Hepatocellular Carcinoma Stem Cells Research Articles

M6A-Mediated Upregulation of LINC00106 Promotes Stemness and Metastasis Properties of Hepatocellular Carcinoma via Sponging Let7f.

Background: Hepatocellular carcinoma (HCC) cells exhibit the stemness property, which makes the patient with HCC prone to tumor recurrence and metastasis. Despite the prominent regulatory role of long non-coding RNAs (lncRNAs) in tumor stemness, the roles and molecular mechanisms of LINC00106 in HCC are poorly understood. Methods: LINC00106, let7f and periostin expression levels in tissue specimens and cell lines were assessed through qRT-PCR and immunohistochemistry (IHC). Various in vivo and in vitro assays, namely sphere/colony formation, proportion of side population cells (SP%), invasion, migration, western blot, and murine xenograft model were employed for assessing the stemness and metastatic properties of HCC cells. Luciferase reporter assays, RNA-seq, RNA pull-down, RNA immunoprecipitation (RIP) were conducted to clarificate the target gene and analyze the underlying mechanisms. Results: LINC00106 was prominently upregulated in tissues and cell lines of HCC. Patients having a high LINC00106 level exhibited a poor outcome. Under in vivo and in vitro conditions, the stemness and metastatic properties of HCC cells were augmented by LINC00106. Additionally, LINC00106 was found to sponge let7f to upregulate periostin, which lead to the activation of periostin-associated PI3K-AKT signaling pathway. Moreover, m6A methylation was found to cause LINC00106 upregulation while maintaining LINC00106 RNA transcript stability. Conclusion: m6A methylation triggers the upregulation of LINC00106, which promotes the stemness and metastasis properties in HCC cells by sponging let7f, thereby resulting in periostin activation. The findings indicate the potential of LINC00106 as a diagnostic marker and therapeutic target for HCC.

The Natural Pigment Violacein Potentially Suppresses the Proliferation and Stemness of Hepatocellular Carcinoma Cells In Vitro.

Hepatocellular carcinoma (HCC) is a malignant type of primary liver cancer with high incidence and mortality, worldwide. A major challenge in the treatment of HCC is chemotherapeutic resistance. It is therefore necessary to develop novel anticancer drugs for suppressing the growth of HCC cells and overcoming drug resistance for improving the treatment of HCC. Violacein is a deep violet-colored indole derivative that is produced by several bacterial strains, including Chromobacterium violaceum, and it possesses numerous pharmacological properties, including antitumor activity. However, the therapeutic effects of violacein and the mechanism underlying its antitumor effect against HCC remain to be elucidated. This study is the first to demonstrate that violacein inhibits the proliferation and stemness of Huh7 and Hep3B HCC cells. The antiproliferative effect of violacein was attributed to cell cycle arrest at the sub-G1 phase and the induction of apoptotic cell death. Violacein induced nuclear condensation, dissipated mitochondrial membrane potential (MMP), increased generation of reactive oxygen species (ROS), activated the caspase cascade, and upregulated p53 and p21. The anticancer effect of violacein on HCC cells was also associated with the downregulation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2 signaling. Violacein not only suppressed the proliferation and formation of tumorspheres of Huh7 and Hep3B cancer stem-like cells but also reduced the expression of key markers of cancer stemness, including CD133, Sox2, Oct4, and Nanog, by inhibiting the signal transducer and activator of transcription 3 (STAT3)/AKT/ERK pathways. These results suggest the therapeutic potential of violacein in effectively suppressing HCC by targeting the proliferation and stemness of HCC cells.

