Promoted Hepatocellular Carcinoma Proliferation Research Articles

Role of Serum Leptin in the Diagnosis and Prognosis of Hepatocellular Carcinoma in Egyptian Cirrhotic Patients

Abstract Background: Hepatocellular carcinoma is the most com-mon primary hepatic malignancy with an average survival rate of 6-20 months. The primary etiology of hepatocellular carcinoma in Egyptian patients is liver cirrhosis. Alfa fetopro-tein is the most commonly used biomarker for HCC diagnosis, in spite of its low sensitivity and specificity in HCC screening; it is usually combined with ultrasound for early detection of HCC. Leptin is a multifunctional peptide hormone that is produced by adipocytes with neuroendocrine activity. Leptin is postulated to increase the hepatic response to different stimuli of liver fibrosis. The actions of leptin are mediated via hepatic stellate cell activation, and liver fibrosis occurs through its indirect effect on the Kupffer cells. Moreover, it regulates the phosphoinositide 3-kinase/protein kinase B (PI-3K/Pkt) pathway, which plays an essential step in the cell motility, proliferation, survival, and angiogenesis in tumor cells including Hepatocellular Carcinoma (HCC). Thus, leptin can promote HCC proliferation, migration, and invasion. Aim of Study: The present study aimed to evaluate the diagnostic and prognostic value of serum Leptin as a tumor marker in Hepatocellular Carcinoma (HCC) in comparison to Alpha-Fetoprotein (AFP). Subjects and Methods: This study included 180 Egyptian patients that were divided into 4 groups (Group 1: 60 patients with untreated HCC, Group 2: 60 patients with treated HCC, Group 3: 30 patients with end-stage chronic liver disease without HCC) in addition to Group 4: 30 age-matched healthy subjects serving as a healthy control group. All patients and controls were subjected to full history, clinical examination, laboratory investigation, and abdominal ultrasonography (U/S). Serum leptin and alpha-fetoprotein were assayed by using Enzyme-Linked Immunosorbent Assay (ELISA) tech-nique. Results: The results of the present study revealed that serum leptin levels were significantly increased in all the patients' groups when compared to controls; besides, it was significantly higher in untreated and treated HCC patients groups when compared to patient’s group without HCC.Furthermore, serum leptin levels were significantly higher in the patients group with untreated HCC compared to the treated HCC patients group with positive correlation with AFP. Conclusion: AFP was more sensitive and specific in differentiation between cirrhotic patients with or without HCC than Leptin. However, Leptin has a good sensitivity and specificity in differentiating between untreated and treated HCC patients, and it is superior to AFP in the specificity of the differentiation between these two categories. Therefore, serum leptin can be used as a useful screening marker for the diagnosis and prognosis of HCC patients combined with Alfa fetoprotein.

MicroRNA‐802 accelerates hepatocellular carcinoma growth by targeting RUNX3

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Prognosis is often unfavorable. In this study, the effects of microRNA-802 (miR-802) on HCC progression were assessed in vivo and in vitro. miR-802 was found to be significantly upregulated in HCC tumor tissue compared to paired adjacent nontumor tissue. In vitro, transfection with a miR-802 mimic accelerated SMMC-7721 cellular proliferation, increased accumulation of the cell-cycle S-phase cell populations, as well as cell migration. In vivo injection of a miR-802 agomir promoted HCC proliferation in nude mice. Targets of miR-802 were predicted by miRWalk, miRanda, RNA22, and Targetscan. By luciferase reporter assay RUNX3 was identified as a direct target of miR-802. As judged by western blot analysis, RUNX3 was upregulated when miR-802 was inhibited. These data demonstrate increased miR-802 expression in patients with HCC and that miR-802 overexpression promotes tumor cell growth, in a RUNX3-dependent manner.

