学研究中心，浙江大学医学院附属第一医院，中国杭州，310003 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China 象山县中医医院医疗健康集团，中国宁波，315700 象山县中医医院医疗健康集团，中国宁波，315700 Xiangshan Hospital of TCM Medical and Health Group, Ningbo 315700, China Xiangshan Hospital of TCM Medical and Health Group, Ningbo 315700, China 浙江省细胞药物与应用技术研究重点实验室，中国杭州，311121 浙江省细胞药物与应用技术研究重点实验室，中国杭州，311121 Zhejiang Provincial Key Laboratory of Cell-Based Drug and Applied Technology Development, Hangzhou 311121, China Zhejiang Provincial Key Laboratory of Cell-Based Drug and Applied Technology Development, Hangzhou 311121, China Accumulating evidence has confirmed the links between transfer RNA (tRNA) modifications and tumor progression. The present study is the first to explore the role of tRNA methyltransferase 5 (TRMT5), which catalyzes the m1G37 modification of mitochondrial tRNAs in hepatocellular carcinoma (HCC) progression. Here, based on bioinformatics and clinical analyses, we identified that TRMT5 expression was upregulated in HCC, which correlated with poor prognosis. Silencing TRMT5 attenuated HCC proliferation and metastasis both in vivo and in vitro, which may be partially explained by declined extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Mechanistically, we discovered that knockdown of TRMT5 inactivated the hypoxia-inducible factor-1 (HIF-1) signaling pathway by preventing HIF-1α stability through the enhancement of cellular oxygen content. Moreover, our data indicated that inhibition of TRMT5 sensitized HCC to doxorubicin by adjusting HIF-‍1α. In conclusion, our study revealed that targeting TRMT5 could inhibit HCC progression and increase the susceptibility of tumor cells to chemotherapy drugs. Thus, TRMT5 might be a carcinogenesis candidate gene that could serve as a potential target for HCC therapy. 越来越多研究表明转运RNA（tRNA）修饰与肿瘤进程有关。本研究首次探索了线粒体tRNA G37位甲基化修饰酶TRMT5（tRNA甲基转移酶5）在肝细胞癌发生发展中的作用。生物信息学和临床分析发现TRMT5在肝癌组织中高表达且与预后不良相关。体内外实验表明TRMT5敲低可诱导肝癌细胞代谢重编程，减弱肝癌细胞的增殖和转移能力。进一步研究发现TRMT5敲低降低了肝癌细胞内HIF-1α的稳定性，进而抑制肝癌细胞生长与转移。此外，TRMT5敲低还导致肝癌细胞对阿霉素的敏感性增加。综上所述，本研究表明靶向TRMT5可以抑制肝癌进程并提升肝癌细胞对化疗药物的敏感性。因此，TRMT5是一个新的致癌候选基因，可以作为肝癌治疗的潜在靶点。 转运RNA（tRNA） tRNA甲基转移酶5（TRMT5） 肝细胞癌 缺氧诱导因子1α（HIF-1α） Transfer RNA (tRNA) tRNA methyltransferase 5 (TRMT5) Hepatocellular carcinoma (HCC) Hypoxia-inducible factor-1α (HIF-1α) 1 Introduction 1 Hepatocellular carcinoma (HCC) is the most common type of liver cancer worldwide ( Forner et al., 2012 Forner et al., 2012 ) and the third leading cause of cancer-related deaths based on the latest GLOBOCAN data ( https://gco. https://gco. ‍iarc.‍fr). Despite advances in diagnosis and treatment options for HCC during the past decades, the prognosis of HCC patients is still unfavorable, with a low five-year survival rate ( Ji and Wang, 2012 Ji and Wang, 2012 ; Zuo et al., 2021 Zuo et al., 2021 ). Studies have proved that hepatocarcinogenesis is a complex multistep process that involves the deregulation of various signaling pathways ( McCubrey et al., 2016 McCubrey et al., 2016 ; Akula et al., 2019 Akula et al., 2019 ; Dimri and Satyanarayana, 2020 Dimri and Satyanarayana, 2020 ), which leads to increased difficulties in targeted therapies. In recent years, emerging immunotherapies have revolutionized the therapeutic landscape for various solid tumors ( Yoest, 2017 Yoest, 2017 ; King et al., 2018 King et al., 2018 ; Bergman, 2019 Bergman, 2019 ). Nevertheless, only a few HCC patients benefit from this therapy ( Liu and Qin, 2019 Liu and Qin, 2019 ; Giraud et al., 2021 Giraud et al., 2021 ; Zhong et al., 2021 Zhong et al., 2021 ). Therefore, there is an urgent need to explore the underlying mechanisms of HCC