Progression Of Human Hepatocellular Carcinoma Research Articles

Long non-coding RNA 657 suppresses hepatocellular carcinoma cell growth by acting as a molecular sponge of miR-106a-5p to regulate PTEN expression.

Previous study has identified the aberrant expression of LINC00657, a long non-coding RNA (lncRNA), in human breast cancer. However, the expression pattern, biological function and underlying mechanism of LINC00657 in human hepatocellular carcinoma (HCC) remain obscure. The expression levels of LINC00657 in HCC tissues and cell lines were determined by quantitative real-time PCR. CCK-8 assay, cell colony formation assay, cell cycle analysis, Transwell assay were performed to determine whether LINC00657 could affect HCC progression. Luciferase reporter assay was used to assess the target of LINC00657. Expressions of the relevant proteins were analyzed by Western blot. Herein, we found that LINC00657 was downregulated in HCC tissue specimens as well as in malignant HCC cell lines. LINC00657 overexpression inhibited the proliferation, migration and invasion of HCC cells, while LINC00657 depletion promoted both cell viability and cell invasion in vitro. We also found that LINC00657 could inhibit tumor growth in vivo. Further experiments demonstrated that down-regulated LINC00657 increased the expression of miR-106a-5p. miR-106a-5p decreased the abundances of PTEN protein, while had no impact on PTEN mRNA. Moreover, we identified that both LINC00657 and PTEN mRNA were targets of miR-106a-5p by using dual-luciferase reporter assay. Our results provide the new evidence supporting the tumor-suppressive role of LINC00657 in HCC, suggesting that LINC00657 might play a role in HCC and can be a novel therapeutic target for treating HCC.

Circular RNA circMTO1 acts as the sponge of microRNA-9 to suppress hepatocellular carcinoma progression.

circMTO1 suppresses HCC progression by acting as the sponge of oncogenic miR-9 to promote p21 expression, suggesting that circMTO1 is a potential target in HCC treatment. The decrease of circMTO1 in HCC tissues may serve as a prognosis predictor for poor survival of patients. (Hepatology 2017;66:1151-1164).

Silencing of solute carrier family 13 member 5 disrupts energy homeostasis and inhibits proliferation of human hepatocarcinoma cells

The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays a key role in importing citrate from the circulation into liver cells. Recent evidence has revealed that SLC13A5 deletion protects mice from high-fat diet-induced hepatic steatosis and that mutation of the SLC13A5 orthologues in Drosophila melanogaster and Caenorhabditis elegans promotes longevity. However, despite the emerging importance of SLC13A5 in energy homeostasis, whether perturbation of SLC13A5 affects the metabolism and malignancy of hepatocellular carcinoma is unknown. Here, we sought to determine whether SLC13A5 regulates hepatic energy homeostasis and proliferation of hepatoma cells. RNAi-mediated silencing of SLC13A5 expression in two human hepatoma cell lines, HepG2 and Huh7, profoundly suppressed cell proliferation and colony formation, and induced cell cycle arrest accompanied by increased expression of cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin B1. Furthermore, such suppressive effects were also observed on the growth of HepG2 cell-derived xenografts expressing SLC13A5-shRNA in nude mice. Metabolically, knockdown of SLC13A5 in HepG2 and Huh7 cells was associated with a decrease in intracellular levels of citrate, the ratio of ATP/ADP, phospholipid content, and ATP citrate lyase expression. Moreover, both in vitro and in vivo assays demonstrated that SLC13A5 depletion promotes activation of the AMP-activated protein kinase, which was accompanied by deactivation of oncogenic mechanistic target of rapamycin signaling. Together, our findings expand the role of SLC13A5 from facilitating hepatic energy homeostasis to influencing hepatoma cell proliferation and suggest a potential role of SLC13A5 in the progression of human hepatocellular carcinoma.

DlgR2 knockdown boosts dendritic cell activity and inhibits hepatocellular carcinoma tumor in-situ growth.

