Bmi1 represses HLF to drive the formation and development of intrahepatic cholangiocarcinoma

Bmi1 represses HLF to drive the formation and development of intrahepatic cholangiocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bmi1 represses HLF to drive the formation and development of intrahepatic cholangiocarcinoma Jun Guo, Xiabing Shi, Ruitao Long, Hua Wu, Feng Ye, Chuanrui Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6059499/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Intrahepatic cholangiocarcinoma (ICC) is the second most prevalent type of primary liver cancer and lacks effective targeted therapy. Previously, we reported that B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) drives the formation and development of ICC independent of Ink4a/Arf; however the underlying mechanism remains unclear. Here, we report that hepatic leukemia factor (HLF) acts as a tumor suppressor gene in ICC and Bmi1 represses HLF to drive ICC initiation and progression. Results In ICC, HLF expression levels were inversely correlated with Bmi1. Overexpression of HLF inhibited the growth of ICC both in vitro and in vivo , whereas HLF knockout promoted ICC development in ICC mouse models. Importantly, HLF repression reversed the inhibitory effects of Bmi1 knockdown on cell survival, proliferation and colony formation. Luciferase reporter assay results indicated that Bmi1 represses HLF by directly binding to its promoter. Conclusion These findings revealed the molecular mechanism through which Bmi1 promotes ICC formation and development and uncovered the role of HLF as a tumor suppressor in ICC. ICC Bmi1 HLF Molecular mechanism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Background Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer, accounting for 10–20% of all hepatic malignancies and 3% of all gastrointestinal malignancies ( 1 , 2 ). In recent years, the incidence of ICC has increased, with a 5-year overall survival rate of approximately 9% ( 3 ). Owing to its long-term asymptomatic progression and high metastatic potential, ICC is often diagnosed at an advanced stage when it is unresectable ( 4 ). Advanced ICC are insensitive to conventional chemotherapy and immunotherapy. Gemcitabine (Gem) combined with cisplatin (Cis) is considered the most effective first-line regimen; however, patient survival is poor ( 5 ). In addition, the molecular mechanisms underlying ICC formation and development remain poorly understood, and effective targeted therapies are limited. B-cell-specific Molony murine leukemia virus integration site 1 (Bmi1) is a core member of the polycomb repressive complex (PRC1) ( 6 ), and has been identified as an oncogene in several types of cancer, including ovarian cancer, hepatocellular carcinoma (HCC), colorectal cancer, prostate cancer and gastric cancer ( 7 – 12 ). Mechanistically, Bmi1 functions in a manner dependent or independent of the Ink4A/Arf locus, the canonical target of Bmi1. Bmi1 reportedly inhibits INK4a/Arf in non-small cell lung cancer and prostate cancer ( 13 , 14 ) but does not act on INK4a/Arf in pancreatic cancer and gliomas ( 15 , 16 ). In a previous study, we reported that Bmi1 promoted ICC initiation and progression independent of the Ink4A/Arf locus ( 17 ). However, the precise role of Bmi1 in the carcinogenesis and progression of ICC remains elusive. Hepatic leukemia factor (HLF) is a transcription factor that belongs to the proline and acidic amino acid-rich basic leucine zipper (PAR bZIP) family ( 18 ). HLF was initially identified as an E2A-HLF fusion gene that functioned as a circadian modulator ( 19 ). It is also involved in regulating of hematopoietic stem cells and hematopoietic malignancies ( 20 – 22 ). In ovarian cancer, HLF was found to promote tumor progression and chemoresistance ( 23 ). In HCC, HLF reportedly promotes the production of tumor-initiating cells (TICs), thereby favoring disease development and progression ( 24 ). In non-small cell lung cancer (NSCLC), Chen et al. reported that HLF promotes NSCLC lung colonization and metastasis to bone, liver and brain i n vivo ( 25 ). Taken together, these studies indicate that HLF functions as an oncogene in various cancers. In the current study, we investigated the molecular mechanism through which Bmi1 drives ICC and found that HLF is a target of Bmi1 in ICC and functions as a tumor suppressor. 2. Results 2.1 RNA-Seq analysis of HLF as a potential target of Bmi1 To identify the possible downstream targets of Bmi1, we performed RNA-Seq using Bmi1 knockdown ICC cells and Bmi1/NRas ICC tissues. We identified 981 and 4985 genes that were significantly upregulated (fold change > 2, p-value < 0.05) in Bmi1 KD QBC-939 and RBE cells, respectively. We also identified 1783 genes that were significantly downregulated (fold change < 0.5, p < 0.05) in Bmi1/NRas ICC tissues. Among these genes, 23 were up-regulated or down-regulated in both cell lines and mouse ICC tissues (Fig. 1 A, 1 B and 1 C, Supplementary Table 3). RT-qPCR results indicated that BCO2, CCT6B, CROT, GANC, PAG1 and HLF were consistently up-regulation in Bmi1 KD RBE and QBC-939 cells (Fig. 1 D). Conversely, AADAT, BCO2, CROT, FAM184A, GANC, NREP, PMDN2 and HLF were consistently downregulated in the liver tissues of Bmi1/NRas and NRas mice (Fig. 1 E). Considering the regulatory role of HLF in tumorigenesis, we selected HLF as a potential target of Bmi1 in subsequent investigations. 2.2 HLF level is negatively correlated with Bmi1 in ICC In 1992, HLF was reported to be expressed in livers and kidney tissues but not in lymphoid cells ( 19 ). In 1999, E2a-Hlf was shown to disrupt the differentiation of T-lymphoid progenitors in vivo, leading to profound postnatal thymic depletion and rendering B- and T-cell progenitors susceptible to malignant transformation ( 26 ). However, according to the GEPIA (Gene Expression Profiling Interactive Analysis) database, HLF expression is reportedly reduced in many cancer types. This decrease was particularly significant in cholangiocarcinoma, with a fold change > 10 (Fig. 2 A). RNA-Seq data of ICC from GEO (Gene Expression Omnibus) indicated that HLF mRNA levels in ICC were reduced by 0.6-fold (40%) when compared with those in biliary epithelial cells (Fig. 2 B). However, the role and mechanism of action of HLF in human ICC remain unclear. Therefore, we examined the expression of HLF mRNA in Bmi1/NRas mouse ICC tissues and human ICC specimens. Consistently, RT-qPCR and western blotting results indicated that the expression of Bmi1 was increased in tumors, whereas that of HLF was decreased. In addition, we detected a negative correlation between HLF and Bmi1 expression in mice and human samples (Fig. 2 C- 2 F). Subsequently, we performed an experiment with HLF KD and verified whether suppression of Bmi1 increased HLF levels. In ICC cells, HLF expression was negatively correlated with Bmi1 (Fig. 2 G and 2 H). In addition, IHC staining results demonstrated negative expression levels of HLF and Bmi1 in ICC mouse and human tissues (Fig. 2 I and 2 J). These data demonstrated that HLF and Bmi1 levels were inversely correlated in ICC cell lines and tissues, indicating a potential causal relationship between Bmi1 and HLF expression during ICC development. 2.3 Overexpression of HLF inhibits the proliferation of ICC both in vitro and in vivo We confirmed that HLF was downregulated in ICC and investigated its function in ICC. After preparing the LV15-HLF lentivirus, the two ICC cell lines, RBE and QBC-939, were infected with the lentivirus to overexpress HLF. Based on the RT-qPCR and western blotting, the expression of HLF was increased at the mRNA (Fig. 3 A) and protein levels (Fig. 3 B), respectively. Cell counting showed that HLF overexpression significantly inhibited the growth of the two ICC cell lines (Fig. 3 C). The CCK8 cell viability assay revealed that HLF overexpression significantly suppressed the viability of ICC cells (Fig. 3 D). The colony formation assay showed that the colony generation ability of ICC cells was significantly impaired upon HLF overexpression (Fig. 3 E). These results indicated that HLF inhibited ICC cell growth. Given that Bmi1 was overexpressed in an ICC subcutaneous xenograft mouse model, we examined whether HLF overexpression could block ICC development in BR subcutaneous xenografts in mice. Initially, BR cells were amplificated, propagated and transfected with LV15-HLF or LV15-SC. Different groups of BR cells were then selected by treatment with 2µg/mL puromycin for 48 h, and the overexpression of HLF, compared with that in the control group, was confirmed by RT-qPCR and western blotting results (Fig. 4 A and 4 B). Next, two groups of BR cells were injected into the subcutaneous tissues of mice. Tumor size and mouse weights were measured simultaneously every alternate day. We found that the size and weight of tumors derived from the HLF-overexpression group were significantly lower than those of tumors from the control group (Fig. 4 C and 4 D). This indicated that overexpression of HLF could inhibit ICC development in a background of Bmi1 overexpression. Although body weight showed no significant difference, we found that tumor volume was significantly lower than that in the HLF-overexpressing group (Fig. 4 E and 4 F). Finally, HLF protein expression was confirmed by western blotting analysis (Fig. 4 G). We have previously reported that Bmi1 cooperates with NRas to induce ICC in mice( 17 ). Therefore, we further investigated whether ectopic expression of HLF is responsible for Bmi1-induced ICC formation and development. First, we cloned HLF downstream of the plasmid pT3-EF1a-Bmi1, with an independent internal ribosome entry site (IRES) (Fig. 5 A). We then injected the Bmi1/NRas or Bmi1-HLF/NRas along with a sleeping beauty transposase plasmid (SB) into mice. The mice were monitored weekly and subsequently sacrificed 18 weeks post-injection. The results revealed that liver volumes was significantly higher in Bmi1/NRas-injected mice than in Bmi1-HLF/NRas-injected mice. Bmi1/NRas injection resulted in multiple ICC nodules spread over the liver surface, whereas the Bmi1-HLF/NRas group had fewer nodules (Fig. 5 B and 5 C). Bmi1-HLF/NRas mice exhibited a substantially lower liver-to-body weight ratio, indicating reduced tumour burden (Fig. 5 D). Furthermore, mice in the Bmi1-HLF/NRas group had a significantly longer survival time than mice in the Bmi1/NRas group, which was delayed by 8 weeks (Fig. 5 E). Finally, slower tumor growth was confirmed by attenuated Ki67 staining in Bmi1-HLF/NRas ICC tissue (Fig. 5 F). According to the western blot analysis, Bmi1-HLF/NRas mice had a significantly higher level of HLF expression than Bmi1/NRas mice, with no significant difference in Bmi1 levels (Fig. 5 G). IHC also indicated a negative correlation between Bmi1 and HLF expression (Fig. 5 H). Collectively, these data indicated that HLF may function as a tumor suppressor gene in ICC. 2.4 HLF knockout