NF90 stabilizes cyclin E1 mRNA through phosphorylation of NF90-Ser382 by CDK2

Nuclear factor 90 (NF90), an RNA-binding protein, has been implicated in regulating interleukin-2 (IL-2) and the immune response. It was recently reported that NF90 is upregulated in hepatocellular carcinoma (HCC) tissues and promotes HCC proliferation through upregulating cyclin E1 at the posttranscription level. However, the regulation of NF90 in HCC remains unclear. We demonstrate here that cyclin-dependent kinase (CDK) 2 interacts with NF90 and phosphorylated it at serine382. Mechanistically, phosphorylation of NF90-Ser382 determines the nuclear export of NF90 and stabilization of cyclin E1 mRNA. We also demonstrate that the phosphorylation deficient mutant NF90-S382A inhibits cell growth and induces cell cycle arrest at the G1 phase in HCC cells. Moreover, an NF90-S382A xenograft tumor had a decreased size and weight compared with the wildtype NF90. The NF90-S382A xenograft contained a significantly lower level of the proliferation marker Ki-67. Additionally, in HCC patients, NF90-Ser382 phosphorylation was stronger in tumor than in non-tumor tissues. Clinically, phosphorylation of NF90-Ser382 is significantly associated with larger tumor sizes, higher AFP levels, and shorter overall survival rates. These results suggest NF90-Ser382 phosphorylation serves as a potential diagnosis and prognostic marker and a promising pharmacological target for HCC.

O-GlcNAcylation of SIX1 enhances its stability and promotes Hepatocellular Carcinoma Proliferation.

It is universally accepted that aberrant metabolism facilitates tumor growth. However, how cancer cells coordinate glucose metabolism and tumor proliferation is largely unknown. Sine oculis homeobox homolog 1 (SIX1) is a transcription factor that belongs to the SIX family and is believed to play an important role in the regulation of the Warburg effect in tumors. However, whether the role of SIX1 and the molecular mechanisms that regulate its activity are similar in hepatocellular carcinoma (HCC) still needs further investigation.Methods: Western blotting was performed to determine the levels of SIX1 and O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) in HCC tissues. Cell Counting Kit 8 (CCK8), colony formation and mouse tumor model assays were used to establish the role of SIX1 and O-GlcNAcylation in HCC processes. Mass spectrometry, immunoprecipitation and site-directed mutagenesis were performed to confirm the O-GlcNAcylation of SIX1.Results: Here, we demonstrated that SIX1, the key transcription factor regulating the Warburg effect in cancer, promotes HCC growth in vitro and in vivo. Furthermore, we revealed that SIX1 could also enhance the levels of a posttranslational modification called O-GlcNAcylation. Importantly, we found that SIX1 was also highly modified by O-GlcNAcylation and that O-GlcNAcylation inhibited the ubiquitination degradation of SIX1. In addition, site-directed mutagenesis at position 276 (T276A) decreased the O-GlcNAcylation level and reversed the protumor effect of SIX1.Conclusions: We conclude that O-GlcNAcylation of SIX1 enhances its stability and promotes HCC proliferation. Our findings illustrate a novel feedback loop of SIX1 and O-GlcNAcylation and show that O-GlcNAcylation of SIX1 is an important way to coordinate glucose metabolism and tumor progression.

Upregulation of amphiregulin by retinoic acid and Wnt signalling promotes liver cancer cell proliferation.

Activated hepatic stellate cells promote hepatocellular carcinoma (HCC) progression. Hepatic stellate cells play a key role in retinoid metabolism, and activation of stellate cells increases retinoic acid (RA) in the liver. However, the role of RA in HCC proliferation remains unclear. We aimed to analyse the mechanism of RA in HCC proliferation. Thirty-eight patients who had undergone hepatic resection for HCCs were recruited. Paired non-tumour tissues, adjacent and distal to HCCs, were collected, and the RA levels in the tissues were analysed. The mechanisms of RA and HCC proliferation were assessed in liver cancer cell lines by protein and gene expression analyses. Early recurrence of HCC was significantly higher in patients with a higher RA concentration than in those with a lower RA concentration in tissues adjacent to HCCs (61.1% vs. 20%, p = .010). RA promoted HCC cell proliferation and activated the expression of Amphiregulin, a growth factor in hepatocarcinogenesis. The promoter of Amphiregulin contained the binding sites of the RA receptor, RXRα. Wnt signalling also activated the expression of Amphiregulin, and the RA and Wnt pathways acted synergistically to increase the expression of Amphiregulin. Furthermore, RXRα interacted with β-catenin and then translocated to the nucleus to activate Amphiregulin. An increased RA concentration in the tissues adjacent to the tumour was associated with an early recurrence of HCC. RA activated the expression of Amphiregulin, and then promoted HCC proliferation, which might partly contribute to early recurrence of HCC after hepatic resection.