Tumor-specific hepatic stellate cells (tHSCs) positively participate in human hepatocellular carcinoma (HCC) tumorigenesis and progression. Our previous studies have shown that tHSCs co-culture with dendritic cells (DCs) induced DIgR2 (dendritic cell-derived immunoglobulin receptor 2) expression. The latter is a member of IgSF inhibitory receptor suppressing DCs-initiated antigen-specific T-cell responses. In the current study, we show that hepatic artery injection of DlgR2 siRNA significantly inhibited in-situ HCC xenograft growth in rat livers. Further, 5-FU-medied inhibition of in-situ HCC growth was dramatically sensitized with DlgR2 silence. DlgR2 siRNA injection indeed downregulated DlgR2 in ex-vivo cultured tumor-derived DCs (tDCs). More importantly, tDCs activity was boosted following DlgR2 siRNA. These cells presented with upregulated CD80, CD86 and MHC-II. Production of interleukin-12 and tumor necrosis factor-α was also increased in the DlgR2-silenced tDCs. We propose that DlgR2 knockdown likely boosts the activity of tumor-associated DCs, and inhibits growth of in-situ HCC xenografts.

Epidermal growth factor receptor transactivation is involved in the induction of human hepatoma SMMC7721 cell proliferation by insufficient radiofrequency ablation

Our previous study revealed that insufficient radiofrequency ablation (RFA) promotes the malignancy of human hepatocellular carcinoma (HCC) SMMC7721 cells via the Ca2+/calmodulin-dependent protein kinase II (CaMKII)/extracellular signal-regulated kinase (ERK)-induced overexpression of vascular endothelial growth factor (VEGF). The aims of the present study were to address the involvement of epidermal growth factor receptor (EGFR) transactivation in the enhanced SMMC7721 cell proliferation induced by insufficient RFA, in addition to its association with the CaMKII/ERK/VEGF signaling cascade. SMMC7721 cells were subjected to a 47°C treatment regimen to simulate insufficient RFA. Cell proliferation was determined using MTT and colony formation assays. The expression levels of VEGF, CaMKII, phosphorylated (phospho)-CaMKII, ERK, phospho-ERK, EGFR and phospho-EGFR were analyzed using western blotting. The results demonstrated that the enhancement of SMMC7721 cell proliferation by the 47°C treatment regimen was significantly inhibited by exposure of the cells to AG178 (a specific inhibitor of EGFR). Furthermore, AG1478 exposure prevented the overexpression of VEGF and phosphorylation of ERK, but had no significant effects on CaMKII phosphorylation. By contrast, 47°C treatment-induced EGFR phosphorylation was inhibited by treatment with KN93 (a specific inhibitor of CaMKII). Overall, the results of the present study have suggested a role for EGFR transactivation in the RFA-promoted growth of residual HCC. Thus, targeting EGFR may represent a useful preventive and therapeutic strategy for RFA-induced HCC progression and recurrence.

Knockout of the Nogo-B Gene Attenuates Tumor Growth and Metastasis in Hepatocellular Carcinoma

Human hepatocellular carcinoma (HCC) is a malignant cancer. It is a challenge to develop anti-HCC drugs due to HCC's extreme aggressiveness and with the sensitivity of the liver to show severe adverse effects. More importantly, the precise mechanisms causing HCC pathogenicity are not known. Our previous study disclosed Nogo-B as a reticulon 4 (Rtn4) family member. In the present study, we first identified that Nogo-B played a critical role in HCC progression. We found, via in vitro and in vivo assays, that Nogo-B was expressed aberrantly in primary HCC tumor tissues and immortal HCC cells but was relatively scarce in the normal liver tissues or cells. Nogo-B knockout, via the CRISPR-Cas9 technique, resulted in significant suppression of HCC cell proliferation and tumor growth. Next-generation sequencing analysis showed that Nogo-B knockout have effects on interleukin-6 (IL-6) signaling pathway. Furthermore, we observed that IL-6 induced phosphorylation of STAT3 (pSTAT3) in wild-type HCC cells, but Nogo-B knockout could reduce IL-6–induced increase of pSTAT3, supporting that Nogo-B affects HCC tumor progression possibly via regulating the IL-6/STAT3 signaling pathway. In conclusion, Nogo-B is expressed aberrantly in HCCs and plays an oncogenic role. These findings support that Nogo-B may be a novel anti-HCC therapeutic target.