promotes the development of ICC induced by AKT/NICD in mice AKT/NICD-induced ICC is another primary mouse model using hydrodynamic transfection, capable of developing a lethal burden of ICC around 4 weeks post-injection. To further explore the role of HLF in ICC development, we generated HLF liver-specific knockout mice by injecting the Alb-Cre plasmid into the HLF fl/fi mice. Plasmids containing AKT/NICD/Alb-Cre or AKT/NICD combined with sleeping beauty transposase were hydrodynamically injected into HLF fl/fi mice. All mice were euthanized 25 days post-injection, and the results revealed that the tumor burden in HLF KO mice was more severe than that in HLF WT mice (Fig. 6 A). The liver weight ratio was higher in the HLF KO group than in the WT group (Fig. 6 B). In addition, the Kaplan-Meier survival curve showed that mice in the HLF KO group died within 24 days, whereas HLF WT mice survived until 32 days post injection (Fig. 6 C). Western blotting results revealed that HLF levels were higher in the normal HLF fl/fl group than in the AKT/NICD-induced ICC group. However, there was no significant difference between AKT/NICD/Alb-Cre- and AKT/NICD-injected mice (Fig. 6 D); this could be attributed to the markedly low baseline HLF level in ICC nodules; thus, HLF protein expression may not be further downregulated, although HLF mRNA levels were inconsistent (Fig. 6 E). Collectively, these data confirmed that HLF is a tumor suppressor gene in ICC by a reverse side. 2.5 HLF knockdown rescues the inhibitory effect of Bmi1 silencing on ICC cell growth Next, we investigated whether the suppression of HLF could restored the tumor-suppressive function of Bmi1 knockdown in ICC cells using an HLF shRNA lentivirus. Using RT-qPCR analysis, we found that both HLF shRNA1 and HLF shRNA2 knocked down HLF expression in RBE cells, with HLF shRNA1 exerting a more robust effect (Fig. 7 A). To examine the restorative function of HLF, RBE cells were treated with 10 µm of Bmi1 inhibitor PTC-209 for 48 h, followed by immediate transfection with HLF shRNA; we also set up the only HLF shRNA transfected group. Next, we examined the changes in Bmi1 and HLF mRNA expression in these four groups of RBE cells and found that the knockdown of Bmi1 significantly upregulated HLF expression, which was inhibited in the PTC-209 + HLF shRNA group (Fig. 7 B). Western blotting analysis also demonstrated a negative correlation between Bmi1 and HLF; however, HLF knockdown was not detected in RBE cells treated with HLF shRNA alone (Fig. 7 C). This was because HLF was originally expressed at low levels in ICC. Furthermore, we found that Bmi1 silencing contributed to cell death in RBE cells, whereas HLF silencing substantially accelerated cell proliferation. Simultaneous HLF silencing restored ICC cell growth inhibited by Bmi1 KD, indicating that Bmi1 promotes cell growth by repressing HLF (Fig. 7 D and 7 E). The colony formation assay results showed that colony formation was reduced in Bmi1-KD RBE cells, whereas HLF suppression reversed this effect (Fig. 7 F and 7 G). Taken together, these results revealed that Bmi1 promotes ICC cell proliferation by repressing HLF. 2.6 Bmi1 represses HLF transcription by binding to its promoter We investigated how Bmi1 repressed HLF in ICC cells. As a core protein of PRC1, Bmi1 generally acts on its target genes as a transcriptional repressor ( 27 – 29 ). Therefore, we examined whether Bmi1 directly binds to the HLF promoter using luciferase reporter assays. To ensure luciferase expression, we cloned both the full (− 2000 to + 1) and core (+ 670 to + 155, promoter 1) HLF promoters into the luciferase reporter pGL3 (Fig. 8 A). Both promoter sequences effectively induced luciferase expression (Fig. 8 B). Then, 0.5 µg pGL3 luciferase vector-expression HLF, along with Bmi1 shRNA or pcDNA-Bmi1 plasmids, were co-transfected into RBE cells. We performed RT-qPCR to confirm the transfection efficiency of Bmi. The results revealed the downregulation of Bmi1 in the Bmi1 shRNA group and upregulation of Bmi1 in the pcDNA-Bmi1 group (Fig. 8 C). We found that Bmi1 knockdown in RBE cells significantly increased luciferase expression under the control of the HLF promoter, whereas Bmi1 overexpression exerted the opposite effect (Fig. 8 D and 8 E). These results indicated that Bmi1 binds to the HLF promoter. Knockdown of Bmi1 significantly reduced the binding of Bmi1 to the promoter region of HLF, whereas overexpression of Bmi1 had the opposite effect. 3. Discussion Currently, the diagnosis and treatment of ICC are limited, while morbidity and mortality rates of ICC continue to increase. Therefore, the mechanisms underlying ICC tumorigenesis warrant further investigation. In our previous report, we demonstrated that Bmi1 drives the formation and development of ICC and that the blockade of Bmi1 inhibits ICC growth ( 17 ). Mechanistically, Bmi1 does not regulate Ink4A/Arf, as observed in several types of cancers ( 13 , 30 – 33 ). In the current study, we investigated the targets of Bmi1 in ICC pathogenesis, identifying HLF as a target of Bmi1 in ICC. Therefore, our findings revealed the mechanism through which Bmi1 promotes the initiation and development of ICC. This study had several implications. First, we discovered that HLF functions as a tumor suppressor in ICC. HLF is a transcription factor that belongs to the proline-and acidic amino acid-rich family and functions as a circadian modulator ( 34 ). HLF regulates the growth of stem cells and hematopoietic malignancies ( 35 – 38 ). Accordingly, HLF can function either as an oncogene or a a tumor suppressor gene. Chromosomal translocations fusing portions of HLF with the E2A gene were shown to lead to a subset of childhood B-lineage acute lymphoid leukemia ( 35 – 38 ). In ovarian cancer, HLF reportedly promotes tumor progression and chemoresistance [23]. In HCC, HLF can promote the production of tumor-initiating cells (TICs), thereby favoring HCC development and progression of HCC [24]. In non-small cell lung cancer (NSCLC), Chen et al reported that HLF promotes NSCLC lung colonization and metastasis to bone, liver and brain in vivo [25]. Taken together, these studies indicate that HLF functions as an oncogene in various cancers. However, recent studies have also reported the tumor-suppressive effect of HLF in some tumors. Chen et al. showed that the upregulation of HLF inhibited the tumorigenesis of H1975 cells in the lungs of mice, whereas silencing HLF had the opposite effect and promoted the metastasis of tumor cells. Wang et al . reported that HLF transactivates c-Jun to enhance the TIC-like properties of hepatoma cells, thereby promoting HCC progression and sorafenib resistance ( 39 ). However, the role of HLF in the ICC remains elusive. In this study, we showed that ICC tissues and cells had reduced levels of HLF. Functional experiments revealed that HLF overexpression could suppress ICC development, whereas HLF knockout accelerates ICC progression. Thus, our study identified HLF as a tumor suppressor in ICC. Second, our study identified HLF as a target of Bmi1 and thus expanded the working mechanism of Bmi1 in the regulation of cancer development. As a member of the polycomb family of transcriptional repressors, Bmi1 binds to polycomb response elements in the genome to silence the transcription of the downstream targets. Bmi1 is reportedly overexpressed in several types of tumors, including ovarian cancer, HCC, colorectal cancer, prostate cancer and gastric cancer ( 7 – 12 ). Mechanistically, the classic downstream target of Bmi1 is Ink4A/Arf, which encodes p16 Ink4A and p14 Arf (p19Arf in mice). For example, in human laryngeal carcinoma, Bmi1 maintains the viability of cancer cells by repressing Ink4A/Arf ( 40 ). Similar mechanisms have been reported in cancers such as colorectal cancer, gallbladder carcinoma and lung cancer ( 41 – 43 ). Nevertheless, several studies have shown that Bmi1 does not regulate Ink4A/Arf in other types of cancers, and numerous new targets of Bmi1 have been identified. In ovarian cancer cells, Bmi1 was shown to activate the PI3K/mTOR/4EBP1 signaling pathway to promote cell proliferation ( 44 ). In HCC, Bmi1 drives HCC formation and development by regulating CDKN2A and NF-κB signaling pathways ( 45 ). In our previous study, we also demonstrated that Bmi1 drives hepatocarcinogenesis by repressing the TGFβ2/SMAD signaling axis ( 46 ). Other targets regulated by Bmi1 include PTEN/AKT/GSK3β axis (regulating cell proliferation and migration) ( 47 ), proapoptotic BH3-only protein Noxa gene (regulating cell survival) ( 48 ), tumor suppressor WW Domain Containing Oxidoreductase (WWOX, regulating cell proliferation) ( 49 ), Smgc and Gcnt3 gene (regulating mucin backbone and mucin-type O-glycosylation) ( 50 ), and the IDAX/Wnt signaling pathway (regulating cell growth) ( 51 ). However, there are few reports on the role of Bmi1 in ICC, particularly its underlying mechanism of Bmi1 in ICC. In our previous study, we demonstrated that Bmi1 drives ICC formation and development independent of Ink4A/Arf ( 17 ). In the present study, we identified HLF as a novel target of Bmi1 in ICC. Bmi1 inhibited HLF expression to maintain ICC growth. Therefore, our study elucidated the mechanism of Bmi1’s function in tumorigenesis and development. Nevertheless, the limitations of this study need to be addressed. First, we did not identify the long-term effects of HLF on ICC development; therefore, we could not evaluate its function in advanced ICC. For example, our experiment focused on the early stages of ICC formation; however, we did not evaluate the role of HLF in tumor suppression in advanced ICC. We plan to evaluate the effect of HLF overexpression on ICC development and progression in a follow-up study using HLF agonists or lentivirus mimics. Furthermore, we observed that HLF expression was not reduced in any of the ICC samples, and no negative correlation was detected between Bmi1 and HLF, indicating that HLF could also be regulated by other factors. Conversely, Bmi1 may have targets other than HLF in the ICC. Therefore, to further confirm the mechanism through which Bmi1 regulates HLF in ICC, additional clinical specimens and mouse models should be employed in future studies. Second, we demonstrated that overexpression of HLF could inhibit ICC progression. However, the specific biological functions of HLF have not yet been clarified. We plan to further explore the role of HLF in ICC, including cell cycle arrest, apoptosis promotion, autophagy, and other biological phenotypes. Finally, although HLF has been reported as an oncogene in many studies, including those on HCC, we did not evaluate the function of HLF in HCC, given that HLF was recently reported to transactivate c-Jun to promote HCC development and sorafenib resistance ( 39 ). In conclusion, our findings demonstrate that Bmi1/HLF signalling promotes the formation and development of ICC and that targeting this signaling pathway is a potential therapeutic strategy for ICC. In addition, this study revealed the tumor- suppressive effect of HLF. Based on current findings, HLF’s function as an oncogene in other cancer types can also be examined. In the future, we plan to uncover novel functions of Bmi1 and HLF signaling in other cancer types in which Bmi1 is overexpressed and does not regulate Ink4A/ Arf. 4. Methods 4.1 RNA sequencing Total RNA of Bmi1/NRas and NRas liver tissues or ICC cell lines was isolated using RNeasy mini kit (Qiagen, Germany). Paired-end libraries were synthesized by using the TruSeq® RNA Sample Preparation Kit (Illumina, USA) following TruSeq® RNA Sample Preparation Guide. Purified libraries were quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. The library construction and sequencing were performed at Beijing Genomics institution (BGI). The raw data were uploaded in the NCBI SRA Submission platform (Accession number: PRJNA1202219). 