Gly-tRF enhances LCSC-like properties and promotes HCC cells migration by targeting NDFIP2

BackgroundAccumulating evidence demonstrates that tRFs (tRNA-derived small RNA fragments) and tiRNAs (tRNA-derived stress-induced RNA), an emerging category of regulatory RNA molecules derived from transfer RNAs (tRNAs), are dysregulated in in various human cancer types and play crucial roles. However, their roles and mechanisms in hepatocellular carcinoma (HCC) and liver cancer stem cells (LCSCs) are still unknown.MethodsThe expression of glycine tRNA-derived fragment (Gly-tRF) was measured by qRT-PCR. Flow cytometric analysis and sphere formation assays were used to determine the properties of LCSCs. Transwell assays and scratch wound assays were performed to detect HCC cell migration. Western blotting was conducted to evaluate the abundance change of Epithelial-mesenchymal transition (EMT)-related proteins. Dual luciferase reporter assays and signalling pathway analysis were performed to explore the underlying mechanism of Gly-tRF functions.ResultsGly-tRF was highly expressed in HCC cell lines and tumour tissues. Gly-tRF mimic increased the LCSC subpopulation proportion and LCSC-like cell properties. Gly-tRF mimic promoted HCC cell migration and EMT. Loss of Gly-tRF inhibited HCC cell migration and EMT. Mechanistically, Gly-tRF decreased the level of NDFIP2 mRNA by binding to the NDFIP2 mRNA 3′ UTR. Importantly, overexpression of NDFIP2 weakened the promotive effects of Gly-tRF on LCSC-like cell sphere formation and HCC cell migration. Signalling pathway analysis showed that Gly-tRF increased the abundance of phosphorylated AKT.ConclusionsGly-tRF enhances LCSC-like cell properties and promotes EMT by targeting NDFIP2 and activating the AKT signalling pathway. Gly-tRF plays tumor-promoting role in HCC and may lead to a potential therapeutic target for HCC.

Mesenchymal stem cell-derived exosomes block malignant behaviors of hepatocellular carcinoma stem cells through a lncRNA C5orf66-AS1/microRNA-127-3p/DUSP1/ERK axis.

Mesenchymal stem cell (MSCs)-derived exosomes have been frequently used as useful tools in disease control. This research aimed to study the function of MSC-derived exosomes (Exo) in the stemness of cancer stem cells (CSCs) of hepatocellular carcinoma (HCC) and the molecular mechanism. Exo from the procured human bone marrow-MSCs were extracted and identified. CSCs from HCC cell lines were collected. The CSCs were treated with Exo, and then the proliferation, migration, invasion, angiogenesis-stimulating and self-renewal abilities of the Hep3B-CSCs and HuH7-CSCs were significantly reduced. C5orf66-AS1 was found as the most upregulated long noncoding RNAs (lncRNAs) in CSCs after Exo treatment. The integrated bioinformatic analyses and luciferase assays suggested that C5orf66-AS1 upregulated DUSP1 expression through sequestering microRNA-127-3p (miR-127-3p). Either artificial overexpression of miR-127-3p or silencing of DUSP1 blocked the inhibitory functions of Exo in the CSCs. DUSP1 inhibition increased the phosphorylation of ERK. Similar results were reproduced in vivo where Exo reduced the growth of xenograft formed by CSCs in nude mice, and this reduction was blocked upon miR-127-3p overexpression or DUSP1 silencing. To conclude, this research reported that MSC-derived Exo block malignant behaviors of HCC-sourced CSCs through a C5orf66-AS1/miR-127-3p/DUSP1/ERK axis.

Silencing PFKP restrains the stemness of hepatocellular carcinoma cells

BackgroundGlycolysis reprogramming is deeply involved in the progression of hepatocellular carcinoma (HCC), in which HCC cells with stemness traits play important roles as well. Thus, whether platelet isoform of phosphofructokinase 1 (PFKP), a rate-limiting enzyme in glycolysis, contributes to the maintenance of stemness of HCC cells is worth investigation. MethodsPFKP levels were compared between human hepatocellular carcinoma and adjacent normal tissues by Western blotting and immunohistochemistry. The relationship between PFKP expression and clinic pathological features was also analyzed. Furthermore, the colony formation capabilities and the levels of stemness markers (ALDH1, CD44, CD133, Sox-2) as well as β-catenin were compared between HCC cells either undergoing PFKP silencing or overexpression. ResultsPFKP levels were higher in HCC as compared to normal hepatic tissues. Silencing PFKP decreased HCC proliferation, colony formation capabilities, and levels of stemness markers and β-catenin; whereas overexpressing PFKP demonstrated the opposite effects. ConclusionPFKP promoted HCC proliferation and contributed to the maintenance of HCC stemness. Silencing PFKP could restrain the stemness of HCC, suggesting that PFKP may be a potential therapeutic target for HCC treatment.