MicroRNA-143 inhibits tumorigenesis in hepatocellular carcinoma by downregulating GATA6.

MicroRNAs serve a critical role in human hepatocellular carcinoma (HCC) progression. However, the exact role of microRNA-143 (miR-143) in HCC remains unclear. The current study investigates the molecular mechanism of miR-143 in HCC. In cultured HepG2 and Bel7402 cell lines, miR-143 levels were raised by lentivirus transduction. This significantly inhibited HCC progression in terms of cell invasion and proliferation in both HepG2 and Bel7402 cell lines (P<0.05). MiR-143 also significantly decreased tumor implantation in vivo (P<0.05). Regulation of miR-143 on its direct target, GATA-binding factor 6 (GATA6), was investigated by multiple strategies, including dual-luciferase assay, quantitative polymerase chain reaction and western blot analysis. The results indicated that miR-143 was downregulated in both HCC cell lines and human tumors. GATA6 was identified as the downstream target of miR-143 in HCC, and overexpressing GATA6 was able to counter the tumor-suppressive effect of miR-143 on HCC in HepG2 and Bel7402 cells by significantly increasing proliferation and invasion rates (P<0.05). Therefore, a novel epigenetic pathway was identified in which miR-143 may suppress the malignancy of HCC by targeting GATA6.

ZKSCAN1 gene and its related circular RNA (circZKSCAN1) both inhibit hepatocellular carcinoma cell growth, migration, and invasion but through different signaling pathways.

There is increasing evidence that circular RNA (circRNA) are involved in cancer development, but the regulation and function of human circRNA remain largely unknown. In this study, we demonstrated that ZKSCAN1, a zinc finger family gene, is expressed in both linear and circular (circZKSCAN1) forms of RNA in human hepatocellular carcinoma (HCC) tissues and cell lines. Here, we analyzed a cohort of 102 patients and found that expression of both ZKSCAN1 mRNA and circZKSCAN1 was significantly lower (P < 0.05) in the HCC samples compared with that in matched adjacent nontumorous tissues by reverse transcription PCR (RT‐PCR). The low expression level of ZKSCAN1 was only associated with tumor size (P = 0.032), while the cirZKSCAN1 levels varied in patients with different tumor numbers (P < 0.01), cirrhosis (P = 0.031), vascular invasion (P = 0.002), or microscopic vascular invasion (P = 0.002), as well as with the tumor grade (P < 0.001). Silencing both ZKSCAN1 mRNA and circZKSCAN1 promoted cell proliferation, migration, and invasion. In contrast, overexpression of both forms of RNA repressed HCC progression in vivo and in vitro. Silencing or overexpression of both forms of RNA did not interfere with each other. RNA‐seq revealed a very different molecular basis for the observed effects; ZKSCAN1 mRNA mainly regulated cellular metabolism, while circZKSCAN1 mediated several cancer‐related signaling pathways, suggesting a nonredundant role for ZKSCAN1 mRNA and circRNA. In conclusion, our results revealed two post‐translational products (ZKSCAN1 mRNA and circZKSCAN1) that cooperated closely with one another to inhibit growth, migration, and invasion of HCC. cirZKSCAN1 might be a useful marker for the diagnosis of HCC.

PGE2 synthesis and signaling in malignant transformation and progression of human hepatocellular carcinoma

Prostaglandin E 2 (PGE2), which is the most abundant prostaglandin produced in hepatocellular carcinoma (HCC), may be involved in hepatocarcinogenesis. Here, the amount of PGE2 was significantly increased in HCC tissue and adjacent noncancerous tissues relative to normal liver tissue (P<.001). In addition, the expression of EP2 receptor was considerably upregulated in HCC tissue compared with the expression of EP1 (P<.05), EP3 (P<.01), and EP4 (P<.01) receptor. The expression of EP2 receptor was positively correlated with the level of PGE2 in HCC tissue (P<.001). Furthermore, PGE2 significantly increased proliferation and invasion potential of human HCC cells. However, antagonism of EP2 signaling suppressed PGE2-induced growth and invasion in human HCC cells. Taken together, upregulation of PGE2 level was associated with proliferation and invasion potential of HCC, and EP2 receptor predominately mediated the function of PGE2 in the transformation and progression of HCC.