progression and develop new therapeutic agents for effective prevention and treatment. Transfer RNAs (tRNAs), the critical components of translation, are the most abundant molecules in a cell ( Kirchner and Ignatova, 2015 Kirchner and Ignatova, 2015 ). Defects in tRNA expressions and modifications have been linked to various tumorigenesis and progression types. For instance, Yamamoto et al. (2019) Yamamoto et al. (2019) reported that the 2-methylthio- N 6 -isopentenyl modification of adenosine (ms 2 i 6 A) in mitochondrial (mt)‍-tRNAs by Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) promoted the maintenance of glioma-initiating cells. Rosselló-Tortella et al. (2020) proved that the epigenetic inactivation of tRNA-modifying enzyme TYW2 promoted primary colorectal tumor progression and resulted in poor clinical outcome by inducing ribosome frameshift. He et al. (2020) He et al. (2020) demonstrated that FtsJ RNA 2'- O -methyltransferase 1 (FTSJ1) regulated tRNA 2'- O -methyladenosine modification and suppressed the malignancy of non-small cell lung cancer. Thus, tRNA-modifying enzymes represent promising novel targets for drug discovery. The tRNA methyltransferase 5 ( TRMT5 ) is a nuclear gene (MIM*611023) that encodes a mitochondrial protein responsible for the m1G37 modification of various mitochondrial tRNAs to prevent ribosomal frameshift errors and enhance translational efficiency/fidelity ( Suzuki et al., 2011 Suzuki et al., 2011 ). Accumulating studies have identified the abnormal expression of tRNA methyltransferases contributing to various types of tumorigenesis and progression ( Endres et al., 2019 Endres et al., 2019 ; Ma et al., 2021 Ma et al., 2021 ). In this study, for the first time, we revealed that TRMT5 was highly expressed in HCC tumors and this correlated with poor prognosis. Furthermore, we proved that targeting TRMT5 not only suppresses HCC growth but also improves the efficacy of doxorubicin-mediated chemotherapy. 2 Results 1 2.1 Upregulation of TRMT5 in HCC 2 In order to evaluate whether TRMT5 is involved in HCC progression, the expression profile of TRMT5 in HCC was assessed in published databases and clinical tissues. We first analyzed the messenger RNA (mRNA) expression of TRMT5 in HCC by The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. The TCGA data showed that the mRNA of TRMT5 was overexpressed in HCC tumor tissues compared to adjacent non-tumor tissues ( Fig. 1 Fig. 1 a). Consistent results were observed in three GEO datasets ( Figs.1 Figs.1 b‍–‍1d). Moreover, we found that the mRNA expression level of TRMT5 in metastatic HCC was higher than that in primary HCC according to the data from GSE40367 ( Fig. 1 Fig. 1 e). The survival data from the Gene Expression Profiling Interactive Analysis (GEPIA) demonstrated that patients with high TRMT5 levels exhibited worse overall survival ( Fig. 1 Fig. 1 f). Besides, the TRMT5 protein expression in clinical tissues was evaluated by IHC. Consistent with the previous findings for open databases, we showed that TRMT5 expression was upregulated in HCC tumor tissues (Figs. 1g and S1). 