A positive feedback loop between Periostin and TGF\u03b21 induces and maintains the stemness of hepatocellular carcinoma cells via AP-2\u03b1 activation

BackgroundLiver cancer stem cells (LCSCs) play key roles in the metastasis, recurrence, and chemotherapeutic resistance of hepatocellular carcinoma (HCC). Our previous research showed that the POSTN gene is closely related to the malignant progression and poor prognosis of HCC. This study aimed to elucidate the role of POSTN in generating LCSCs and maintaining their stemness as well as the underlying mechanisms.MethodsHuman HCC tissues and matched adjacent normal tissues were obtained from 110 patients. Immunohistochemistry, western blotting (WB), and RT-PCR were performed to detect the expression of POSTN and stemness factors. The roles of transforming growth factor (TGF)-β1 and AP-2α in the POSTN-induced stemness transformation of HCC cells were explored in vitro and in vivo using LCSCs obtained by CD133+ cell sorting.ResultsThe high expression of POSTN was correlated with the expression of various stemness factors, particularly CD133, in our HCC patient cohort and in TCGA and ICGC datasets. Knockdown of POSTN expression decreased the abilities of HCC cell lines to form tumours in xenograft mouse models. Knockdown of POSTN expression also suppressed cell viability and clone formation, invasion, and sphere formation abilities in vitro. Knockdown of AP-2α attenuated the generation of CD133+ LCSCs and their malignant behaviours, indicating that AP-2α was a critical factor that mediated the POSTN-induced stemness transformation and maintenance of HCC cells. The role of AP-2α was verified by using a specific αvβ3 antagonist, cilengitide, in vitro and in vivo. Activation of POSTN could release TGFβ1 from the extracellular matrix and initiated POSTN/TGFβ1 positive feedback signalling. Furthermore, we found that the combined use of cilengitide and lenvatinib suppressed the growth of HCC cells with high POSTN expression more effectively than the use of lenvatinib alone in the patient-derived xenograft (PDX) mouse model.ConclusionsThe POSTN/TGFβ1 positive feedback pathway regulates the expression of stemness factors and the malignant progression of HCC cells by regulating the transcriptional activation of AP-2α. This pathway may serve as a new target for targeted gene therapy in HCC.

Long non-coding RNA DUXAP9 promotes hepatocellular carcinoma cell stemness via directly interacting with sox9.

Long non-coding RNA (LncRNA) DUXAP9 expression was recently found to be higher in hepatocellular carcinoma (HCC) tissues and cells, and correlated with a shorter overall survival of HCC patients. However, its roles in HCC progression have never been revealed. Here, the roles of DUXAP9 in HCC cell stemness are explored as cancer stem cells (CSCs) contribute to one of the root of cancer progression. We found that DUXAP9 positively regulated HCC cell stemness, as characterized by the change of sphere-formation ability, ALDH activity and stemness marker expression. Further luciferase reporter, mRNA stability and RNA-RNA in vitro interaction assays indicated that DUXAP9 directly bound to the 3' untranslated region (UTR) of sox9, enhanced the mRNA stability of sox9 and thus increased sox9 expression. Notably, the effects induced by DUXAP9 on HCC cell stemness depended on sox9 expression. Therefore, this work identifies a novel DUXAP9/sox9 axis essential for HCC cell stemness.

Isovitexin Suppresses Stemness of Lung Cancer Stem-Like Cells through Blockage of MnSOD/CaMKII/AMPK Signaling and Glycolysis Inhibition.