MiR-1247-5p functions as a tumor suppressor in human hepatocellular carcinoma by targeting Wnt3.

Increasing evidence suggests that aberrant expression of certain microRNAs (miRNAs) may participate in the genesis and progression of tumors. Several studies have indicated that miR-1247-5p plays different roles in various types of cancer cells. The effects of miR-1247-5p on human hepatocellular carcinoma (HCC) cells are elusive. In the present study, we investigated the effects of miR-1247-5p on the progression of HCC. The transcript of miR-1247-5p was markedly downregulated in clinical samples of patients with HCC and HCC cell lines, and ectopic overexpression of miR‑1247-5p markedly inhibited the proliferation and invasion of HepG2 cells, induced cell apoptosis invitro, and suppressed the growth of transplanted tumors invivo. Wnt3 was found to be a potential target of miR-1247-5p and overexpression of miR-1247-5p was able to significantly downregulate the expression of Wnt3 by directly targeting the 3'UTR of this gene, which was verified by luciferase reporter assay and western blotting. Furthermore, we found that the miR-1247-5p gene was hypermethylated in HepG2 cells, and the transcript of miR-1247-5p was increased significantly after treatment with the demethylation drug 5-azacytidine. These findings demonstrated that miR-1247-5p functions as a tumor suppressor in human HCC by targeting Wnt3 and that the expression of miR-1247-5p can be regulated by DNA methylation, which indicates that miR-1247-5p has the potential to be a therapeutic target as well as a diagnostic marker of HCC.

PS-143 - Chk2 DNA damage response protein mislocalization further enhances chromosomal instability and human hepatocellular carcinoma progression

PS-143 - Chk2 DNA damage response protein mislocalization further enhances chromosomal instability and human hepatocellular carcinoma progression

Ectopic overexpression of filamin C scaffolds MEK1/2 and ERK1/2 to promote the progression of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) invasion and metastasis are mediated by a complicated signal transduction network and downstream cytoskeletal and adhesion molecules. In this study, a microarray-based analysis revealed a dramatic increase in filamin C (FLNC), which is commonly expressed in muscle rather than in liver cells, in the two metastatic HCC cell lines MHCC97L and HCCLM3. Clinicopathological studies showed that increased FLNC expression was associated with microvascular invasion and poor prognosis. Specific hypomethylation was identified within the FLNC promoter region in HCC cell lines and patient tumor samples, which might contribute to the ectopic overexpression of FLNC. FLNC downregulation inhibited cell migration and impaired cell proliferation and promoted apoptosis. Mechanistic studies suggested that FLNC interacts with mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) and that FLNC downregulation inhibited MEK1/2 and ERK1/2 activation. Xenographic tumor transplantation experiments in nude mice further confirmed the role of FLNC in HCC progression and metastasis. Our results reveal a novel mechanism by which the cytoskeletal protein FLNC enhances the mitogen-activated protein kinase signaling pathway during tumorigenesis.

Keratin 19 as a key molecule in progression of human hepatocellular carcinomas through invasion and angiogenesis.

