PURPOSE:Terminal nucleotidyltransferase 5A (TENT5A), recently predicted as a non-canonical poly(A) polymerase, is critically involved in several human disorders including retinitis pigmentosa, cancer and obesity. However, the exact biological role of TENT5A in hepatocellular carcinoma (HCC) has not been elucidated. METHODS:The transcription level of TENT5A and clinical correlation were analyzed using the LIRI-JP cohort, the TCGA-LIHC cohort, and clinical tissue samples of HCC patients in our laboratory. Proliferation, migration, and invasion were detected with stably TENT5A overexpressing and knockdown HCC cells in vitro and in vivo. Chromatin immunoprecipitation and dual-luciferase reporter assay were performed to verify the binding of the target protein to DNA. Co-immunoprecipitation and GST pull-down assay combined with mass spectrometry (MS) were used to identify protein interactions. RESULTS:Our study presented here shows that TENT5A is downregulated in HCC tissues, suggesting a shorter overall survival for patients. Gain- and loss-of-function experiments reveal that TENT5A suppresses the proliferation and metastasis, and the residue Gly122 is of great importance to the role of TENT5A in HCC. More importantly, EGR1 (Early growth response 1) directly binds to the TENT5A promoter and promotes TENT5A expression. By interacting with RPL35, TENT5A is involved in ribosome biogenesis and exerts a negative regulatory effect on the mTOR pathway. CONCLUSIONS:Our findings illustrate the role of the oncosuppressive function of TENT5A in HCC and suggest that the EGR1/TENT5A/RPL35 regulatory axis may be a promising target for therapeutic strategies in HCC.

Hepatocellular carcinoma (HCC) is one of the most lethal malignant cancers across the world. It has a poor prognosis and lacks effective therapies, especially for patients with advanced-stage cancer, indicating an urgent need for new therapies and novel therapeutic targets. Here, by screening the U.S. Food and Drug Administration drug library against HCC cell lines, we identified that flubendazole, a traditional anthelmintic drug, could prominently suppress HCC cells in vivo and in vitro. RNA sequence analysis and cellular thermal shift assays showed that flubendazole reduced the expression of PCSK9 protein by direct targeting. The increased expression of PCSK9 in HCC tissues was demonstrated to be correlated with poor prognosis, and the inhibitory ability of flubendazole was selectively dependent on PCSK9 expression. PCSK9 knockdown abolished the antitumor effects of flubendazole in HCC. Mechanistically, flubendazole inhibited the Hedgehog signaling pathway induced by PCSK9, resulting in the downregulation of smoothened (SMO) and GLI Family Zinc Finger 1 (Gli1). Moreover, combining flubendazole with lenvatinib was found more effective than administering lenvatinib only for HCC treatment in vivo and in vitro. These findings reveal the therapeutic potential of flubendazole against HCC and provide clues on new repurposed drugs and targets for cancer treatment.

BACKGROUND:As a small G protein of Ras family, Ras-like-without-CAAX-1 (RIT1) plays a critical role in various tumors. Our previous study has demonstrated the involvement of RIT1 in promoting malignant progression of hepatocellular carcinoma (HCC). However, its underlying mechanism remains unclear.METHODS:Gene set enrichment analysis (GSEA) was conducted in the TCGA LIHC cohort to investigate the underlying biological mechanism of RIT1. Live cell imaging, immunofluorescence (IF) and flow cytometry assays were used to verify biological function of RIT1 in HCC mitosis. Subcutaneous xenografting of human HCC cells in BALB/c nude mice was utilized to assess tumor proliferation in vivo. RNA-seq, co-immunoprecipitation (Co-IP), mass spectrometry analyses, western blot and IF assays were employed to elucidate the mechanisms by which RIT1 regulates mitosis and promotes proliferation in HCC.RESULTS:Our findings demonstrate that RIT1 plays a crucial role in regulating mitosis in HCC. Knockdown of RIT1 disrupts cell division, leading to G2/M phase arrest, mitotic catastrophe, and apoptosis in HCC cells. SMC3 is found to interact with RIT1 and knockdown of SMC3 attenuates the proliferative effects mediated by RIT1 both in vitro and in vivo. Mechanistically, RIT1 protects and maintains SMC3 acetylation by binding to SMC3 and PDS5 during mitosis, thereby promoting rapid cell division and proliferation in HCC. Notably, we have observed an upregulation of SMC3 expression in HCC tissues, which is associated with poor patient survival and promotion of HCC cell proliferation. Furthermore, there is a significant positive correlation between the expression levels of RIT1, SMC3, and PDS5. Importantly, HCC patients with high expression of both RIT1 and SMC3 exhibit worse prognosis compared to those with high RIT1 but low SMC3 expression.CONCLUSIONS:Our findings underscore the crucial role of RIT1 in regulating mitosis in HCC and further demonstrate its potential as a promising therapeutic target for HCC treatment.