Background Manganese superoxide dismutase (MnSOD) has been reported to promote stemness of lung cancer stem-like cells (LCSLCs) which had higher glycolytic rates compared with non-CSLCs. Isovitexin exhibited an inhibitory effect on the stemness of hepatocellular carcinoma cells. However, whether isovitexin could inhibit the promotion of stemness of LCSLCs mediated by MnSOD through glycolysis remains unclear. Objective Our study was aimed at investigating whether isovitexin inhibits lung cancer stem-like cells (LCSLCs) through MnSOD signaling blockage and glycolysis suppression. Methods Sphere formation and soft agar assays were conducted to determine self-renewal ability. The migration and invasion of LCSLCs were determined by wound healing and transwell assay. The glycolytic activity was assessed by determination of L-lactate metabolism rate. The influences of isovitexin on MnSOD, CaMKII, and AMPK activations as well as the metabolic shift to glycolysis were determined by manipulating MnSOD expression. Results It was found that MnSOD and glycolysis enhanced simultaneously in LCSLCs compared with parental H460 cells. Overexpression of MnSOD activated CaMKII/AMPK signaling and glycolysis in LCSLCs with increased self-renewal, migration, invasion, and expression of stemness-associated markers in vitro and elevated carcinogenicity in vivo. Knockdown of MnSOD induced an inverse effect in LCSLCs. Isovitexin blocked MnSOD/CaMKII/AMPK signaling axis and suppressed glycolysis in LCSLCs, resulting in inhibition of stemness features in LCSLCs. The knockdown of MnSOD significantly augmented isovitexin-associated inhibition of CaMKII/AMPK signaling, glycolysis, and stemness in LCSLCs. However, the overexpression of MnSOD could attenuate the inhibition of isovitexin on LCSLCs. Importantly, isovitexin notably suppressed tumor growth in nude mice bearing LCSLCs by downregulation of MnSOD expression. Conclusion MnSOD promotion of stemness of LCSLCs derived from H460 cell line is involved in the activation of the CaMKII/AMPK pathway and induction of glycolysis. Isovitexin-associated inhibition of stemness in LCSLCs is partly dependent on blockage of the MnSOD/CaMKII/AMPK signaling axis and glycolysis suppression.

2-Methoxyestradiol synergizes with Erlotinib to suppress hepatocellular carcinoma by disrupting the PLAGL2-EGFR-HIF-1/2α signaling loop

Erlotinib, an EGFR tyrosine kinase inhibitor has been introduced into cancer chemotherapy. However, the therapeutic effects of erlotinib in hepatocellular carcinoma (HCC) remain vaguely understood. Our previous study found that a hypoxia-mediated PLAGL2-EGFR-HIF-1/2α signaling loop in HCC decreased response to erlotinib. The current study has demonstrated that the combination of erlotinib and 2ME2 exerted synergistic antitumor effects against HCC. Further investigation showed that erlotinib increased the expression level of EGFR, HIF-2α, and PLAGL2, which contributes to the insensitivity of hypoxic HCC cells to erlotinib. The simultaneous exposure to 2ME2 effectively inhibited the expression level of EGFR, HIF-2α, and PLAGL2 that was induced by erlotinib. This contributes to the synergistic effect of the two therapeutic agents. Furthermore, the combination of erlotinib and 2ME2 induced apoptosis and inhibited the stemness of hypoxic HCC cells. Our findings potentially explain the mechanism of HCC insensitivity to erlotinib and provide a new strategy of combining EGFR and HIF1/2α inhibitors for HCC treatment.

PHAROH lncRNA regulates Myc translation in hepatocellular carcinoma via sequestering TIAR.

Hepatocellular carcinoma, the most common type of liver malignancy, is one of the most lethal forms of cancer. We identified a long non-coding RNA, Gm19705, that is overexpressed in hepatocellular carcinoma and mouse embryonic stem cells. We named this RNA Pluripotency and Hepatocyte Associated RNA Overexpressed in HCC, or PHAROH. Depletion of PHAROH impacts cell proliferation and migration, which can be rescued by ectopic expression of PHAROH. RNA-seq analysis of PHAROH knockouts revealed that a large number of genes with decreased expression contain a Myc motif in their promoter. MYC is decreased in knockout cells at the protein level, but not the mRNA level. RNA-antisense pulldown identified nucleolysin TIAR, a translational repressor, to bind to a 71-nt hairpin within PHAROH, sequestration of which increases MYC translation. In summary, our data suggest that PHAROH regulates MYC translation by sequestering TIAR and as such represents a potentially exciting diagnostic or therapeutic target in hepatocellular carcinoma.