BackgroundKeratin (K) 19-positive hepatocellular carcinoma (HCC) is well known to have a higher malignant potential than K19-negative HCC: However, the molecular mechanisms involved in K19-mediated progression of HCC remain unclear. We attempted to clarify whether K19 directly affects cell survival and invasiveness in association with cellular senescence or epithelial-mesenchymal transition (EMT) in K19-positive HCC.MethodsK19 expression was analysed in 136 HCC surgical specimens. The relationship of K19 with clinicopathological factors and survival was analysed. Further, the effect of K19 on cell proliferation, invasion, and angiogenesis was examined by silencing K19 in the human HCC cell lines, HepG2, HuH-7, and PLC/PRF/5. Finally, we investigated HCC invasion, proliferation, and angiogenesis using K19-positive HCC specimens.ResultsAnalysis of HCC surgical specimens revealed that K19-positive HCC exhibited higher invasiveness, metastatic potential, and poorer prognosis. In vitro experiments using the human HCC cell lines revealed that K19 silencing suppressed cell growth by inducting apoptosis or upregulating p16 and p27, resulting in cellular senescence. In addition, transfection with K19 siRNA upregulated E-cadherin gene expression, significantly inhibited the invasive capacity of the cells, downregulated angiogenesis-related molecules such as vasohibin-1 (VASH1) and fibroblast growth factor 1 (FGFR1), and upregulated vasohibin-2 (VASH2). K19-positive HCC specimens exhibited a high MIB-1 labelling index, decreased E-cadherin expression, and high microvessel density around cancer foci.ConclusionK19 directly promotes cancer cell survival, invasion, and angiogenesis, resulting in HCC progression and poor clinical outcome. K19 may therefore be a novel drug target for the treatment of K19-positive HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2949-y) contains supplementary material, which is available to authorized users.

MiR-199a-3p inhibits cell proliferation and induces apoptosis by targeting YAP1, suppressing Jagged1-Notch signaling in human hepatocellular carcinoma.

BackgroundmiR-199a-3p was significantly downregulated in the majority of human hepatocellular carcinoma (HCC) tissues and HCC cell lines. Yes associated protein 1 (YAP1) was overexpressed in human HCC, which promoted HCC development and progression by upregulating Jagged1 and activating the Notch pathway. We searched potential targets of miR-199a-3p with DIANA, TargetScan and PicTar tools, and found that YAP1 is one of the potential targets. Based on these findings, we speculated that miR-199a-3p might suppress HCC growth by targeting YAP1, downregulating Jagged1 and suppressing the Notch pathway.ResultsWe determined the expression of miR-199a-3p and YAP1 by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found downregulation of miR-199a-3p and upregulation of YAP1 in HCC cell lines. Cell proliferation and apoptosis assays showed that miR-199a-3p suppresses HCC cell proliferation and promotes apoptosis, and knockdown of YAP1 has similar role. Furthermore, we verified that miR-199a-3p can directly target YAP1. We further investigated and confirmed that miR-199a-3p and YAP1 regulate HCC cell proliferation and apoptosis through Jagged1-Notch signaling.ConclusionmiR-199a-3p targets YAP1, downregulates Jagged1 and suppresses the Notch signaling to inhibit HCC cell proliferation and promote apoptosis. These findings provide new insights into the mechanism by which miR-199a-3p suppresses HCC cell proliferation and induces apoptosis.

CD200R, a co-inhibitory receptor on immune cells, predicts the prognosis of human hepatocellular carcinoma

The inhibitory CD200:CD200 receptor axis is essential in preventing inflammatory responses during early microbial infection. It was reported in several tumor models that CD200 expression is closely associated to tumor progression and the blockade of this pathway may restore anti-tumor responses. Our study for the first time investigates the role of CD200:CD200R axis in relation to tumor progression and prognosis of human hepatocellular carcinoma. CD200 and CD200R protein expressions were evaluated by immunostaining on liver tissue specimens and we found higher expressions of CD200 and CD200R in HCC patients comparing to healthy controls. CD200 expresses in peritumoral, peritumoral stroma and intratumoral regions of HCC while CD200R predominantly expresses in peritumoral stroma. Furthermore, protein intensity of CD200R is positively associated to the diameter of tumor and alpha-fetoprotein level, in addition, patients with higher pathological grade and absence of tumor capsule exhibit higher CD200R expression. CD200R predominantly expresses on infiltrating macrophages and may associate with liver injury. Moreover, both overall and recurrence-free survival rates are significantly lower in patients with high CD200R expression comparing to those with low CD200R expression. Our findings suggest a promising role of CD200R as a prognostic marker in predicting elevated recurrence and reduced survival, and a potential therapeutic target in treating hepatocellular carcinoma.