5-Hydroxytryptamine Receptor 1D Aggravates Hepatocellular Carcinoma Progression Through FoxO6 in AKT-Dependent and Independent Manners.

Serotonin and its receptors have been shown to play critical regulatory roles in cancer biology. Nevertheless, the contributions of 5-hydroxytryptamine 1D (5-HT1D), an indispensable member of the serotonergic system, to hepatocellular carcinoma (HCC) remain unknown. The present study demonstrated that the 5-HT1D expression level was significantly up-regulated in HCC tissues and cell lines. The 5-HT1D expression level was closely correlated with unfavorable clinicopathological characteristics. Survival analyses show that elevated 5-HT1D expression level predicts poor overall survival and high recurrence probability in HCC patients. Functional studies revealed that 5-HT1D significantly promoted HCC proliferation, epithelial-mesenchymal transition, and metastasis in vitro and in vivo. Mechanistically, 5-HT1D could stabilize PIK3R1 by inhibiting its ubiquitin-mediated degradation. The interaction between 5-HT1D and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) enhanced the expression of FoxO6 through the PI3K/Akt signaling pathway; FoxO6 could also be directly transcriptionally activated by 5-HT1D in an Akt-independent manner. MicroRNA-599 was found to be an upstream suppressive modulator of 5-HT1D. Additionally, 5-HT1D could attenuate tryptophan hydroxylase 1 expression through the PI3K/Akt/cut-like homeobox 1 axis in HCC. Conclusion: Herein, we uncovered the potent oncogenic effect of 5-HT1D on HCC by interacting with PIK3R1 to activate the PI3K/Akt/FoxO6 pathway, and provided a potential therapeutic target for HCC.

Stimulated hepatic stellate cell promotes progression of hepatocellular carcinoma due to protein kinase R activation.

Hepatic stellate cells (HSCs) were reported to promote the progression of hepatocellular carcinoma (HCC), however its mechanism is uncertain. We previously reported that protein kinase R (PKR) in hepatocytes regulated HCC proliferation. In this study, we focused on the role of PKR in HSCs, and clarified the mechanism of its association with HCC progression. We confirmed the activation of PKR in a human HSC cell line (LX-2 cell). IL-1β is produced from HSCs stimulated by lipopolysaccharide (LPS) or palmitic acid which are likely activators of PKR in non-alcoholic steatohepatitis (NASH). Production was assessed by real-time PCR and ELISA. C16 and small interfering RNA (siRNA) were used to inhibit PKR in HSCs. The HCC cell line (HepG2 cell) was cultured with HSC conditioning medium to assess HCC progression, which was evaluated by proliferation and scratch assays. Expression of PKR was increased and activated in stimulated HSCs, and IL-1β production was also increased molecular. Key molecules of the mitogen-activated protein kinase pathway were also upregulated and activated by LPS. Otherwise, PKR inhibition by C16 and PKR siRNA decreased IL-1β production. HCC progression was promoted by HSC-stimulated conditioning medium although it was reduced by the conditioning medium from PKR-inhibited HSCs. Moreover, palmitic acid also upregulated IL-1β expression in HSCs, and conditioning medium from palmitic acid-stimulated HSCs promoted HCC proliferation. Stimulated HSCs by activators of PKR in NASH could play a role in promoting HCC progression through the production of IL-1β, via a mechanism that seems to be dependent on PKR activation.

RRAD suppresses the Warburg effect by downregulating ACTG1 in hepatocellular carcinoma.