4.2 Human ICC specimens and clinical database analysis ICC tissue samples were obtained from the Union Hospital of Huazhong University of Science and Technology between 2017–2018. The use of clinical specimens was approved by Medical Ethics Committees of Huazhong University of Science and Technology. Written informed consent was obtained from all patients before surgery. Clinical specimen database of ICC was downloaded from gene expression omnibus (GEO) dataset (Series accession: GSE32225, ID: 200032225), including 149 human ICC specimens. HLF expression profile across multiple types of tumor samples was from gene expression profiling interactive analysis (GEPIA) database (Ensembl ID: ENSG00000108924.13). Bioinformatics analysis of genes was referred to the protocol as followed. First, gene expression profile data is collected from a public database, such as a GEO database. These data often include levels of gene expression under different conditions, such as disease states compared with normal states. Then appropriate statistical methods, such as T-tests or ANOVA, were used to screen out genes that were significantly differentially expressed across different conditions. These differentially expressed genes (DEGs) were the role genes for subsequent analyses. ‌Next, functional enrichment analysis of the selected DEGs was conducted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. GO analysis can reveal the biological processes, cellular components and molecular functions of genes, while KEGG analysis indicates the signaling pathways involved in these genes. Finally, ‌ the role genes and signaling pathways predicted by bioinformatics were verified by real-time quantitative PCR, western blot or other experimental methods to ensure the reliability of the results. 4.3 Quantitative reverse-transcription (q-RT) PCR and Western blotting Total RNA or proteins from ICC tissues or cell lines using TRIzol™ Plus RNA Purification Kit (Thermo Fisher Scientific, Rockford, IL, USA) and M-PER Mammalian Protein Extraction Buffer (Thermo Fisher Scientific) respectively. For qRT-PCR, SYBR Green Master Mix (Thermo Fisher Scientific) was used. Primer pairs used are listed in Supplementary Table 1. For western blotting, proteins were separated by sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and transferred onto polyvinylidene membranes (Bio-Rad) and then performed with primary and secondary antibody incubation. Antibodies used are listed in Supplementary Table 2. The raw blot bands for verification were listed in Supplementary Fig. 1. 4.4 Cell culture and lentiviral infection Human ICC cell line RBE and QBC-939 were purchased from China Center for Type Culture Collection Cell bank in Shanghai, China. Both cell lines were authenticated by STR profiling and tested clear of mycoplasma contamination. Mouse primary ICC cell line BR was extracted from Bmi1/NRas ICC liver tissues and authenticated by xenograft tumor model. In brief, Bmi1/NRas mouse ICC tissue was mechanically disintegrated using a sterile scalpel and enzymatically digested with collagenase I at 37℃. Digested tissues were filtered through 70-µm and 40-µm strainers, respectively. Suspended single cells were cultured in 24-well plates in a series dilution to obtain single clones. ICC single clones were picked and passaged 10 times to establish a stable cell line, named BR cells. Cells were cultured separately in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C. Lentivirus containing Bmi1 shRNA sequence, HLF shRNA sequence or HLF overexpression sequence was synthesized respectively by GenePharma Gene (Suzhou, China). In brief, 293T cells were packaged at the density of 1.3–1.5×10 5 cell/ml and incubated for 24h with a 70% confluent. Then a mixture of the transfection plasmids with Opti-MEM 250µl/well was performed (Mix Plasmid 1.8µg/well + PLKO.1-plasmid 1.8µg/well or Plenti-plasmid 1.8µg/well). The transfection reagent Lipofecamine 2000 was also diluted with Opti-MEM 250µl/well. After that, the two ingredients were mixed and incubated for 20-30min at room temperature. Next, the transfection mix (500µl) was transfered to 293T cells in seeding media (2ml per well of 6 well plate) and cells were incubated for 18h. Then change media to remove the transfection reagent and replace with harvest media (2-4ml per well of 6 well plate) for viral harvests. Cells were incubated continually and 24h later, the media containing virus was retrieved to a storage tube. Continue incubating cells with harvest media (2-4ml per well of 6 well plate), repeat viral harvesting every 12-24h and replace with harvest media. Finally, the media containing virus was centrifuged at 1250 rpm for 5 min and the supernatant was filtered through a 45µm filter and transfer to a sterile storage tube. The lentivirus was then transfected into cells and two days post infection, cells were selected with 2µg/mL puromycin for 48h and then harvested for further experiments. Sequences used for lentivirus synthesis were listed as follows: Sequences used for lentivirus synthesis were listed as follows: Bmi1 shRNA (T: 5’-CAGATTGGATCGGAAAGTA-3’, B: 5’-TACTTTCCGATCCAATCTG-3’), HLF shRNA1 (T: 5’-GGCGCAGAAAGAACAACAT-3’, B: 5’-ATGTTGTTCTTTCTGCG CC-3’), HLF shRNA2 (T: 5’-GAACAAACAAGCCAAGAAA-3’, B: 5’-TTTCTTGG CTTGTTTGTTC-3’), NC shRNA (T: 5’-TTCTCCGAACGTGTCACGT-3’, B: 5’-ACGTGACACGTTCGGAGAA-3’), For overexpression experiments, HLF overexpression lentivirus was synthesized by GenePharma Co and the scrambled sequence with the same plasmid system was used as a negative control. 4.5 Cell proliferation, growth and colony formation assay For cell viability analysis, cells were seeded in 96-well plates at a density of 2000 cells per well and cultured for 2, 6 and 10 days, respectively. Then, cell viability was determined using a Cell Counting Kit-8 (CCK-8). To assay cell proliferation, 5000 cells were seeded in 12-well plates and recounted at different time points. For colony formation assay, 8000 cells were seeded in 10-cm plates and cultured for 14 days. Then, colonies were fixed with methanol and stained with 0.1% crystal violet. The number of colonies was counted and quantified with Image J (NIH, Bethesda, MD, USA). 4.6 Plasmids and ICC mouse models Plasmids used in this study were constructed by professor Xin Chen in University of Hawaii Cancer Center, including pT3-EF1α-Bmi1, pCaggsN-RasV12, pT3-EF1α-myr-AKT, pT3-EF1α-NICD1, pCMV-Cre and pCMV-SB transposase. Human HLF sequence was cloned into pT3-EF1α-Bmi1 to generate pT3-EF1α-Bmi1-IRES-HLF plasmid. All plasmids were extracted and purified using the Endotoxin-free Maxi Prep Kit (Omega Bio-Tek, Norcross, GA, USA). Wild-type FVB/N mice and BALB/c nude mice were purchased from Charles River Technology Corporation (Beijing, China). HLF fl/fl mice in a C57BL/6 background were obtained from Cyagen Biosciences (Guangzhou, China). All mice were housed, fed and monitored in accordance with protocols approved by Medical Ethics Committees and the Committee for Animal Research at Huazhong University of Science and Technology. For ICC primary mouse model, hydrodynamic transfection was performed as described previously ( 52 ). Plasmids were mixed and diluted in 2 mL of saline and injected into the tail vein of FVB/N mice. Mice were monitored at the indicated time points for growth evaluation and sacrificed at the humanistic endpoint. For ICC allograft tumor model, 5 × 10 6 BR or BR-HLF cells in 100 µL of RPMI-1640 medium were injected into the left front armpit of BALB/c nude mice. Tumor diameters were measured every 3 days and tumor volume (V) was calculated as V = (L ×W 2 ) × 0.52. At the end of the experiment, mice were sacrificed and tumors were excised, weighed and photographed. We used a mixture of 50% CO 2 and 50% O 2 to anesthetize mice. The experimental animals were placed in a CO 2 anesthesia tank mixed with O 2 . Then the valve was opened, and after the animals gradually lost consciousness, the CO 2 concentration was increased to 100%. When animals showed an unconscious state, including no pinch reflex, they were continued to ventilate for 2 minutes to determine the death of the animals. Animals will maintain an unconscious state for 20–30 seconds. Trained personnel will conduct euthanasia for cervical dislocation to ensure the death of the mice. For xenograft mouse model, we set the humane endpoints when tumor maximum diameter is close to 2cm and tumor volume is less than 2000mm3. For ICC primary tumor mouse model, the technology using hydrodynamic tail vein injection has been mature in our laboratory and the probable time of tumor formation was stable. Based on this, we observed all mice at least 3 times a week, and when tumors have reached 80% of the maximum, the frequency of observation increased to everyday. When mice exhibited mental depression without anesthesia or sedation, mice were euthanized. For mice survival studies, we set the human endpoints based on BCS (Body Condition Scoring) and clinical observation evaluation. When the BCS was evaluated as 1, mice were euthanized and the survival time was recorded. 4.7 Luciferase reporter assay The regions − 2000 to + 1 or + 155 to + 670 of the human HLF promoter were cloned into pGL3 luciferase vector. For luciferase reporter assay, 0.5 µg pGL3 luciferase vector expression HLF (or indicated mutant) and an internal control reporter plasmid, pRL-TK (Promega, Madison, WI, USA), along with Bmi1 shRNA or pcDNA-Bmi1 plasmid were co-transfected in triplicates into RBE cells using lipofectamine 2000. Two days after transfection, luciferase activities were measured using Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Firefly luciferase activities were normalized to luciferase control values and shown as an average of triplicates. 4.8 Histology and immunohistochemistry Tissue samples were fixed with 4% cold paraformaldehyde at 4 ℃ overnight. Then the samples were paraffin-embedded for hematoxylin and eosin (H&E) or immunohistochemical (IHC) staining. Immunohistochemistry staining was performed as reported previously. Briefly, paraffin slides were stained with primary antibody at 4 ℃ overnight followed by the avidin–biotin-peroxidase protocol. All antibodies used for IHC are listed in Supplementary Table 2. 