Histone acetyl transferase GCN5 promotes human hepatocellular carcinoma progression by enhancing AIB1 expression.

BackgroundGeneral control non-depressible 5 (GCN5) is a crucial catalytic component of a transcriptional regulatory complex that plays important roles in cellular functions from cell cycle regulation to DNA damage repair. Although GCN5 has recently been implicated in certain oncogenic roles, its role in liver cancer progression remains vague.ResultsIn this study, we report that GCN5 was overexpressed in 17 (54.8 %) of 31 human hepatocellular carcinoma (HCC) specimens. Down-regulation of GCN5 inhibited HCC cell proliferation and xenograft tumor formation. GCN5 knockdown decreased the protein levels of the proliferation marker proliferating cell nuclear antigen (PCNA) and amplified in breast cancer 1 (AIB1), but increased the protein levels of cell cycle inhibitor p21Cip1/Waf1 in HepG2 cells. GCN5 regulated AIB1 expression, at least in part, by cooperating with E2F1 to enhance AIB1 transcription. Consistently, GCN5 expression was positively correlated with AIB1 expression in human HCC specimens in two GEO profile datasets.ConclusionSince AIB1 plays a promoting role in HCC progression, our results propose that GCN5 promotes HCC progression at least partially by regulating AIB1 expression. This study implicates that GCN5 might be a potential molecular target for HCC diagnosis and treatment.

Arginine Methylation of SREBP1a via PRMT5 Promotes De Novo Lipogenesis and Tumor Growth.

Dysregulation of the sterol regulatory element-binding transcription factors sterol regulatory element-binding protein (SREBP) and SREBF activates de novo lipogenesis to high levels in cancer cells, a critical event in driving malignant growth. In this study, we identified an important posttranslational mechanism by which SREBP1a is regulated during metabolic reprogramming in cancer cells. Mass spectrometry revealed protein arginine methyltransferase 5 (PRMT5) as a binding partner of SREBP1a that symmetrically dimethylated it on R321, thereby promoting transcriptional activity. Furthermore, PRMT5-induced methylation prevented phosphorylation of SREBP1a on S430 by GSK3β, leading to its disassociation from Fbw7 (FBXW7) and its evasion from degradation through the ubiquitin-proteasome pathway. Consequently, methylation-stabilized SREBP1a increased de novo lipogenesis and accelerated the growth of cancer cells in vivo and in vitro. Clinically, R321 symmetric dimethylation status was associated with malignant progression of human hepatocellular carcinoma, where it served as an independent risk factor of poor prognosis. By showing how PRMT5-induced methylation of SREBP1a triggers hyperactivation of lipid biosynthesis, a key event in tumorigenesis, our findings suggest a new generalized strategy to selectively attack tumor metabolism.

Reversal of liver cancer-associated stellate cell-induced stem-like characteristics in SMMC-7721 cells by 8-bromo-7-methoxychrysin via inhibiting STAT3 activation.

Hepatic stellate cells (HSCs) that are activated by human hepatocellular carcinoma (HCC) cells secrete a variety of cytokines, which are the main component of the HCC microenvironment. We aimed to determine whether 8-bromo-7-methoxychrysin (BrMC) could interfere in cross-talk of the human hepatic stellate cell line LX-2 and liver cancer stem-like cells (LCSLCs) to inhibit the characteristics of LCSLCs endowed with the capacity of sustaining human hepatocellular carcinoma (HCC) self-renewal and progression, and to identify its potential mechanism of action. We found that the levels of fibroblast activation protein (FAP) were augmented in LX-2 cells treated with the conditioned medium of LCSLCs (LCSLC-CM) compared to those cultured with routine medium, indicating that the LCSLC-CM can activate LX-2 cells to become liver cancer-associated stellate cells (LCAHSCs). Furthermore, sphere forming capability of SMMC-7721 cells was enhanced and stem cell-related protein expression was significantly increased following treatment with the conditioned medium of LCAHSCs (LCAHSC-CM). Moreover, the level of p-STAT3 was increased in LX-2 cells treated with LCSLC-CM and BrMC reduced expression of p-STAT3. Combination of BrMC and the selective inhibitor of STAT3 cucurbitacin I (JSI-124) synergistically suppressed the LCSLC characteristics in SMMC-7721 cells. Collectively, our data showed that BrMC inhibited the interaction between LX-2 cells and HCC-derived CSCs, and did so potentially through modulation of the STAT3 pathway. Future therapeutic strategies employing anti-CSC therapy should confirm the potential of cucurbitacin I (JSI-124) and BrMC as potent therapeutic agents.