2.2 Attenuated HCC cell proliferation by knockdown of TRMT5 2 In order to explore the function of TRMT5 in HCC cells, we knocked down TRMT5 in the human HCC cell line HepG2 and the mouse HCC cell line Hepa1-6 via lentivirus-mediated short hairpin RNA (shRNA). The knockdown efficiency was demonstrated by western blotting. As shown in Figs. 2 Figs. 2 a and S2, both shRNA1 and shRNA2 effectively inhibited the expression of TRMT5 in HepG2, and only shRNA1 was effective in Hepa1-6. Then, we compared the cell proliferation between these cell lines using cell counting kit-8 (CCK8) assay and colony formation assay, as well as Ki67 immunofluorescence staining ( Figs. 2 Figs. 2 b‍–2d). The results suggested that cell proliferation was attenuated in these TRMT5 -knockdown HCC cells. Furthermore, we detected the cell cycle distribution by flow cytometry. It turned out that TRMT5 knockdown significantly decreased the proportion of cells in the S phase but increased the proportion of cells in the G1 phase ( Fig. 2 Fig. 2 e), confirming the role of TRMT5 in promoting cell growth by regulating cell cycle progression in HCC cells. 2.3 Metabolic reprogramming induced by TRMT5 deficiency 2 In order to characterize the mechanism of TRMT5 regulation in HCC cell growth, we further assessed the effect of TRMT5 on the bioenergetic profiling of HCC by examining both extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in knockdown cells and control cells. The results demonstrated that the knockdown of TRMT5 led to reduced ECAR and OCR in both HepG2 and Hepa1-6 cells (Figs. 3 3 a and 3 3 b), which also indicated a reduced metabolic transition from glycolysis to mitochondrial oxidative phosphorylation in TRMT5 knockdown cells. Moreover, lactate production was measured to evaluate the function of TRMT5 in glycolytic metabolism. As shown in Fig. 3 Fig. 3 c, lactate production was declined in TRMT5 knockdown cells compared to controls. We found that adenosine triphosphate (ATP) production was also decreased in knockdown cells ( Fig. 3 Fig. 3 d). These findings confirmed that TRMT5 deficiency in HCC impaired glycolysis, which is the primary method for cancer cells to produce energy. Thus, we concluded that TRMT5 knockdown induced metabolic reprogramming in HCC cells and resulted in insufficient energy for HCC progression. 2.4 HCC cell migration and invasion inhibited by TRMT5 knockdown 2 Next, the effects of TRMT5 on the in vitro migration and invasion of HCC cells were evaluated. The wound healing assay showed that TRMT5 knockdown significantly reduced the efficiency of wound healing in both HepG2 and Hepa1-6 cells ( Fig. 4 Fig. 4 a). The cell transwell migration and invasion assays revealed that the migrative and invasive cell numbers were markedly decreased in TRMT5 -knockdown cells compared to control cells (Figs. 4 4 b and 4 4 c). Additionally, the effect of TRMT5 on epithelial-mesenchymal transition (EMT) in vitro was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting (Figs. 4 4 d and 4 4 e). The levels of mesenchymal regulators N -cadherin and Vimentin were downregulated, while the epithelial regulator E-cadherin was upregulated in TRMT5 -knockdown cells relative to control cells. Meanwhile, matrix metalloproteinase 13 (MMP13), which may play a role in cell migration and invasion in tumor cells, was also decreased in TRMT5 -knockdown cells. 2.5 HCC tumorigenic progression inhibited by TRMT5 deficiency 2 In order to further validate the effect of TRMT5 on HCC tumorigenic progression in vivo, Hepa1-6 cells with stable transfection of shRNA negative control (shNC) and shRNA1 were subcutaneously injected into the left and right flanks of Balb/c nude mice, respectively. Tumor volume was determined for each mouse in Days 3, 5, 8, 10, 12, and 14 after subcutaneous injection. As shown in Fig. 5 Fig. 5 a, the tumor sizes in the shRNA1 cell group were smaller than those in the shNC group, and this difference increased over time. The mice in each group were then sacrificed, and the tumors were removed and weighed (Figs. 5 5 b and 5 5 c), which further confirmed that the shRNA1 cell group grew slower than the