Hepatocellular carcinoma (HCC) is a highly malignant tumor and its progression is associated with altered lipid metabolism in precancerous lesions, such as non-alcoholic fatty liver disease. Here, we identified sperm associated antigen 4 (SPAG4), and explored its oncogenic role in HCC progression. Database analysis and immunohistochemistry indicated increased level of SPAG4 in HCC tissues which was of prognostic value. Gain/loss-offunction experiments showed that SPAG4 exerted oncogenic roles in HCC growth both in vitro and in vivo. RNA sequencing revealed activation of a lipogenic state and SREBP1-mediated pathway following SPAG4 overexpression. Mechanistically, the N-terminal region of SPAG4 bound to lamin A/C, which increased SREBP1 expression, nuclear translocation, and transcriptional activity. Treatment with orlistat, a lipid synthesis inhibitor, reversed SPAG4-mediated oncogenic effects, and its efficacy varied with SPAG4 level. The effect of orlistat was further amplified when combined with sorafenib in tumor xenograft mouse models. Our study provides evidence that SPAG4 mediates HCC progression by affecting lipid metabolism. Administration of orlistat combined with sorafenib reverses SPAG4-mediated oncogenesis in HCC cells and ectopic xenograft tumors in mice, suggesting that this pathway represents a potential target for HCC treatment.

Hepatocellular carcinoma (HCC) is one of the most lethal cancer worldwide, characterized with high heterogeneity and inclination to metastasize. Emerging evidence suggests that BAP31 gets involved in cancer progression with different kinds. It still remains unknown whether and how BAP31 plays a role in HCC metastasis. Epithelial–mesenchymal transition (EMT) has been a common feature in tumor micro-environment, whose inducer TGF-β increased BAP31 expression in this research. Elevated expression of BAP31 was positively correlated with tumor size, vascular invasion and poor prognosis in human HCC. Ectopic expression of BAP31 promoted cell migration and invasion while BAP31 knockdown markedly attenuated metastatic potential in HCC cells and mice orthotopic xenografts. BAP31 induced EMT process, and enhanced the expression level of EMT-related factor Snail and decreased contents and membrane distribution of E-cadherin. BAP31 also activated AKT/β-catenin pathway, which mediated its promotional effects on HCC metastasis. AKT inhibitor further counteracted the activated AKT/β-catenin/Snail upon BAP31 over-expression. Moreover, silencing Snail in BAP31-overexpressed cells impaired enhanced migratory and invasive abilities of HCC cells. In HCC tissues, BAP31 expression was positively associated with Snail. In conclusion, BAP31 promotes HCC metastasis by activating AKT/β-catenin/Snail pathway. Thus, our study implicates BAP31 as potential prognostic biomarker, and provides valuable information for HCC prognosis and treatment.