PurposeHepatocellular carcinoma (HCC) is a common malignancy with poor prognosis and limited therapeutic options. Ras-related associated with diabetes (RRAD) belongs to the subfamily of Ras-related GTPases and is associated with several types of cancer, including HCC, although the mechanisms involving RRAD in HCC remains unknown.Patients and methodsWe aimed to elucidate the role of RRAD and whether it affects glucose metabolism in HCC by immunohistochemically examining tissue samples from HCC patients and assessing the effect of RRAD overexpression and knockdown on the glucose metabolism, proliferation, cell cycle, and apoptosis of HCC cell lines SK-Hep-1 and Huh7, as well as on tumor progression in vivo.ResultsWe demonstrated that RRAD binds to actin gamma 1 (ACTG1). RRAD suppressed aerobic glycolysis in HCC by downregulating ACTG1. On the other hand, ACTG1 promoted HCC proliferation by regulating the cell cycle via downregulation of cyclins and cyclin-dependent kinases and inhibited apoptosis through the mitochondrial apoptosis pathway in vitro. In addition, RRAD retarded tumor growth by downregulating ACTG1 in vivo. ACTG1 was overexpressed in HCC tissues compared with adjacent normal tissues, whereas the expression of RRAD was low in tumor tissues. Low RRAD levels were significantly correlated with large tumor size and advanced tumor stage; high ACTG1 levels were significantly correlated with advanced tumor stage. Furthermore, Kaplan–Meier survival curves showed that HCC patients with high RRAD and low ACTG1 expression may have a better prognosis.ConclusionWe have shown that RRAD exhibits a tumor-suppressing role in HCC by downregulating glucose metabolism and ACTG1 expression, thus lowering cell proliferation, arresting the cell cycle, and increasing apoptosis. These findings indicate that ACTG1 may act as a downstream effector of RRAD and open a new avenue for potential HCC treatment.

MicroRNA‑9‑5p downregulates Klf4 and influences the progression of hepatocellular carcinoma via the AKT signaling pathway.

Krüppel-like factor 4 (Klf4) is a transcriptional factor involved in the progression of hepatocellular carcinoma (HCC). However, the underlying regulatory mechanisms associated with the Klf4 gene as a tumor suppressor in HCC remain unclear. microRNAs (miRNAs or miRs) are a series of small non-coding RNAs that serve a vital role in regulating gene expression via their influence on protein translation and the associated degradation of mRNA. The mRNA expression levels of the miRNA, miR-9-5p, and Klf4 were measured using reverse transcription-quantitative polymerase chain reaction. The protein expression levels of Klf4, protein kinase B (AKT), phosphorylated (p-)AKT, mechanistic target of rapamycin (mTOR), p-mTOR, B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were determined by western blot analysis. Dual luciferase reporter assay was used to confirm a direct interaction between miR-9-5p and the 3′-untranslated region (3′-UTR) sequence of Klf4. Cell counting kit-8 assay, wound healing assay, Transwell migration assay and flow cytometric analysis were performed to evaluate the proliferative, migratory and apoptotic capabilities of the HCC cells. In the present study, miR-9-5p was revealed to be overexpressed in HCC as a novel upstream gene of Klf4. miR-9-5p expression was inversely associated with Klf4 expression in clinical samples. Additionally, Kaplan-Meier analysis revealed a markedly poor prognosis of HCC in the miR-9-5p high-expression group. Bioinformatics analysis revealed that miR-9-5p bound directly to the 3′-UTR of Klf4, which reduced the expression levels of Klf4. The miR-9-5p/Klf4 axis promoted HCC proliferation and migration, and inhibited HCC apoptosis. Furthermore, miR-9-5p upregulated the Bcl-2/Bax protein ratio and activated AKT/mTOR signaling. Taken together, these data demonstrated that the miR-9-5p/Klf4 axis was able to promote HCC progression, which may occur via regulation of the AKT signaling pathway, highlighting a potential novel target in HCC treatment.