shNC group. Moreover, immunohistochemical analysis revealed that tumors of the shRNA1 group displayed a significantly reduced number of positive Ki67 cells (approximate 34% reduction; Fig. 5 Fig. 5 d). These data suggested that depressed TRMT5 inhibited HCC tumor growth in vivo. For the HCC metastasis assay, the stably transfected cell lines were injected into the lateral tail veins of nude mice. The mice were sacrificed after eight weeks, and the number of metastatic lung nodules was counted. As shown in Fig. 5 Fig. 5 e, the metastatic nodules significantly declined in the shRNA1 group compared to the shNC group. Besides, the hematoxylin-eosin (HE) staining results confirmed our findings ( Fig. 5 Fig. 5 f). Taken together, TRMT5 deficiency was confirmed to inhibit HCC tumorigenic progression in vivo. 2.6 Effect of TRMT5 knockdown on the HIF-1 signaling pathway 2 In order to further investigate the underlying mechanism of TRMT5 on HCC progression, we compared the global gene expression profiles between HepG2 cells transfected with either shRNA1 or shNC by RNA sequencing (RNA-seq) analysis. A cluster of genes differentially expressed between shRNA1 and shNC cells was identified based on fold change of > 1.5 and adjusted P -value of < 0.05 criteria. Among them, 329 genes were downregulated and 204 were upregulated in HepG2-shRNA1 cells ( Figs. 6 Figs. 6 a and S3a). Furthermore, the assessment of biological function by Gene Ontology (GO) analysis indicated that various GO anno‑tations were significantly different between the two groups related to biological processes, which included response to decreased oxygen levels, response to oxygen levels, response to hypoxia, and cellular component and molecular function (Figs. S3b‍–‍S3d). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes was also performed. The top 20 KEGG pathways were listed in Fig. 6 Fig. 6 b. Among them, the hypoxia-inducible factor-1 (HIF-1) signaling pathway attracted our attention, which has been proven to regulate various biological processes, including angiogenesis, cellular metabolism, proliferation, and migration ( Masoud and Li, 2015 Masoud and Li, 2015 ; Blatchley et al., 2019 Blatchley et al., 2019 ). The genes with dysregulated expression enriched in the HIF-1 signaling pathway were shown in Fig. 6 Fig. 6 c, and included pyruvate dehydrogenase kinase 1 ( PDK1 ), vascular endothelial growth factor-A ( VEGFA ), Egl-9 family hypoxia inducible factor 3 ( EGLN3 ), serine protease serpin protein E1 ( SERPINE1 ), MAP kinase interacting serine/threonine kinase 2 ( MKNK2 ), phosphofructokinase ( PFKL ), phosphofructokinase-2/fructose-2,‍6-bisphosphatase 3 ( PFKFB3 ), transferrin ( TF ), epidermal growth factor receptor ( EGFR ), TIMP metallopeptidase inhibitor 1 ( TIMP1 ), and nitric oxide synthase 3 ( NOS3 ). Then, we confirmed the mRNA expression of some dysregulated genes by qRT-PCR, and the results were consistent with those by RNA sequencing (Fig. S3e). Moreover, we analyzed the correlation between these genes and TRMT5 according to data from GEPIA. As shown in Fig. S4, TRMT5 was closely correlated with PDK1 and VEGFA expression ( R =0.32, P =3.3×10 -10 and R =0.33, P =9.4×10 -11 , respectively; Pearson’s correlation test). Therefore, we further examined the protein expression of PDK1 and VEGFA , which were the target genes of HIF-1. Indeed, the protein expression levels of these two genes were downregulated in HepG2 and Hepa1-6 shRNA cells relative to control cells ( Fig. 6 Fig. 6 d). These data suggested that knockdown of TRMT5 might affect the HIF-1 signaling pathway and then inhibit HCC progression. HIF-1 