4.9 Statistical analysis All statistical analysis were performed using SPSS 16.0 software (SPSS Software, Chicago, IL, USA). Experiments were repeated independently at least three times and data are expressed as the mean ± standard deviation (SD). The results were consistent across all three experiments, indicating high reproducibility. Student’s t-test was used to compare means of two groups. The correlation between two variables was analyzed by the Pearson correlation method. A two-side P value < 0.05 was considered statistically significant and P < 0.01 was considered highly significant. Declarations Acknowledgments Not applicable. Funding This work was supported by the National Science Foundation of China (82372667, 82273059, and 82073091) and Guizhou Province Science and Technology Project (ZK-2024-Key-097). Availability of data and materials The authors confirm that the data supporting the findings of this study are available within the article [and/or its supplementary materials]. The raw data of RNA sequence were uploaded in the NCBI SRA Submission platform (Accession number: PRJNA1202219) and data will be released until the reception of this article. Authors' contributions CX contributed to the conception and the design of the study. JG, XS and RL performed the experiments and contributed to the acquisition of data. HW and ND to the analysis and interpretation of data. FY contributed to manuscript drafting or critical revisions on the intellectual content. YX finalized the manuscript. JG and CX confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript. Ethics declarations The research protocols were approved by Medical Ethics Committees and the Committee for Animal Research of Huazhong University of Science and Technology. - Informed Consent: N/A. - Registry and the Registration No. of the study/trial: N/A. - Animal Studies: All mice were housed, fed and monitored in accordance with protocols approved by the Committee for Animal Research at Huazhong University of Science and Technology (the animal ethics approval was provided but the accession number was not available). Supplementary Material Supporting Data. 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Kameyama A, Nishijima R and Yamakoshi K: Bmi-1 regulates mucin levels and mucin O-glycosylation in the submandibular gland of mice. PLoS One 16: e0245607, 2021. Yu F, Zhou C, Zeng H, Liu Y and Li S: BMI1 activates WNT signaling in colon cancer by negatively regulating the WNT antagonist IDAX. Biochem Biophys Res Commun 496: 468–474, 2018. Tward AD, Jones KD, Yant S, et al : Distinct pathways of genomic progression to benign and malignant tumors of the liver. Proc Natl Acad Sci U S A 104: 14771–14776, 2007. Additional Declarations No competing interests reported. Supplementary Files HLFSupportingInformation2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6059499","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":417947211,"identity":"7e77577c-f238-40c9-beee-7e2431cac5f5","order_by":0,"name":"Jun Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIie3RsQrCMBCA4UjguhzOgUB8AqElIBSLiy/SINSl3R0rQlZXB9GHEDoHBCcfoGNdnDrYFxA76pS4Cebfv9wdIcTn+8Eg2D6MeiYCKDVuZIgmah6QyWGgUzciWCqjHZzVAa+h42IkzTjiWQLLu7olMzEurcRcOLKlAFac4j1ZyImxkcFacwyn/ZSi4kiMqqyEUuCYUqVZfnckADTambnSeAVHgjhoujKTEGgZ70OHW0bHtn+5TMRoQ291u5oJK/mIoePXvJNvhc/n8/1FL+zBO0dMRVr3AAAAAElFTkSuQmCC","orcid":"","institution":"Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Guo","suffix":""},{"id":417947212,"identity":"215daaaf-34f4-44b7-843e-9bc1fb4f3f5e","order_by":1,"name":"Xiabing Shi","email":"","orcid":"","institution":"The Central Hospital of Wuhan, Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiabing","middleName":"","lastName":"Shi","suffix":""},{"id":417947214,"identity":"a7040f7f-44d5-43a7-87bf-516ab0767a3f","order_by":2,"name":"Ruitao Long","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ruitao","middleName":"","lastName":"Long","suffix":""},{"id":417947218,"identity":"931f02bf-e0d0-4872-9e86-bc4df96bd3a4","order_by":3,"name":"Hua Wu","email":"","orcid":"","institution":"The Central Hospital of Wuhan, Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Wu","suffix":""},{"id":417947220,"identity":"293a6965-c472-4d2e-a8ed-e9ae4314bf27","order_by":4,"name":"Feng Ye","email":"","orcid":"","institution":"Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Ye","suffix":""},{"id":417947221,"identity":"ea48f159-9f08-4d9c-b67c-c0ed870c681f","order_by":5,"name":"Chuanrui Xu","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Chuanrui","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2025-02-19 01:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6059499/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6059499/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76829989,"identity":"26ed96f1-eccf-4ebb-9906-1e00352b46f1","added_by":"auto","created_at":"2025-02-21 08:11:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":409315,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of Bmi1 target genes in Bmi1/NRas mouse ICC tissues and human REB and QBC-939 ICC cell lines using RNA sequencing. (A) Enrichment of potential target genes in 3 groups of samples. (B) Clustering heat maps of 23 downregulated genes in Bmi1/NRas induced mouse ICC tissues compared with NRas liver tissues. (C) Cluster of 23 upregulated genes in ICC cells after knockdown of Bmi1. (D and E) Verification of 23 target genes using RT-qPCR. Data were shown as mean ± SD. ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/c6025e08da3c0de7ce550c6e.png"},{"id":76831640,"identity":"46a4eeb9-de1d-45b8-b905-1c9b9e61a613","added_by":"auto","created_at":"2025-02-21 08:35:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1270131,"visible":true,"origin":"","legend":"\u003cp\u003eHLF expression is low in ICC and negatively correlates with Bmi1. (A) Expression of HLF in a variety of tumor tissues. Data were from the GEPIA database. (B) HLF level in human ICC samples. Data were from the GEO database (Series accession: GSE32225, ID: 200032225). (C-F) Correlation between Bmi1 and HLF levels in mouse and human ICC tissues detected using RT-qPCR or western blotting analysis, respectively. (G and H) Bmi1 and HLF mRNA and protein levels in ICC cells infected with Bmi1 shRNA lentivirus. (I and J) H\u0026amp;E and IHC staining of mice or human liver tissues from each group. Data were shown as mean ± SD. ** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. Scale bar =100 μm.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/3e1be62041c0a87d909aeb30.png"},{"id":76828509,"identity":"9f493d65-e247-40cb-89d4-3934b70106ef","added_by":"auto","created_at":"2025-02-21 08:03:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":592236,"visible":true,"origin":"","legend":"\u003cp\u003eHLF overexpression inhibits ICC cell growth \u003cem\u003ein vitro\u003c/em\u003e. (A and B) mRNA or protein levels of HLF in ICC cells transfected with HLF overexpression lentivirus LV15-HLF. (C, D and E) Viability, growth and colony formation ability of QBC-939 and RBE cells after HLF overexpression. Data are the mean ± SD. n = 3 per group. ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/8d1a6a98d7a189d8c0b84b98.png"},{"id":76828516,"identity":"b201da14-b908-42a7-b5e6-f486bfa87271","added_by":"auto","created_at":"2025-02-21 08:03:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":619094,"visible":true,"origin":"","legend":"\u003cp\u003eHLF overexpression inhibits the growth of ICC allograft in nude mice. (A and B) HLF mRNA and protein levels in BR cells treated with LV15-HLF. (C) Gross images of BR tumor allografts with or without HLF overexpression. (D, E and F) Tumor weight, body weight and tumor volume of BR tumor allografts with or without HLF overexpression. (G) HLF protein levels in BR cells after HLF overexpression. Data are shown as mean ± SD. n = 6 mice per group. ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/011eafdb61aee15946155e9e.png"},{"id":76831411,"identity":"0abe6e7e-2177-4634-8465-b5131424e666","added_by":"auto","created_at":"2025-02-21 08:27:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1013292,"visible":true,"origin":"","legend":"\u003cp\u003eHLF overexpression delays ICC tumor formation induced by Bmi1 and NRas in mice. (A) Schematic diagram illustrating the constructs used for hydrodynamic injections. (B) Representative gross morphological images of livers from Bmi1/NRas and Bmi1-HLF/NRas mice. (C) Tumor nodule numbers of mouse liver tissues injected with Bmi1/NRas or Bmi1-HLF/NRas. (D) Liver/body weight ratio of different groups of mice. (E) Survival curve of the two groups of mice. (F) Ki67 IHC staining of liver tissues from each group. (G) Bmi1 and HLF levels in liver tissues. (H) H\u0026amp;E and IHC staining of mice liver tissues from each group. Data are shown as mean ± SD. n = 6 mice per group. For survival experiment, n= 15 per group. Scale bar =100 μm.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/6680c2ce6944890885bdcbca.png"},{"id":76828511,"identity":"130f32a9-cc7a-4d50-9cb4-007e41bcbed3","added_by":"auto","created_at":"2025-02-21 08:03:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":703722,"visible":true,"origin":"","legend":"\u003cp\u003eHLF knockout promotes the development of ICC. (A) Representative gross morphological images of livers from AKT/NICD/Alb-Cre and AKT/NICD injected mice. (B) Liver/body weight ratio of different groups of mice. (C) Survival curve of the two groups of mice. (D and E) HLF protein and mRNA levels in liver tissues of different groups of mice. Data are shown as mean ± SD. n = 6 mice per group and 12 mice per group for survival experiment. ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/e72d71e5b1830c42e9c79af3.png"},{"id":76829994,"identity":"0888d3ca-a987-4a54-8f26-7072e67dcdfd","added_by":"auto","created_at":"2025-02-21 08:11:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":835549,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of HLF restores the ICC cell growth inhibited by Bmi1 KD. (A) HLF levels in RBE cells infected with HLF shRNA lentivirus. (B and C) Bmi1 and HLF mRNA and protein levels in ICC cells treated with Bmi1 inhibitor PTC-209 (10 μm) and/or HLF shRNA lentivirus. (D) Viability of RBE cells treated with PTC-209 and/or HLF shRNA lentivirus for various periods. (E) Growth curve of RBE cells treated with PTC-209 and/or HLF shRNA lentivirus. (F and G) Colony formation assay and quantity of RBE cells in different groups. Data were shown as mean ± SD. n= 3. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/6ff47b094949b7f248b4a2b8.png"},{"id":76828521,"identity":"22d05844-d8d4-4da8-b558-206ffafdc162","added_by":"auto","created_at":"2025-02-21 08:03:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":351291,"visible":true,"origin":"","legend":"\u003cp\u003eBmi1 inhibits HLF transcription in ICC by binding to its promoter. (A) Schematic diagram illustrating the two luciferase reporter constructs used. (B) Luciferase activities in RBE cells transfected with luciferase reporter constructs containing entire or core promoter for 48 h. (C) Bmi1 mRNA level after transfection with Bmi1 shRNA or pcDNA-Bmi1. (D \u0026amp; E) Luciferase activities in RBE cells transfected with luciferase reporter plasmids and Bmi1 shRNA or pcDNA-Bmi1 plasmid for 48 h. Mock: cells were transfected with luciferase reporter plasmids and blank vector. Data were shown as mean ± SD (n= 3). ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/12da0b10a0e15081bbd4cd5d.png"},{"id":77884211,"identity":"6f983b92-6f25-4060-85d6-1eeb8fceb722","added_by":"auto","created_at":"2025-03-06 12:47:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7114272,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/e422b3fc-3ed0-4dc2-a57c-d031a13733cc.pdf"},{"id":76828507,"identity":"78a627a1-2a9d-458b-a990-1aa408395cf4","added_by":"auto","created_at":"2025-02-21 08:03:47","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":31819,"visible":true,"origin":"","legend":"","description":"","filename":"HLFSupportingInformation2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6059499/v1/f3c6ed42b6f15f25ffac4cc5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bmi1 represses HLF to drive the formation and development of intrahepatic cholangiocarcinoma","fulltext":[{"header":"1. Background","content":"\u003cp\u003eIntrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer, accounting for 10\u0026ndash;20% of all hepatic malignancies and 3% of all gastrointestinal malignancies (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In recent years, the incidence of ICC has increased, with a 5-year overall survival rate of approximately 9% (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Owing to its long-term asymptomatic progression and high metastatic potential, ICC is often diagnosed at an advanced stage when it is unresectable (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Advanced ICC are insensitive to conventional chemotherapy and immunotherapy. Gemcitabine (Gem) combined with cisplatin (Cis) is considered the most effective first-line regimen; however, patient survival is poor (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In addition, the molecular mechanisms underlying ICC formation and development remain poorly understood, and effective targeted therapies are limited.\u003c/p\u003e \u003cp\u003eB-cell-specific Molony murine leukemia virus integration site 1 (Bmi1) is a core member of the polycomb repressive complex (PRC1) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), and has been identified as an oncogene in several types of cancer, including ovarian cancer, hepatocellular carcinoma (HCC), colorectal cancer, prostate cancer and gastric cancer (\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Mechanistically, Bmi1 functions in a manner dependent or independent of the Ink4A/Arf locus, the canonical target of Bmi1. Bmi1 reportedly inhibits INK4a/Arf in non-small cell lung cancer and prostate cancer (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) but does not act on INK4a/Arf in pancreatic cancer and gliomas (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In a previous study, we reported that Bmi1 promoted ICC initiation and progression independent of the Ink4A/Arf locus (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, the precise role of Bmi1 in the carcinogenesis and progression of ICC remains elusive.\u003c/p\u003e \u003cp\u003eHepatic leukemia factor (HLF) is a transcription factor that belongs to the proline and acidic amino acid-rich basic leucine zipper (PAR bZIP) family (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). HLF was initially identified as an E2A-HLF fusion gene that functioned as a circadian modulator (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). It is also involved in regulating of hematopoietic stem cells and hematopoietic malignancies (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). In ovarian cancer, HLF was found to promote tumor progression and chemoresistance (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In HCC, HLF reportedly promotes the production of tumor-initiating cells (TICs), thereby favoring disease development and progression (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In non-small cell lung cancer (NSCLC), Chen \u003cem\u003eet al.\u003c/em\u003e reported that HLF promotes NSCLC lung colonization and metastasis to bone, liver and brain i\u003cem\u003en vivo\u003c/em\u003e (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Taken together, these studies indicate that HLF functions as an oncogene in various cancers. In the current study, we investigated the molecular mechanism through which Bmi1 drives ICC and found that HLF is a target of Bmi1 in ICC and functions as a tumor suppressor.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 RNA-Seq analysis of HLF as a potential target of Bmi1\u003c/h2\u003e \u003cp\u003eTo identify the possible downstream targets of Bmi1, we performed RNA-Seq using Bmi1 knockdown ICC cells and Bmi1/NRas ICC tissues. We identified 981 and 4985 genes that were significantly upregulated (fold change\u0026thinsp;\u0026gt;\u0026thinsp;2, \u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in Bmi1 KD QBC-939 and RBE cells, respectively. We also identified 1783 genes that were significantly downregulated (fold change\u0026thinsp;\u0026lt;\u0026thinsp;0.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in Bmi1/NRas ICC tissues. Among these genes, 23 were up-regulated or down-regulated in both cell lines and mouse ICC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Supplementary Table\u0026nbsp;3). RT-qPCR results indicated that BCO2, CCT6B, CROT, GANC, PAG1 and HLF were consistently up-regulation in Bmi1 KD RBE and QBC-939 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Conversely, AADAT, BCO2, CROT, FAM184A, GANC, NREP, PMDN2 and HLF were consistently downregulated in the liver tissues of Bmi1/NRas and NRas mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Considering the regulatory role of HLF in tumorigenesis, we selected HLF as a potential target of Bmi1 in subsequent investigations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 HLF level is negatively correlated with Bmi1 in ICC\u003c/h2\u003e \u003cp\u003eIn 1992, HLF was reported to be expressed in livers and kidney tissues but not in lymphoid cells (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In 1999, E2a-Hlf was shown to disrupt the differentiation of T-lymphoid progenitors in vivo, leading to profound postnatal thymic depletion and rendering B- and T-cell progenitors susceptible to malignant transformation (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, according to the GEPIA (Gene Expression Profiling Interactive Analysis) database, HLF expression is reportedly reduced in many cancer types. This decrease was particularly significant in cholangiocarcinoma, with a fold change\u0026thinsp;\u0026gt;\u0026thinsp;10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). RNA-Seq data of ICC from GEO (Gene Expression Omnibus) indicated that HLF mRNA levels in ICC were reduced by 0.6-fold (40%) when compared with those in biliary epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, the role and mechanism of action of HLF in human ICC remain unclear. Therefore, we examined the expression of HLF mRNA in Bmi1/NRas mouse ICC tissues and human ICC specimens. Consistently, RT-qPCR and western blotting results indicated that the expression of Bmi1 was increased in tumors, whereas that of HLF was decreased. In addition, we detected a negative correlation between HLF and Bmi1 expression in mice and human samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Subsequently, we performed an experiment with HLF KD and verified whether suppression of Bmi1 increased HLF levels. In ICC cells, HLF expression was negatively correlated with Bmi1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). In addition, IHC staining results demonstrated negative expression levels of HLF and Bmi1 in ICC mouse and human tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). These data demonstrated that HLF and Bmi1 levels were inversely correlated in ICC cell lines and tissues, indicating a potential causal relationship between Bmi1 and HLF expression during ICC development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Overexpression of HLF inhibits the proliferation of ICC both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eWe confirmed that HLF was downregulated in ICC and investigated its function in ICC. After preparing the LV15-HLF lentivirus, the two ICC cell lines, RBE and QBC-939, were infected with the lentivirus to overexpress HLF. Based on the RT-qPCR and western blotting, the expression of HLF was increased at the mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), respectively. Cell counting showed that HLF overexpression significantly inhibited the growth of the two ICC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The CCK8 cell viability assay revealed that HLF overexpression significantly suppressed the viability of ICC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The colony formation assay showed that the colony generation ability of ICC cells was significantly impaired upon HLF overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These results indicated that HLF inhibited ICC cell growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that Bmi1 was overexpressed in an ICC subcutaneous xenograft mouse model, we examined whether HLF overexpression could block ICC development in BR subcutaneous xenografts in mice. Initially, BR cells were amplificated, propagated and transfected with LV15-HLF or LV15-SC. Different groups of BR cells were then selected by treatment with 2\u0026micro;g/mL puromycin for 48 h, and the overexpression of HLF, compared with that in the control group, was confirmed by RT-qPCR and western blotting results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Next, two groups of BR cells were injected into the subcutaneous tissues of mice. Tumor size and mouse weights were measured simultaneously every alternate day. We found that the size and weight of tumors derived from the HLF-overexpression group were significantly lower than those of tumors from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). This indicated that overexpression of HLF could inhibit ICC development in a background of Bmi1 overexpression. Although body weight showed no significant difference, we found that tumor volume was significantly lower than that in the HLF-overexpressing group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Finally, HLF protein