Novel role of LINC01013/miR-6795-5p/FMNL3 axis in the regulation of hepatocellular carcinoma stem cell features.

Cancer stem cells (CSCs) are major contributors to tumor initiation, recurrence, and metastasis of hepatocellular carcinoma (HCC). Some long non-coding RNAs have been reported as modulators of stem-like properties in cancer cells. However, the role of LINC01013 in liver CSCs has not yet been clarified. In this study, we aimed to elucidate the expression pattern and functions of LINC01013 in HCC. HCC tissues and normal controls were collected, and the expression pattern of LINC01013 and miR-6795-5p was identified by quick real-time polymerase chain reaction. Cell counting kit-8 assay, colony formation, and spheroid formation were performed to measure cell viability, proliferation, and self-renewal of HCC cell lines. The expression of stem markers was detected by western blot analysis. The effect of LINC01013 on viability, proliferation, and stem-like properties was detected through gain-of-function and loss-of-function experiments. The direct interaction among LINC01013, miR-6795-5p, and FMNL3 was testified by dual-luciferase reporter gene assay. Tumor-bearing mice were constructed to ascertain the functions of LINC01013 in vivo. HCC tissues showed increased LINC01013 and FMNL3 expression, while it showed a decreased miR-6795-5p expression as compared to the relative controls. Moreover, the high level of LINC01013 was closely related to the poor prognosis of HCC patients. LINC01013 directly binds to miR-6795-5p and subsequently relieves FMNL3. Silencing LINC01013, FMNL3, or overexpression of miR-6795-5p could suppress spheroid and colony formation, proliferation, as well as expression of stemness markers in HepG2 and SNU-182 cells. LINC01013 knockdown suppressed growth and stem-like traits of HCC cells in vivo by reducing FMNL3 expression. LINC01013/miR-6795-5p/FMNL3 axis may be a novel therapeutic target for HCC.

Bie Jia Jian Pill Combined with Bone Mesenchymal Stem Cells Regulates microRNA-140 to Suppress Hepatocellular Carcinoma Stem Cells.

Background and ObjectivesCancer stem cells (CSCs) with tumorigenic potential are reported as the crucial factors of hepatocellular carcinoma (HCC) recurrence and therapy resistance. Bone mesenchymal stem cells (BMSCs) are documented to play an important role in the protection of hepatocytes. Bie Jia Jian pill (BJJP), a Traditional Chinese Medicine, has been used to treat liver fibrosis and liver cancer. This study aimed to explore the potential role of combined use of BJJP with BMSCs in HCC cell lines.Methods and ResultsFlow cytometry was used to identify BMSCs isolated from BALB/c mice and CSCs enriched from Huh7 cells by measuring CD24, CD133, CD44, CD73, CD105, CD166, CD29, CD14 and CD34. Differentiation potential of BMSCs was also determined. Cell viability and proliferation ability of CSCs were determined by CCK-8 assay and clone formation assay. The expressions of CSCs biomarkers and Wnt/β-catenin signal pathway related proteins were determined by PCR and western blot. TOP-Flash/FOP-Flash luciferase assay was applied to measure the activity of β-catenin/TCF. Compared with untreated CSCs, BJJP or BMSCs treatment alone on CSCs lead to increased miR-140 expression and cell apoptosis, as well as decreased expressions of CD24, CD133, EpCAM and cell viability. Downregualted expressions of Wnt/β-catenin signal pathway related proteins, Wnt3a and β-catenin were found in response to BJJP or BMSCs treatment alone. The combination of BJJP＋BMSCs treatment on CSCs could further enhance the suppressive effect on CSCs. Down-regulation of miR-140 in CSCs partially blocked the effects of BMSCs or BMSCs＋BJJP on the expressions of Wnt3a and β-catenin as well as the cell viability and apoptosis of CSCs. Reversed expression pattern was found in CSCs transfected with miR-140 overexpression.ConclusionsTaken together, we demonstrate that BJJP＋BMSCs together could further enhance the suppressive effect on CSCs through regulating miR-140 and suppressing Wnt/β-catenin signal pathway. This study demonstrated the potential of BJJP＋BMSCs in therapeutic treatment of HCC.