目的:探讨肝细胞癌中缺氧对肝配蛋白A3(ephrin A3,EFNA3)的调控作用,以及沉默EFNA3基因表达对缺氧诱导的肝细胞癌细胞迁移和侵袭的影响.方法:肝细胞癌HCC-LY10和MHCC-97L细胞缺氧条件下培养0、12、24和48 h,采用实时荧光定量PCR法检测2株细胞中EFNA3 mRNA和长链非编码RNA (long non-coding RNA,LncRNA) EFNA3的表达水平,蛋白质印迹法检测EFNA3和缺氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)蛋白的表达水平.采用脂质体法将miR-210-3p-mimic和阴性对照(NC-mimic)转入HCC-LY10和MHCC-97L细胞,实时荧光定量PCR检测miR-210-3p的转染效率,以及miR-210-3p过表达后常氧和缺氧条件下EFNA3 mRNA和LncRNA EFNA3表达水平的变化,同时采用蛋白质印迹法检测HIF-1α和EFNA3蛋白表达水平的变化.采用慢病毒感染法将靶向HIF-1α和HIF-2α基因的shRNA慢病毒重组载体GV248-shHIF-1α-1、GV248-shHIF-1α-2、GV248-shHIF-2α-1和GV248-shHIF-2α-2及阴性对照GV248-NC转入HCC-LY10和MHCC-97L细胞,实时荧光定量PCR检测HIF-1α和HIF-2α的干扰效率,以及沉默HIF-1α和HIF-2α表达后缺氧条件下EFNA3 mRNA和LncRNA EFNA3表达水平的变化;采用慢病毒感染法将靶向EFNA3基因的shRNA重组慢病毒载体LVRU6GP-shEFNA3-1和LVRU6GP-shEFNA3-2及阴性对照LVRU6GP-NC转入HCC-LY10和MHCC-97L细胞,采用蛋白质印迹法检测EFNA3基因的干扰效率,Transwell小室法检测沉默EFNA3表达后缺氧条件下对肝细胞癌细胞HCC-LY10和MHCC-97L迁移和侵袭的影响.结果:在缺氧培养条件下,肝细胞癌HCC-LY10和MHCC-97L细胞中EFNA3 mRNA的表达水平无明显变化(P值均＞0.05),LncRNA EFNA3和EFNA3蛋白的表达水平均明显上调(P值均＜0.01);HCC-LY10和MHCC-97L细胞中稳定转染miR-210-3p mimic后,miR-210-3p 的表达水平显著增加(P值均＜ 0.001),miR-210-3p对EFNA3 mRNA和LncRNA EFNA3的表达无明显影响(P值均＞0.05),但能够下调缺氧诱导的EFNA3蛋白表达水平(P值均＜0.05);缺氧条件下在HCC-LY1O和MHCC-97L细胞中稳定干扰HIF-1α和HIF-2α表达后,HIF-1α mRNA和HIF-2α mRNA的表达水平均明显下调(P值均＜ 0.01);稳定干扰HIF-1α表达后对EFNA3 mRNA的表达无明显影响(P值均＞ 0.05),稳定干扰HIF-1α表达可下调LncRNA EFNA3的表达水平(P值均＜0.001);稳定干扰HIF-2α对EFNA3 mRNA和LncRNA EFNA3的表达水平均无明显影响(P值均＞0.05);稳定干扰EFNA3表达后显著抑制缺氧诱导的HCC-LY10和MHCC-97L细胞的迁移和侵袭能力的增加(P值均＜0.01).结论:缺氧条件下,HIF-1α可上调LncRNA EFNA3的水平,竞争性结合miR-210-3p,从而上调EFNA3蛋白的表达水平,干扰EFNA3的表达能抑制肝细胞癌细胞的迁移和侵袭能力.