is a heterodimeric protein that consists of HIF-‍1α and HIF-‍1‍β ( Vadlapatla et al., 2013 Vadlapatla et al., 2013 ). Since HIF-‍1α is a master transcription factor regulated in an oxygen-dependent manner, we wondered whether TRMT5 regulates HIF-1α. Therefore, we determined the protein expression of HIF-1α under hypoxia. The results showed that the level of HIF-1α was markedly increased under hypoxic conditions. In contrast, the HIF-1α expression level was significantly lower in knockdown cells compared to shNC cells under hypoxia ( Fig. 6 Fig. 6 e), indicating that TRMT5 plays an essential role in HIF-1α stabilization. HIF-1α is oxygen-sensitive, and oxygen consumption is related to the stabilization of HIF-1α in hypoxia ( Ellinghaus et al., 2013 Ellinghaus et al., 2013 ; Wheaton et al., 2014 Wheaton et al., 2014 ). According to our above findings, TRMT5 deficiency reduced OCR, which could increase cellular oxygen viability. Therefore, we hypothesized that deficient TRMT5 might prevent HIF-1α stabilization by increasing cellular oxygen viability. HCC cells were preincubated with pimonidazole for 3 h, a standard hypoxia marker that forms adducts under hypoxic conditions ( Urtasun et al., 1986 Urtasun et al., 1986 ; Doege et al., 2005 Doege et al., 2005 ). Then, the cells were visualized by immunofluorescence detection of pimonidazole using mouse monoclonal antibody. As anticipated, TRMT5 -knockdown cells attenuated the accumulation of pimonidazole adducts with a lower fluorescent intensity of hypoxyprobe-1 compared to control cells ( Figs. 6 Figs. 6 f and S5), which indicated that the cellular oxygen tension was increased in these TRMT5 -knockdown cells. In conclusion, the deficiency of TRMT5 reduced the OCR and increased the cellular oxygen content, which prevented HIF-1α stabilization. 2.7 HCC sensitivity to doxorubicin increased by inhibition of TRMT5 2 In order to investigate whether the inhibition of TRMT5 contributed to chemoresistance, we treated HCC cells with doxorubicin, a common antitumor drug and widely used chemotherapeutic agent in HCC treatment. First, we examined the viability of HCC cells treated with doxorubicin for 48 h using CCK8. As shown in Fig. 7 Fig. 7 a, the viability of TRMT5 knockdown cells was lower compared to control cells. Besides, the long-term colony formation assays revealed that TRMT5 downregulation increased the sensitivity to doxorubicin in both HepG2 and Hepa1-6 cells ( Fig. 7 Fig. 7 b). Since doxorubicin is an apoptosis inducer, we further detected the apoptosis rate of HCC cells treated with doxorubicin for 48 h by annexin V-fluorescein isothiocyanate (FITC) assay. As shown in Fig. 7 Fig. 7 c, the knockdown of TRMT5 in HCC cells increased the sensitivity to doxorubicin (in HepG2 (shNC vs. shRNA1 vs. shRNA2): 1 μmol/L, (10.52±3.31)% vs. (28.14±2.79)% vs. (20.11±2.11)% annexin V +‍ , and 2 μmol/L, (21.44±3.92)% vs. (35.97±5.48)% vs. (29.32±2.38)% annexin V + ; in Hepa1-6 (shNC vs. shRNA1): 1 μmol/L, (17.05±0.89)% vs. (26.09±2.76)% annexin V + , and 2 μmol/L, (36.82±2.67)% vs. (65.02±3.66)% annexin V + ; P < 0.05 for both cells). The western blotting results revealed that doxorubicin reduced the protein level of HIF-1α in a dose-dependent manner in HCC cells under hypoxia mimicked by CoCl 2 (Fig. S6). Furthermore, the dose-dependent upregulation of apoptosis marker cleaved-caspase 3 was observed. These data were consistent with previous research, in which doxorubicin inhibited the accumulation of HIF-1α in HepG2 under hypoxia ( Bowyer et al., 2017 Bowyer et al., 2017 ). Of note, when we treated HCC cells with doxorubicin (5 μmol/L, 