Methylation-associated silencing of miR-200b facilitates human hepatocellular carcinoma progression by directly targeting BMI1.

This study aims to investigate the biological function of microRNA-200b and BMI1, predicted target of microRNA-200b in human hepatocellular carcinoma (HCC). MicroRNA-200b and BMI1 expression in HCC tissues were evaluated by qPCR. A luciferase reporter assay was used to validate BMI1 as a direct target of microRNA-200b. The effect of microRNA-200b on HCC progression was studied in vitro and in vivo. Methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) were used to detect the methylation status of the microRNA-200b promoter. Significant downregulation of microRNA-200b was observed in 83.3% of HCC tissues. By contrast, BMI1 was significantly overexpressed in 66.7% of HCC tissues. The results of the luciferase assay confirmed BMI1 as a direct target gene of microRNA-200b. Forced expression of microRNA-200b in HCC cells dramatically repressed proliferation, colony formation, cell cycle progression, and invasion. Moreover, microRNA-200b synergized with 5-fluorouracil to induce apoptosis in vitro and suppressed tumorigenicity in vivo. In addition, MSP analysis and BSP revealed that CpG sites in the promoter region of microRNA-200b were extensively methylated in HCC, with concomitant downregulation of microRNA-200b expression. Furthermore, microRNA-200b was activated in HCC cells after treatment with 5-azacytidine, whereas BMI1 expression was clearly downregulated. Our results indicate that microRNA-200b is partially silenced by DNA hypermethylation and that it can repress tumor progression by directly targeting BMI1 in HCC.

Aspartate β-hydroxylase modulates cellular senescence through glycogen synthase kinase 3β in hepatocellular carcinoma.

Aspartate β-hydroxylase (ASPH) is an enzyme overexpressed in human hepatocellular carcinoma (HCC) tumors that participates in the malignant transformation process. We determined if ASPH was a therapeutic target by exerting effects on cellular senescence to retard HCC progression. ASPH knockdown or knockout was achieved by short hairpin RNAs or the CRISPR/Cas9 system, respectively, whereas enzymatic inhibition was rendered by a potent second-generation small molecule inhibitor of ASPH. Alterations of cell proliferation, colony formation, and cellular senescence were evaluated in human HCC cell lines. The potential mechanisms for activating cellular senescence were explored using murine subcutaneous and orthotopic xenograft models. Inhibition of ASPH expression and enzymatic activity significantly reduced cell proliferation and colony formation but induced tumor cell senescence. Following inhibition of ASPH activity, phosphorylation of glycogen synthase kinase 3β and p16 expression were increased to promote senescence, whereas cyclin D1 and proliferating cell nuclear antigen were decreased to reduce cell proliferation. The mechanisms involved demonstrate that ASPH binds to glycogen synthase kinase 3β and inhibits its subsequent interactions with protein kinase B and p38 upstream kinases as shown by coimmunoprecipitation. In vivo experiments demonstrated that small molecule inhibitor treatment of HCC bearing mice resulted in significant dose-dependent reduced tumor growth, induced phosphorylation of glycogen synthase kinase 3β, enhanced p16 expression in tumor cells, and promoted cellular senescence. We have identified a new mechanism that promotes HCC growth and progression by modulating senescence of tumor cells; these findings suggest that ASPH enzymatic activity is a novel therapeutic target for HCC.