expression was confirmed by western blotting analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe have previously reported that Bmi1 cooperates with NRas to induce ICC in mice(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Therefore, we further investigated whether ectopic expression of HLF is responsible for Bmi1-induced ICC formation and development. First, we cloned HLF downstream of the plasmid pT3-EF1a-Bmi1, with an independent internal ribosome entry site (IRES) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We then injected the Bmi1/NRas or Bmi1-HLF/NRas along with a sleeping beauty transposase plasmid (SB) into mice. The mice were monitored weekly and subsequently sacrificed 18 weeks post-injection. The results revealed that liver volumes was significantly higher in Bmi1/NRas-injected mice than in Bmi1-HLF/NRas-injected mice. Bmi1/NRas injection resulted in multiple ICC nodules spread over the liver surface, whereas the Bmi1-HLF/NRas group had fewer nodules (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Bmi1-HLF/NRas mice exhibited a substantially lower liver-to-body weight ratio, indicating reduced tumour burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, mice in the Bmi1-HLF/NRas group had a significantly longer survival time than mice in the Bmi1/NRas group, which was delayed by 8 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Finally, slower tumor growth was confirmed by attenuated Ki67 staining in Bmi1-HLF/NRas ICC tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). According to the western blot analysis, Bmi1-HLF/NRas mice had a significantly higher level of HLF expression than Bmi1/NRas mice, with no significant difference in Bmi1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). IHC also indicated a negative correlation between Bmi1 and HLF expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Collectively, these data indicated that HLF may function as a tumor suppressor gene in ICC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 HLF knockout promotes the development of ICC induced by AKT/NICD in mice\u003c/h2\u003e \u003cp\u003eAKT/NICD-induced ICC is another primary mouse model using hydrodynamic transfection, capable of developing a lethal burden of ICC around 4 weeks post-injection. To further explore the role of HLF in ICC development, we generated HLF liver-specific knockout mice by injecting the Alb-Cre plasmid into the HLF\u003csup\u003e\u003cem\u003efl/fi\u003c/em\u003e\u003c/sup\u003e mice. Plasmids containing AKT/NICD/Alb-Cre or AKT/NICD combined with sleeping beauty transposase were hydrodynamically injected into HLF\u003csup\u003e\u003cem\u003efl/fi\u003c/em\u003e\u003c/sup\u003e mice. All mice were euthanized 25 days post-injection, and the results revealed that the tumor burden in HLF KO mice was more severe than that in HLF WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The liver weight ratio was higher in the HLF KO group than in the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In addition, the Kaplan-Meier survival curve showed that mice in the HLF KO group died within 24 days, whereas HLF WT mice survived until 32 days post injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Western blotting results revealed that HLF levels were higher in the normal HLF\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e group than in the AKT/NICD-induced ICC group. However, there was no significant difference between AKT/NICD/Alb-Cre- and AKT/NICD-injected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD); this could be attributed to the markedly low baseline HLF level in ICC nodules; thus, HLF protein expression may not be further downregulated, although HLF mRNA levels were inconsistent (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Collectively, these data confirmed that HLF is a tumor suppressor gene in ICC by a reverse side.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 HLF knockdown rescues the inhibitory effect of Bmi1 silencing on ICC cell growth\u003c/h2\u003e \u003cp\u003eNext, we investigated whether the suppression of HLF could restored the tumor-suppressive function of Bmi1 knockdown in ICC cells using an HLF shRNA lentivirus. Using RT-qPCR analysis, we found that both HLF shRNA1 and HLF shRNA2 knocked down HLF expression in RBE cells, with HLF shRNA1 exerting a more robust effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). To examine the restorative function of HLF, RBE cells were treated with 10 \u0026micro;m of Bmi1 inhibitor PTC-209 for 48 h, followed by immediate transfection with HLF shRNA; we also set up the only HLF shRNA transfected group. Next, we examined the changes in Bmi1 and HLF mRNA expression in these four groups of RBE cells and found that the knockdown of Bmi1 significantly upregulated HLF expression, which was inhibited in the PTC-209\u0026thinsp;+\u0026thinsp;HLF shRNA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Western blotting analysis also demonstrated a negative correlation between Bmi1 and HLF; however, HLF knockdown was not detected in RBE cells treated with HLF shRNA alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). This was because HLF was originally expressed at low levels in ICC. Furthermore, we found that Bmi1 silencing contributed to cell death in RBE cells, whereas HLF silencing substantially accelerated cell proliferation. Simultaneous HLF silencing restored ICC cell growth inhibited by Bmi1 KD, indicating that Bmi1 promotes cell growth by repressing HLF (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). The colony formation assay results showed that colony formation was reduced in Bmi1-KD RBE cells, whereas HLF suppression reversed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Taken together, these results revealed that Bmi1 promotes ICC cell proliferation by repressing HLF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Bmi1 represses HLF transcription by binding to its promoter\u003c/h2\u003e \u003cp\u003eWe investigated how Bmi1 repressed HLF in ICC cells. As a core protein of PRC1, Bmi1 generally acts on its target genes as a transcriptional repressor (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Therefore, we examined whether Bmi1 directly binds to the HLF promoter using luciferase reporter assays. To ensure luciferase expression, we cloned both the full (\u0026minus;\u0026thinsp;2000 to +\u0026thinsp;1) and core (+\u0026thinsp;670 to +\u0026thinsp;155, promoter 1) HLF promoters into the luciferase reporter pGL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Both promoter sequences effectively induced luciferase expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Then, 0.5 \u0026micro;g pGL3 luciferase vector-expression HLF, along with Bmi1 shRNA or pcDNA-Bmi1 plasmids, were co-transfected into RBE cells. We performed RT-qPCR to confirm the transfection efficiency of Bmi. The results revealed the downregulation of Bmi1 in the Bmi1 shRNA group and upregulation of Bmi1 in the pcDNA-Bmi1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). We found that Bmi1 knockdown in RBE cells significantly increased luciferase expression under the control of the HLF promoter, whereas Bmi1 overexpression exerted the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). These results indicated that Bmi1 binds to the HLF promoter. Knockdown of Bmi1 significantly reduced the binding of Bmi1 to the promoter region of HLF, whereas overexpression of Bmi1 had the opposite effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eCurrently, the diagnosis and treatment of ICC are limited, while morbidity and mortality rates of ICC continue to increase. Therefore, the mechanisms underlying ICC tumorigenesis warrant further investigation. In our previous report, we demonstrated that Bmi1 drives the formation and development of ICC and that the blockade of Bmi1 inhibits ICC growth (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Mechanistically, Bmi1 does not regulate Ink4A/Arf, as observed in several types of cancers (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In the current study, we investigated the targets of Bmi1 in ICC pathogenesis, identifying HLF as a target of Bmi1 in ICC. Therefore, our findings revealed the mechanism through which Bmi1 promotes the initiation and development of ICC.\u003c/p\u003e \u003cp\u003eThis study had several implications. First, we discovered that HLF functions as a tumor suppressor in ICC. HLF is a transcription factor that belongs to the proline-and acidic amino acid-rich family and functions as a circadian modulator (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). HLF regulates the growth of stem cells and hematopoietic malignancies (\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Accordingly, HLF can function either as an oncogene or a a tumor suppressor gene. Chromosomal translocations fusing portions of HLF with the E2A gene were shown to lead to a subset of childhood B-lineage acute lymphoid leukemia (\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In ovarian cancer, HLF reportedly promotes tumor progression and chemoresistance [23]. In HCC, HLF can promote the production of tumor-initiating cells (TICs), thereby favoring HCC development and progression of HCC [24]. In non-small cell lung cancer (NSCLC), Chen et al reported that HLF promotes NSCLC lung colonization and metastasis to bone, liver and brain in vivo [25]. Taken together, these studies indicate that HLF functions as an oncogene in various cancers. However, recent studies have also reported the tumor-suppressive effect of HLF in some tumors. Chen et al. showed that the upregulation of HLF inhibited the tumorigenesis of H1975 cells in the lungs of mice, whereas silencing HLF had the opposite effect and promoted the metastasis of tumor cells. Wang \u003cem\u003eet al\u003c/em\u003e. reported that HLF transactivates c-Jun to enhance the TIC-like properties of hepatoma cells, thereby promoting HCC progression and sorafenib resistance (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). However, the role of HLF in the ICC remains elusive. In this study, we showed that ICC tissues and cells had reduced levels of HLF. Functional experiments revealed that HLF overexpression could suppress ICC development, whereas HLF knockout accelerates ICC progression. Thus, our study identified HLF as a tumor suppressor in ICC.