Imbalance of TGF-β1/BMP-7 pathways induced by M2-polarized macrophages promotes hepatocellular carcinoma aggressiveness

The transforming growth factor-beta (TGF-β) signaling pathway is the predominant cytokine signaling pathway in the development and progression of hepatocellular carcinoma (HCC). Bone morphogenetic protein (BMP), another member of the TGF-β superfamily, has been frequently found to participate in crosstalk with the TGF-β pathway. However, the complex interaction between the TGF-β and BMP pathways has not been fully elucidated in HCC. We found that the imbalance of TGF-β1/BMP-7 pathways was associated with aggressive pathological features and poor clinical outcomes in HCC. The induction of the imbalance of TGF-β1/BMP-7 pathways in HCC cells could significantly promote HCC cell invasion and stemness by increasing inhibitor of differentiation 1 (ID1) expression. We also found that the microRNA (miR)-17-92 cluster, originating from the extracellular vesicles (EVs) of M2-polarized tumor-associated macrophages (M2-TAMs), stimulated the imbalance of TGF-β1/BMP-7 pathways in HCC cells by inducing TGF-β type II receptor (TGFBR2) post-transcriptional silencing and inhibiting activin A receptor type 1 (ACVR1) post-translational ubiquitylation by targeting Smad ubiquitylation regulatory factor 1 (Smurf1). Invivo, short hairpin (sh)-MIR17HG and ACVR1 inhibitors profoundly attenuated HCC cell growth and metastasis by rectifying the imbalance of TGF-β1/BMP-7 pathways. Therefore, we proposed that the imbalance of TGF-β1/BMP-7 pathways is a feasible prognostic biomarker and recovering the imbalance of TGF-β1/BMP-7 pathways might be a potential therapeutic strategy for HCC.

Research Progress on Regulating LncRNAs of Hepatocellular Carcinoma Stem Cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies around the world. The self-renewal, proliferation, differentiation, and tumorigenic potential of liver cancer stem cells (LCSCs) may account for the high recurrence rate and the refractory feature of HCC. Despite extensive researches, the underlying regulatory mechanism of LCSCs has not been fully disclosed. Long nonprotein coding RNAs (lncRNAs) may exert an essential role in regulating various biological functions of LCSCs, such as maintaining the stemness of cancer stem cells (CSCs) and promoting tumor development. Therefore, it is highly critical to determine which lncRNAs can control LCSCs functions and understand how LCSCs are regulated by lncRNAs. Herein, we summarized lncRNAs and the main signaling pathways involved in the regulation of LCSCs found in recent years. Moreover, we shed light on the existence of the network system of lncRNAs and LCSCs, which may provide valuable clues on targeting LCSCs.

Dichloroacetate attenuates the stemness of hepatocellular carcinoma cells via promoting nucleus‐cytoplasm translocation of YAP

The antitumor effects of dichloroacetate (DCA) have been widely explored, however, its roles in hepatocellular carcinoma (HCC) progression are still unclear. In the current work, we found that DCA had little effects on HCC cell viability, but could attenuate the stemness of HCC cells, which is evident by decreasing the tumor sphere-formation ability, ALDH activity and the expression of stemness critical regulators. Mechanistic studies based on RNA-sequencing data showed that DCA activated the Hippo pathway. Furthermore, we indicated that DCA promoted the nucleus-cytoplasm translocation of YAP, but not TAZ, another critical executor of Hippo pathway. Moreover, suppressing of Hippo pathway using XMU-MP-1, an inhibitor of Hippo pathway, partially abrogated DCA-induced inhibitory effects on HCC cell stemness. This work suggests that DCA might be a potential inhibitor for HCC progression.