24 h), the HIF-1α level was further decreased, and cleaved-caspase 3 was increased in TRMT5 -deficient cells compared to control cells ( Fig. 7 Fig. 7 d). These data indicated that the inhibition of TRMT5 sensitizes HCC to doxorubicin by adjusting HIF-1α. 3 Discussion 1 Recent advances have highlighted the altered epitranscriptomic modifications in tumorigenesis and progression, frequently correlated with poor prognosis ( Barbieri and Kouzarides, 2020 Barbieri and Kouzarides, 2020 ; Suzuki, 2021 Suzuki, 2021 ). TRMT5 is a highly conserved mitochondrial modified enzyme that catalyzes the m1G37 modification of mitochondrial tRNAs ( Christian et al., 2013 Christian et al., 2013 ). Previous research has demonstrated that TRMT5 mutation causes mitochondrial dysfunction and multiple respiratory chain deficiencies ( Powell et al., 2015 Powell et al., 2015 ). In addition, Wing (2020) Wing (2020) identified high TRMT5 expression in pancreatic ductal adenocarcinoma cancer (PDAC) tissue but not in surrounding normal fibroblasts. Herein, for the first time, we provided quantitative evidence that TRMT5 was involved with HCC progression. We conducted bioinformatics and clinical analyses to find that TRMT5 was upregulated in HCC tissues compared to non-tumor tissues, and patients with high levels of TRMT5 exhibited worse overall survival. Consistently, downregulated TRMT5 attenuated HCC proliferation and inhibited metastasis in vitro and in vivo. These results indicated that TRMT5 might be a carcinogenesis candidate gene. Moreover, we identified a series of dysregulated pathways in TRMT5 -deficient HCC cells, which could be the mechanisms linking TRMT5 deficiency to delayed HCC progression. Among these, the HIF-1 signaling pathway is involved in multiple aspects of tumorigenesis and cancer progression, including proliferation, metabolism, angiogenesis, invasion, metastasis, and therapy resistance ( Ke and Costa, 2006 Ke and Costa, 2006 ; Luo et al., 2014 Luo et al., 2014 ; Li et al., 2022 Li et al., 2022 ). This information led to our hypothesis that the activation of HIF-1 signaling pathway is responsible for TRMT5-mediated HCC rapid progression. HIF-1α plays an important role in sensing intratumoral oxygen tension, and its expression is tightly controlled by oxygen tension ( Chun et al., 2002 Chun et al., 2002 ). Mitochondrion, the main oxygen sensor, plays a vital role in regulating HIF-1α stability in hypoxia and responses to hypoxic stress by sending retrograde signals to the nucleus that initiate adaptive metabolic responses and maintain the survival of HCC cells ( Méndez-Blanco et al., 2019 Méndez-Blanco et al., 2019 ; Yang et al., 2020 Yang et al., 2020 ; Yan et al., 2021 Yan et al., 2021 ). Consistent with our hypothesis, when we suppressed TRMT5 in HCC cell lines, the cells showed mitochondrial defects including declined OCR and ECAR, ATP production, and lactate production. Notably, TRMT5 deficiency impaired the stability of HIF-1α under hypoxic condition. Thus, we proposed that targeting TRMT5 inactivates the HIF-1 signaling pathway depending on the attenuation of HIF-1α stabilization. Currently, metastatic HCC is commonly treated by doxorubicin, while drug resistance occurs during therapy, leading to poor prognosis ( Wu et al., 2014 Wu et al., 2014 ). Based on the previous investigation, targeting HIF-1α is one of the useful strategies to improve the effectiveness of doxorubicin in a variety of tumors ( Ward et al., 2013 Ward et al., 2013 ; Arnason and Harkness, 2015 Arnason and Harkness, 2015 ; Prieto-Domínguez et al., 2017 Prieto-Domínguez et al., 2017 ). In our study, the