\u003c/p\u003e \u003cp\u003eSecond, our study identified HLF as a target of Bmi1 and thus expanded the working mechanism of Bmi1 in the regulation of cancer development. As a member of the polycomb family of transcriptional repressors, Bmi1 binds to polycomb response elements in the genome to silence the transcription of the downstream targets. Bmi1 is reportedly overexpressed in several types of tumors, including ovarian cancer, HCC, colorectal cancer, prostate cancer and gastric cancer (\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Mechanistically, the classic downstream target of Bmi1 is Ink4A/Arf, which encodes p16\u003csup\u003eInk4A\u003c/sup\u003e and p14\u003csup\u003eArf\u003c/sup\u003e (p19Arf in mice). For example, in human laryngeal carcinoma, Bmi1 maintains the viability of cancer cells by repressing Ink4A/Arf (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Similar mechanisms have been reported in cancers such as colorectal cancer, gallbladder carcinoma and lung cancer (\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Nevertheless, several studies have shown that Bmi1 does not regulate Ink4A/Arf in other types of cancers, and numerous new targets of Bmi1 have been identified. In ovarian cancer cells, Bmi1 was shown to activate the PI3K/mTOR/4EBP1 signaling pathway to promote cell proliferation (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). In HCC, Bmi1 drives HCC formation and development by regulating CDKN2A and NF-κB signaling pathways (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). In our previous study, we also demonstrated that Bmi1 drives hepatocarcinogenesis by repressing the TGFβ2/SMAD signaling axis (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Other targets regulated by Bmi1 include PTEN/AKT/GSK3β axis (regulating cell proliferation and migration) (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), proapoptotic BH3-only protein Noxa gene (regulating cell survival) (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), tumor suppressor WW Domain Containing Oxidoreductase (WWOX, regulating cell proliferation) (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), Smgc and Gcnt3 gene (regulating mucin backbone and mucin-type O-glycosylation) (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), and the IDAX/Wnt signaling pathway (regulating cell growth) (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). However, there are few reports on the role of Bmi1 in ICC, particularly its underlying mechanism of Bmi1 in ICC. In our previous study, we demonstrated that Bmi1 drives ICC formation and development independent of Ink4A/Arf (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In the present study, we identified HLF as a novel target of Bmi1 in ICC. Bmi1 inhibited HLF expression to maintain ICC growth. Therefore, our study elucidated the mechanism of Bmi1\u0026rsquo;s function in tumorigenesis and development.\u003c/p\u003e \u003cp\u003eNevertheless, the limitations of this study need to be addressed. First, we did not identify the long-term effects of HLF on ICC development; therefore, we could not evaluate its function in advanced ICC. For example, our experiment focused on the early stages of ICC formation; however, we did not evaluate the role of HLF in tumor suppression in advanced ICC. We plan to evaluate the effect of HLF overexpression on ICC development and progression in a follow-up study using HLF agonists or lentivirus mimics. Furthermore, we observed that HLF expression was not reduced in any of the ICC samples, and no negative correlation was detected between Bmi1 and HLF, indicating that HLF could also be regulated by other factors. Conversely, Bmi1 may have targets other than HLF in the ICC. Therefore, to further confirm the mechanism through which Bmi1 regulates HLF in ICC, additional clinical specimens and mouse models should be employed in future studies. Second, we demonstrated that overexpression of HLF could inhibit ICC progression. However, the specific biological functions of HLF have not yet been clarified. We plan to further explore the role of HLF in ICC, including cell cycle arrest, apoptosis promotion, autophagy, and other biological phenotypes. Finally, although HLF has been reported as an oncogene in many studies, including those on HCC, we did not evaluate the function of HLF in HCC, given that HLF was recently reported to transactivate c-Jun to promote HCC development and sorafenib resistance (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, our findings demonstrate that Bmi1/HLF signalling promotes the formation and development of ICC and that targeting this signaling pathway is a potential therapeutic strategy for ICC. In addition, this study revealed the tumor- suppressive effect of HLF. Based on current findings, HLF\u0026rsquo;s function as an oncogene in other cancer types can also be examined. In the future, we plan to uncover novel functions of Bmi1 and HLF signaling in other cancer types in which Bmi1 is overexpressed and does not regulate Ink4A/ Arf.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1 RNA sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA of Bmi1/NRas and NRas liver tissues or ICC cell lines was isolated using RNeasy mini kit (Qiagen, Germany). Paired-end libraries were synthesized by using the TruSeq\u0026reg; RNA Sample Preparation Kit (Illumina, USA) following TruSeq\u0026reg; RNA Sample Preparation Guide. Purified libraries were quantified by Qubit\u0026reg; 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. The library construction and sequencing were performed at Beijing Genomics institution (BGI). The raw data were uploaded in the NCBI SRA Submission platform (Accession number: PRJNA1202219).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Human ICC specimens and clinical database analysis\u003c/h2\u003e \u003cp\u003eICC tissue samples were obtained from the Union Hospital of Huazhong University of Science and Technology between 2017\u0026ndash;2018. The use of clinical specimens was approved by Medical Ethics Committees of Huazhong University of Science and Technology. Written informed consent was obtained from all patients before surgery. Clinical specimen database of ICC was downloaded from gene expression omnibus (GEO) dataset (Series accession: GSE32225, ID: 200032225), including 149 human ICC specimens. HLF expression profile across multiple types of tumor samples was from gene expression profiling interactive analysis (GEPIA) database (Ensembl ID: ENSG00000108924.13).\u003c/p\u003e \u003cp\u003eBioinformatics analysis of genes was referred to the protocol as followed. First, gene expression profile data is collected from a public database, such as a GEO database. These data often include levels of gene expression under different conditions, such as disease states compared with normal states. Then appropriate statistical methods, such as T-tests or ANOVA, were used to screen out genes that were significantly differentially expressed across different conditions. These differentially expressed genes (DEGs) were the role genes for subsequent analyses. \u0026zwnj;Next, functional enrichment analysis of the selected DEGs was conducted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. GO analysis can reveal the biological processes, cellular components and molecular functions of genes, while KEGG analysis indicates the signaling pathways involved in these genes. Finally, \u0026zwnj; the role genes and signaling pathways predicted by bioinformatics were verified by real-time quantitative PCR, western blot or other experimental methods to ensure the reliability of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Quantitative reverse-transcription (q-RT) PCR and Western blotting\u003c/h2\u003e \u003cp\u003eTotal RNA or proteins from ICC tissues or cell lines using TRIzol\u0026trade; Plus RNA Purification Kit (Thermo Fisher Scientific, Rockford, IL, USA) and M-PER Mammalian Protein Extraction Buffer (Thermo Fisher Scientific) respectively. For qRT-PCR, SYBR Green Master Mix (Thermo Fisher Scientific) was used. Primer pairs used are listed in Supplementary Table\u0026nbsp;1. For western blotting, proteins were separated by sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and transferred onto polyvinylidene membranes (Bio-Rad) and then performed with primary and secondary antibody incubation. Antibodies used are listed in Supplementary Table\u0026nbsp;2. The raw blot bands for verification were listed in Supplementary Fig.\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Cell culture and lentiviral infection\u003c/h2\u003e \u003cp\u003eHuman ICC cell line RBE and QBC-939 were purchased from China Center for Type Culture Collection Cell bank in Shanghai, China. Both cell lines were authenticated by STR profiling and tested clear of mycoplasma contamination. Mouse primary ICC cell line BR was extracted from Bmi1/NRas ICC liver tissues and authenticated by xenograft tumor model. In brief, Bmi1/NRas mouse ICC tissue was mechanically disintegrated using a sterile scalpel and enzymatically digested with collagenase I at 37℃. Digested tissues were filtered through 70-\u0026micro;m and 40-\u0026micro;m strainers, respectively. Suspended single cells were cultured in 24-well plates in a series dilution to obtain single clones. ICC single clones were picked and passaged 10 times to establish a stable cell line, named BR cells. Cells were cultured separately in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eLentivirus containing Bmi1 shRNA sequence, HLF shRNA sequence or HLF overexpression sequence was synthesized respectively by GenePharma Gene (Suzhou, China). In brief, 293T cells were packaged at the density of 1.3\u0026ndash;1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cell/ml and incubated for 24h with a 70% confluent. Then a mixture of the transfection plasmids with Opti-MEM 250\u0026micro;l/well was performed (Mix Plasmid 1.8\u0026micro;g/well\u0026thinsp;+\u0026thinsp;PLKO.1-plasmid 1.8\u0026micro;g/well or Plenti-plasmid 1.8\u0026micro;g/well). The transfection reagent Lipofecamine 2000 was also diluted with Opti-MEM 250\u0026micro;l/well. After that, the two ingredients were mixed and incubated for 20-30min at room temperature. Next, the transfection mix (500\u0026micro;l) was transfered to 293T cells in seeding media (2ml per well of 6 well plate) and cells were incubated for 18h. Then change media to remove the transfection reagent and replace with harvest media (2-4ml per well of 6 well plate) for viral harvests. Cells were incubated continually and 24h later, the media containing virus was retrieved to a storage tube. Continue incubating cells with harvest media (2-4ml per well of 6 well plate), repeat viral harvesting every 12-24h and replace with harvest media. Finally, the media containing virus was centrifuged at 1250 rpm for 5 min and the supernatant was filtered through a 45\u0026micro;m filter and transfer to a sterile storage tube. The lentivirus was then transfected into cells and two days post infection, cells were selected with 2\u0026micro;g/mL puromycin for 48h and then harvested for further experiments. Sequences used for lentivirus synthesis were listed as follows: Sequences used for lentivirus synthesis were listed as follows: Bmi1 shRNA (T: 5\u0026rsquo;-CAGATTGGATCGGAAAGTA-3\u0026rsquo;, B: 5\u0026rsquo;-TACTTTCCGATCCAATCTG-3\u0026rsquo;), HLF shRNA1 (T: 5\u0026rsquo;-GGCGCAGAAAGAACAACAT-3\u0026rsquo;, B: 5\u0026rsquo;-ATGTTGTTCTTTCTGCG CC-3\u0026rsquo;), HLF shRNA2 (T: 5\u0026rsquo;-GAACAAACAAGCCAAGAAA-3\u0026rsquo;, B: 5\u0026rsquo;-TTTCTTGG CTTGTTTGTTC-3\u0026rsquo;), NC shRNA (T: 5\u0026rsquo;-TTCTCCGAACGTGTCACGT-3\u0026rsquo;, B: 5\u0026rsquo;-ACGTGACACGTTCGGAGAA-3\u0026rsquo;), For overexpression experiments, HLF overexpression lentivirus was synthesized by GenePharma Co and the scrambled sequence with the same plasmid system was used as a negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Cell proliferation, growth and colony formation assay\u003c/h2\u003e \u003cp\u003eFor cell viability analysis, cells were seeded in 96-well plates at a density of 2000 cells per well and cultured for 2, 6 and 10 days, respectively. Then, cell viability was determined using a Cell Counting Kit-8 (CCK-8). To assay cell proliferation, 5000 cells were seeded in 12-well plates and recounted at different time points. For colony formation assay, 8000 cells were seeded in 10-cm plates and cultured for 14 days. Then, colonies were fixed with methanol and stained with 0.1% crystal violet. The number of colonies was counted and quantified with Image J (NIH, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Plasmids and ICC mouse models\u003c/h2\u003e \u003cp\u003ePlasmids used in this study were constructed by professor Xin Chen in University of Hawaii Cancer Center, including pT3-EF1α-Bmi1, pCaggsN-RasV12, pT3-EF1α-myr-AKT, pT3-EF1α-NICD1, pCMV-Cre and pCMV-SB transposase. Human HLF sequence was cloned into pT3-EF1α-Bmi1 to generate pT3-EF1α-Bmi1-IRES-HLF plasmid. All plasmids were extracted and purified using the Endotoxin-free Maxi Prep Kit (Omega Bio-Tek, Norcross, GA, USA).