Involvement of CDK11B-mediated SPDEF ubiquitination and SPDEF-mediated microRNA-448 activation in the oncogenicity and self-renewal of hepatocellular carcinoma stem cells.

Increasing evidence has suggested the crucial role cyclin-dependent kinases (CDKs) in the biology of hepatocellular carcinoma (HCC), a lethal malignancy with high morbidity and mortality. Hence, this study explored the modulatory effect of the putative cyclin-dependent kinase 11B (CDK11B)-mediated ubiquitination on HCC stem cells. The expression of CDK11B, SAM pointed domain-containing ETS transcription factor (SPDEF) and DOT1-like histone lysine methyltransferase (DOT1L) was determined by RT-qPCR and western blot analysis in HCC tissues and cells. The interaction among CDK11B, SPDEF, miR-448, and DOT1L was analyzed by Co-IP, ubiquitination-IP and ChIP assays, whereas their effects on the biological characteristics of HCC stem cells were assessed by sphere formation and colony formation assays. An in vivo xenograft tumor model was developed for validating the regulation of CDK11B in oncogenicity of HCC stem cells. We characterized the aberrant upregulation of CDK11B and downregulation SPDEF in HCC tissues and cells. CDK11B degraded SPDEF through ubiquitin-proteasome pathway, whereas SPDEF could bind to the miR-448 promoter and inhibit the expression of DOT1L by activating miR-448, whereby promoting self-renewal of HCC stem cells. Knockdown of CDK11B attenuated the self-renewal capability of HCC stem cells and their oncogenicity in vivo. These findings highlighted that blocking the CDK11B-induced degradation of SPDEF and enhancing miR-448-dependent inhibition of DOT1L may delay the progression of HCC by restraining self-renewal capability of HCC stem cells, representing novel targets for HCC management.

SNRPB-mediated RNA splicing drives tumor cell proliferation and stemness in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading malignant diseases worldwide, but therapeutic targets for HCC are lacking. Here, we characterized a significant upregulation of Small Nuclear Ribonucleoprotein Polypeptides B and B1 (SNRPB) in HCC via qRT-PCR, western blotting, tissue microarray and public database analyses. Increased SNRPB expression was positively associated with adjacent organ invasion, tumor size, serum AFP level and poor HCC patient survival. Next, we transfected SNRPB into HCC cells to construct SNRPB-overexpressing cell lines, and short hairpin RNA targeting SNRPB was used to silence SNRPB in HCC cells. Functional studies showed that SNRPB overexpression could promote HCC cell malignant proliferation and stemness maintenance. Inversely, SNRPB knockdown in HCC cells caused inverse effects. Importantly, analysis of alternative splicing by RNA sequencing revealed that SNRPB promoted the formation of AKT3-204 and LDHA-220 splice variants, which activated the Akt pathway and aerobic glycolysis in HCC cells. In conclusion, SNRPB could serve as a prognostic predictor for patients with HCC, and it promotes HCC progression by inducing metabolic reprogramming.

Loss of miR-192-5p initiates a hyperglycolysis and stemness positive feedback in hepatocellular carcinoma