Background: Emerging evidence suggests that Eph/ephrin-system involves different facets of tumorigenesis and cancer progression. Whether ephrin-A3, a cognate ligand to several Eph receptors, plays a role in HCC cell proliferation and metastasis remains unclear.Methods: The expression of ephrin-A3 was detected by immunohistochemistry and qRT-PCR in human HCC tissues. The clinical significance of ephrin-A3 was analyzed using multiple databases. The biological functions of ephrin-A3 were investigated in vitro and in vivo. Immunofluorescence and co-immunoprecipitation were used to detect the interaction between ephrin-A3 and EphA2.Findings: Elevated expression of ephrin-A3 was positively correlated with the histological grade, metastasis and poor prognosis in human HCC. Knockdown of ephrin-A3 led to significantly decreased proliferative activity and metastatic potential, and re-introduction of ephrin-A3 rescued the decreased proliferation, migration, and invasion induced by ephrin-A3 knockdown in HCC cells. Ephrin-A3 interacted with EphA2 and positively regulated EphA2 and AKT phosphorylation. Knockdown of EphA2 attenuated HCC cell proliferation, migration, and invasion induced by ephrin-A3 overexpression. Furthermore, ephrin-A3-induced EphA2 phosphorylation is both a substrate and a positive regulator of AKT. In HCC tissues, ephrin-A3 expression was positively correlated with EphA2, and patients with high co-expression of ephrin-A3 and EphA2 exhibited poorer prognosis.Interpretation: Ephrin-A3 promotes HCC metastasis by interacting with EphA2 and activating EphA2 and AKT. Thus, our study implicates ephrin-A3 and EphA2 as potential prognostic biomarkers, and provides valuable information for HCC prognosis and treatment.Funding Statement: This work was supported in part by grants from the National Natural Science Foundation of China (81672832), Outstanding Subject Leader Training Program of Shanghai Municipal Commission of Health and Family Planning (2018BR20), the National Natural Science Foundation of Shanghai (20ZR1454000), the National Key Sci-Tech Special Project of China (2018ZX10723204-006), the Research Project of Shanghai Municipal Commission of Health and Family Planning (20204Y0093), and the Grants from the State Key Laboratory of Oncogenes and Related Genes (91-17-18).Declaration of Interests: The authors declare that they have no competing interests.Ethics Approval Statement: The study was approved by the Research Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine, and informed consent was obtained from each patient from the Zhejiang University (Hangzhou, China) and the Qidong Liver Cancer Institute (Qidong, China).

目的:探讨C型凝集素域家族4,成员M (C-type lectin domain family 4,member M,CLEC4M)在正常肝、肝硬化、肝细胞癌(hepatocellular carcinoma,HCC)组织及其配对癌旁肝组织中的表达及其临床意义.方法:利用肿瘤基因组图谱(The Cancer Genome Atlas,TCGA)和基因表达综合(Gene Expression Omnibus,GEO)等数据库,筛选各个肝癌数据库中在HCC组织中表达明显下调的基因.利用GEO数据库分析正常肝、肝硬化及HCC组织中CLEC4M mRNA的表达水平.采用实时定量PCR检测83对HCC组织及其配对癌旁肝组织中CLEC4M mRNA的表达水平.利用肝细胞癌癌基因图谱数据库(Integrative Molecular Analysis of Hepatocellular Carcinoma,HCCDB)分析CLEC4M mRNA在多种肿瘤中的表达情况.分别采用免疫组织化学法检测236对HCC及其配对癌旁肝组织、10例正常肝组织和12例肝硬化组织中CLEC4M蛋白的表达水平;蛋白质印迹法检测8对HCC及其配对癌旁肝组织中CLEC4M蛋白的表达水平;再用Kaplan-Meier法分析癌旁肝组织中CLEC4M表达与患者总生存期之间的关系.结果:对GEO和TCGA数据库的检索结果以及对83对临床样品的检测结果显示,HCC组织中CLEC4M mRNA的表达水平明显低于癌旁肝组织(P值均＜ 0.000 1).对GEO数据库的检索结果显示,CLEC4M mRNA在正常肝、肝硬化及HCC组织中的表达呈逐渐下降的趋势(P＜0.05).免疫组织化学法检测结果显示,236例HCC组织中仅6例为CLEC4M蛋白高表达,而配对癌旁肝组织中有157例为CLEC4M高表达.Kaplan-Meier生存期分析显示,癌旁肝组织中CLEC4M高表达患者的总生存期较CLEC4M低表达者明显延长(P＜0.000 1).结论:HCC组织中CLEC4M低表达,而癌旁肝组织中CLEC4M高表达提示患者预后较好.