\u003c/p\u003e \u003cp\u003eWild-type FVB/N mice and BALB/c nude mice were purchased from Charles River Technology Corporation (Beijing, China). HLF\u003csup\u003efl/fl\u003c/sup\u003e mice in a C57BL/6 background were obtained from Cyagen Biosciences (Guangzhou, China). All mice were housed, fed and monitored in accordance with protocols approved by Medical Ethics Committees and the Committee for Animal Research at Huazhong University of Science and Technology. For ICC primary mouse model, hydrodynamic transfection was performed as described previously (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Plasmids were mixed and diluted in 2 mL of saline and injected into the tail vein of FVB/N mice. Mice were monitored at the indicated time points for growth evaluation and sacrificed at the humanistic endpoint. For ICC allograft tumor model, 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e BR or BR-HLF cells in 100 \u0026micro;L of RPMI-1640 medium were injected into the left front armpit of BALB/c nude mice. Tumor diameters were measured every 3 days and tumor volume (V) was calculated as V = (L \u0026times;W\u003csup\u003e2\u003c/sup\u003e) \u0026times; 0.52. At the end of the experiment, mice were sacrificed and tumors were excised, weighed and photographed.\u003c/p\u003e \u003cp\u003eWe used a mixture of 50% CO\u003csub\u003e2\u003c/sub\u003e and 50% O\u003csub\u003e2\u003c/sub\u003e to anesthetize mice. The experimental animals were placed in a CO\u003csub\u003e2\u003c/sub\u003e anesthesia tank mixed with O\u003csub\u003e2\u003c/sub\u003e. Then the valve was opened, and after the animals gradually lost consciousness, the CO\u003csub\u003e2\u003c/sub\u003e concentration was increased to 100%. When animals showed an unconscious state, including no pinch reflex, they were continued to ventilate for 2 minutes to determine the death of the animals. Animals will maintain an unconscious state for 20\u0026ndash;30 seconds. Trained personnel will conduct euthanasia for cervical dislocation to ensure the death of the mice.\u003c/p\u003e \u003cp\u003eFor xenograft mouse model, we set the humane endpoints when tumor maximum diameter is close to 2cm and tumor volume is less than 2000mm3. For ICC primary tumor mouse model, the technology using hydrodynamic tail vein injection has been mature in our laboratory and the probable time of tumor formation was stable. Based on this, we observed all mice at least 3 times a week, and when tumors have reached 80% of the maximum, the frequency of observation increased to everyday. When mice exhibited mental depression without anesthesia or sedation, mice were euthanized. For mice survival studies, we set the human endpoints based on BCS (Body Condition Scoring) and clinical observation evaluation. When the BCS was evaluated as 1, mice were euthanized and the survival time was recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Luciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe regions \u0026minus;\u0026thinsp;2000 to +\u0026thinsp;1 or +\u0026thinsp;155 to +\u0026thinsp;670 of the human HLF promoter were cloned into pGL3 luciferase vector. For luciferase reporter assay, 0.5 \u0026micro;g pGL3 luciferase vector expression HLF (or indicated mutant) and an internal control reporter plasmid, pRL-TK (Promega, Madison, WI, USA), along with Bmi1 shRNA or pcDNA-Bmi1 plasmid were co-transfected in triplicates into RBE cells using lipofectamine 2000. Two days after transfection, luciferase activities were measured using Dual Luciferase Reporter Assay System (Promega) according to the manufacturer\u0026rsquo;s instructions. Firefly luciferase activities were normalized to luciferase control values and shown as an average of triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Histology and immunohistochemistry\u003c/h2\u003e \u003cp\u003eTissue samples were fixed with 4% cold paraformaldehyde at 4 ℃ overnight. Then the samples were paraffin-embedded for hematoxylin and eosin (H\u0026amp;E) or immunohistochemical (IHC) staining. Immunohistochemistry staining was performed as reported previously. Briefly, paraffin slides were stained with primary antibody at 4 ℃ overnight followed by the avidin\u0026ndash;biotin-peroxidase protocol. All antibodies used for IHC are listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analysis were performed using SPSS 16.0 software (SPSS Software, Chicago, IL, USA). Experiments were repeated independently at least three times and data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The results were consistent across all three experiments, indicating high reproducibility. Student\u0026rsquo;s t-test was used to compare means of two groups. The correlation between two variables was analyzed by the Pearson correlation method. A two-side \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was considered highly significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science Foundation of China (82372667, 82273059, and 82073091) and Guizhou Province Science and Technology Project (ZK-2024-Key-097).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article [and/or its supplementary materials]. The raw data of RNA sequence were uploaded in the NCBI SRA Submission platform (Accession number: PRJNA1202219) and data will be released until the reception of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCX contributed to the conception and the design of the study. JG, XS and RL performed the experiments and contributed to the acquisition of data. HW and ND to the analysis and interpretation of data. FY contributed to manuscript drafting or critical revisions on the intellectual content. YX finalized the manuscript. JG and CX confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research protocols were approved by Medical Ethics Committees and\u0026nbsp;the Committee for Animal Research of Huazhong University of Science and Technology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e- Informed Consent: N/A.\u003c/p\u003e\n\u003cp\u003e- Registry and the Registration No. of the study/trial: N/A.\u003c/p\u003e\n\u003cp\u003e- Animal Studies:\u0026nbsp;All mice were housed, fed and monitored in accordance with protocols approved by the Committee for Animal Research at Huazhong University of Science and Technology (the animal ethics approval was provided but the accession number was not available).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting Data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest and no author of this manuscript is a current Editor or Editorial Board Member of Oncology Reports.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Fuchs HE and Jemal A: Cancer statistics, 2022. CA Cancer J Clin 72: 7\u0026ndash;33, 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeal EW, Tumin D, Moris D, \u003cem\u003eet al\u003c/em\u003e: Cohort contributions to trends in the incidence and mortality of intrahepatic cholangiocarcinoma. Hepatobiliary Surg Nutr 7: 270\u0026ndash;276, 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoris D, Palta M, Kim C, Allen PJ, Morse MA and Lidsky ME: Advances in the treatment of intrahepatic cholangiocarcinoma: An overview of the current and future therapeutic landscape for clinicians. CA Cancer J Clin 73: 198\u0026ndash;222, 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuropean Association for the Study of the Liver. Electronic address eee and European Association for the Study of the L: EASL-ILCA Clinical Practice Guidelines on the management of intrahepatic cholangiocarcinoma. 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Previously, we reported that B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) drives the formation and development of ICC independent of Ink4a/Arf; however the underlying mechanism remains unclear. Here, we report that hepatic leukemia factor (HLF) acts as a tumor suppressor gene in ICC and Bmi1 represses HLF to drive ICC initiation and progression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn ICC, HLF expression levels were inversely correlated with Bmi1. Overexpression of HLF inhibited the growth of ICC both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, whereas HLF knockout promoted ICC development in ICC mouse models. Importantly, HLF repression reversed the inhibitory effects of Bmi1 knockdown on cell survival, proliferation and colony formation. Luciferase reporter assay results indicated that Bmi1 represses HLF by directly binding to its promoter.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings revealed the molecular mechanism through which Bmi1 promotes ICC formation and development and uncovered the role of HLF as a tumor suppressor in ICC.\u003c/p\u003e","manuscriptTitle":"Bmi1 represses HLF to drive the formation and development of intrahepatic cholangiocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-21 08:03:42","doi":"10.21203/rs.3.rs-6059499/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11f69296-f58a-46c7-9d8b-6ca224b70201","owner":[],"postedDate":"February 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-06T12:38:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-21 08:03:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6059499","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6059499","identity":"rs-6059499","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}