BackgroundEmerging studies revealed that cancer stem cells (CSCs) possessed peculiar metabolic properties, which however remained largely unknown in hepatocellular carcinoma (HCC). Genetic silencing of liver-abundant miR-192-5p was a key feature for multiple groups of CSC-positive HCCs. We thus aimed to investigate essential metabolic features of hepatic CSCs via using HCCs with miR-192-5p silencing as a model.MethodsDatasets from two independent HCC cohorts were used. Data integration analyses of miR-192-5p with metabolome and mRNA transcriptome data in HCC Cohort 1 were performed to investigate miR-192-5p related metabolic features, which was further validated in Cohort 2. Cellular and molecular assays were performed to examine whether and how miR-192-5p regulated the identified metabolic features. Co-culture systems consisting of HCC cells and LX2 (human hepatic stellate cell line) or THP1 (human monocyte cell line) were established to explore effects of the identified metabolic properties on stemness features of HCC cells via interacting with co-cultured non-tumor cells.ResultsHigh levels of glycolysis-related metabolites and genes were present in HCCs with low miR-192-5p and CSC-positive HCCs in two independent HCC cohorts. miR-192-5p knockout cells displayed CSC features and miR-192-5p loss led to an enhanced glycolytic phenotype via upregulating three bona fide targets, GLUT1 and PFKFB3 (two glycolytic enzymes) and c-Myc (regulating glycolytic genes’ expression). Meanwhile, c-Myc suppressed miR-192-5p transcription, ensuring a low-miR-192-5p/high-c-Myc loop to maintain hyperglycolysis. Moreover, over-produced lactic acid from hyperglycolytic HCC cells stimulated the ERK phosphorylation of co-cultured LX2 and THP1 non-tumor cells partially via NDRG3 and MCT1, which in turn promoted cell malignancy and stemness of HCC cells. Consistently, HCC patients with low level of miR-192-5p in their tumor tissues and high level of NDRG3 or MCT1 in their non-tumor tissues had the shortest overall survival.ConclusionsIn CSC-positive HCCs, miR-192-5p loss enhanced glycolysis and over produced lactate might further increase HCC malignant features via interacting with environmental non-tumor cells.

Glycochenodeoxycholic acid induces stemness and chemoresistance via the STAT3 signaling pathway in hepatocellular carcinoma cells.

The poor prognosis of hepatocellular carcinoma (HCC) is primarily attributed to its high frequency of recurrence and resistance to chemotherapy. Epithelial-to-mesenchymal transition (EMT) and the acquisition of cancer stem cells (CSCs) are the fundamental drivers of chemoresistance in HCC. Glycochenodeoxycholic acid (GCDC), a component of bile acid (BA), has been reported to induce necrosis in primary human hepatocytes. In the present work, we investigated the function of GCDC in HCC chemoresistance. We found that GCDC promoted chemoresistance in HCC cells by down-regulating and up-regulating the expression of apoptotic and anti-apoptotic genes, respectively. Furthermore, GCDC induced the EMT phenotype and stemness in HCC cells and activated the STAT3 signaling pathway. These findings reveal that GCDC promotes chemoresistance in HCC by inducing stemness via the STAT3 pathway and could be a potential target in HCC chemotherapy.

Irradiated mesenchymal stem cells support stemness maintenance of hepatocellular carcinoma stem cells through Wnt/\u03b2-catenin signaling pathway

BackgroundCancer stem cells are the main reason of relapse, metastasis and resistance to anti-cancer therapies of Hepatocellular carcinoma (HCC). Mesenchymal stem cells (MSCs) are an important part of the tumor microenvironment. MSCs have been demonstrated to be involved in drug resistance in tumor. How MSCs contribute to radiotherapy resistance of HCC is still indistinct.MethodsFlow cytometry analysis was performed to isolate CD133+ cells from HCC cell lines Huh7 and PLC. The stemness of Huh7-CD133 and PLC-CD133 those were co-cultured with IR-MSCs were investigated by Colony formation assay. Tumor formation in nude mice was used to explore the tumorigenicity of CD133+ cancer cells. The activating Wnt/β-catenin signaling pathway in CSCs were also detected by RT-PCR and Western blotting.ResultsWe report that irradiated MSCs (IR-MSCs) could increase the ratio of CD133+ cells in hepatocellular carcinoma cells. IR-MSCs could promote stemness maintenance of HCC stem cells. After co-cultured with IR-MSCs, liver cancer stem cells (CSCs) presented increased colony formation ability and tumor formation ability. We also found IR-MSCs promoted Wnt expression of CSCs. Reverse suppression experiment showed that when Wnt inhibitor was added into the culture medium, the effect of IR-MSCs on stemness maintenance was counteracted.ConclusionsThese data showed that IR-MSCs could support stemness maintenance of CSCs by activating Wnt/β-catenin signaling pathway.