knockdown of TRMT5 expression in HCC cells impaired the expression of HIF-1α, and substantially enhanced the sensitivity to doxorubicin. Consequently, TRMT5 might be a potential target to overcome doxorubicin resistance in HCC. In conclusion, our study demonstrated for the first time that TRMT5 is overexpressed in HCC tissues, and this phenomenon is correlated with poor prognosis. RNA-seq analysis revealed that TRMT5 promotes HIF-1α stabilization under hypoxia. Furthermore, targeting TRMT5 in HCC cell lines enhanced their sensitivity to doxorubicin. Thus, our findings provide new insights into the roles of TRMT5 , as well as clues for developing new therapeutic approaches for HCC. Materials and methods 1 The detailed methods are provided in the electronic supplementary materials of this paper. Availability of data and materials RNA-seq data have been deposited to Sequence Read Archive ( https://www.ncbi.nlm.nih.gov/sra https://www.ncbi.nlm.nih.gov/sra ), and the data can be accessed by the BioProject ID PRJNA 747936. References Akula SM , Abrams SL , Steelman LS , et al. , 2019 . RAS/RAF/MEK/ERK, PI3K/PTEN/AKT/mTORC1 and TP53 pathways and regulatory miRs as therapeutic targets in hepatocellular carcinoma . 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Hepatology , 73 ( 2 ): 644 - 660 . https://doi.org/10.1002/hep.31280 https://doi.org/10.1002/hep.31280 PPARγ coactivator-1α suppresses metastasis of hepatocellular carcinoma by inhibiting warburg effect by PPARγ-dependent WNT/β‍-catenin/pyruvate dehydrogenase kinase isozyme 1 axis Qiong ZHAO, Ruolang PAN, and Ye CHEN designed the experiments, and monitored the project progression and interpretation. Qiong ZHAO, Luwen ZHANG, Qiufen HE, Hui CHANG, Zhiqiang WANG, Hongcui CAO, Ying ZHOU, and Ruolang PAN participated in the study’s data acquisition and analysis. Qiong ZHAO, Qiufen HE, Hui CHANG, Zhiqiang WANG, and Ying ZHOU participated in in vitro experiments. Qiong ZHAO, Luwen ZHANG, and Ruolang PAN completed animal experiments. Qiong ZHAO, Luwen ZHANG, and Hongcui CAO were responsible for histology, image analysis, and quantification. Qiong ZHAO prepared the initial draft of the manuscript. Ye CHEN made the final version of the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Key Research and Development Program of China (Nos. 2020YFA0113003 and 2018YFC1004803) and the Fundamental Research Funds for the Central Universities. We thank Dr. Minxin GUAN (Zhejiang University, Hangzhou, China) and Dr. Jianzhong SHAO (Zhejiang University) for their technical support and valuable suggestions. 10.1631/jzus.B2200224 1673-1581（2023）01-0050-14 赵琼,张璐雯,何秋芬等.靶向TRMT5抑制HIF-1α信号通路调控肝癌进程[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(01):50-63. 赵琼,张璐雯,何秋芬等.靶向TRMT5抑制HIF-1α信号通路调控肝癌进程[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(01):50-63. This work was supported by the National Key Research and Development Program of China (Nos. 2020YFA0113003 and 2018YFC1004803) and the Fundamental Research Funds for the Central Universities. 2022-04-18 2022-08-19 2023-01-15 2023-02-06 2022-12-08 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 Qiong ZHAO, Luwen ZHANG, Qiufen HE, Hui CHANG, Zhiqiang WANG, Hongcui CAO, Ying ZHOU, Ruolang PAN, and Ye CHEN declare that they have no conflict of interest. The study protocol was approved by the Institutional Review Board of Zhejiang University and all animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee at Zhejiang University (application ID: 18021). Compliance with ethics guidelines Supplementary information 1 Figs. S1‒S6; Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 1 24