Nanomedicines reveal how PBOV1 promotes hepatocellular carcinoma for effective gene therapy

There exists an urgent medical demand at present to develop therapeutic strategies which can improve the treatment outcome of hepatocellular carcinoma (HCC). Here, we explore the biological functions and clinical significance of PBOV1 in HCC in order to push forward the diagnosis and treatment of HCC. Using theranostical nanomedicines, PBOV1 is verified to be a key oncogene which greatly promotes HCC proliferation, epithelial-to-mesenchymal transition, and stemness by activating the Wnt/β-catenin signaling pathway. Therefore, single-chain antibody for epidermal growth factor receptor (scAb-EGFR)-targeted nanomedicine effectively silencing the PBOV1 gene exhibits potent anticancer effects. In vivo HCC-targeting siRNA delivery mediated by the theranostical nanomedicine remarkably inhibits the tumor growth and metastasis. In addition, the superparamagnetic iron oxide nanocrystals (SPION)-encapsulated nanomedicines possess high MRI detection sensitivity, which endows them with the potential for MRI diagnosis of HCC. This study shows that PBOV1 represents a prognostic biomarker and therapeutic target for HCC.

MiR-190 promotes HCC proliferation and metastasis by targeting PHLPP1

miRNAs regulate gene expression and enable clinicians to distinguish between benign and malignant tissues in cancers. PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1) is known to be a tumour suppressor. A lentiviral overexpression system was used to stably express miR-190, leading to the enhancement of hepatocellular carcinoma (HCC) proliferation and metastasis as a result of inhibited PHLPP1 expression. The results showed that stable miR-190 expression increased the expression of EMT-related proteins (Snail and TCF8/ZEB1) as well as the phosphorylation of Akt at Ser473 and the expression of a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1). However, restoring PHLPP1 expression counteracted the effects of miR-190 on HCC proliferation, migration and invasion. The results of the animal experiments showed that miR-190 improved the HepG2 cell tumour formation and lung metastasis ability. Stable miR-190 overexpression leads to the downregulation of PHLPP1 protein expression. miR-190 has potential as a target in the treatment and diagnosis of HCC.

BMP4 promotes hepatocellular carcinoma proliferation by autophagy activation through JNK1-mediated Bcl-2 phosphorylation

BackgroundAutophagy is a conserved catabolic process with complicated roles in tumor development. Bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor (TGF-β) family of regulatory proteins, plays a crucial role in human malignancies. However, whether BMP4 contributes to the regulation of autophagy in hepatocellular carcinoma (HCC) progression remains elusive.MethodsFunctional analysis of BMP4 on HCC proliferation and autophagy was performed both in vitro and in vivo in HepG2 and HCCLM3 cells. Autophagic activity was estimated by Western blot for autophagic marker proteins and by transmission electron microscopy (TEM). Transfection of mRFP-GFP-LC3 adenovirus was applied to observe autophagic flux and high content screening was used for quantification. The signaling pathway of BMP4-regulated HCC proliferation and autophagy was investigated by Western blot.ResultsBMP4 treatment promoted HCC cells proliferation and induced autophagy. The in vivo xenograft model supported that BMP4 overexpression promoted the growth of HCC cells and autophagy induction while BMP4 knockdown exerted the opposite effect. 3-MA pre-treatment or knockdown of Beclin-1 (BECN1) blocked HCC autophagy by decreasing the expression of LC3-II and subsequently attenuated BMP4-induced autophagy and cells proliferation enhanced by BMP4 in vitro and in vivo. Mechanistic study revealed that the induction of autophagy by BMP4 was mediated through activating the JNK1/Bcl2 pathway. Furthermore, the JNK1 inhibitor and knockdown of JNK1 could attenuate autophagy induced by BMP4 and eliminated BMP4-promoted HCC cells growth.ConclusionsBMP4 promoted HCC proliferation by autophagy activation through JNK1/Bcl-2 signaling.

LncRNA HOXA11-AS promotes hepatocellular carcinoma progression by repressing miR-214-3p.

Accumulating studies supported that lncRNAs played important roles in tumorigenesis. LncRNA HOXA11‐AS was a novel lncRNA that has been proved to involved in several tumours. However, the role of HOXA11‐AS in the development of hepatocellular carcinoma (HCC) remains to be explained. In our study, we showed that HOXA11‐AS expression was up‐regulated in the HCC tissues, and the higher expression of HOXA11‐AS was associated with the advanced stage in the HCC samples. In addition, we indicated that the expression of HOXA11‐AS was up‐regulated in HCC cell lines (Hep3B, SMMC‐7721, MHCC97‐H and BEL‐7402) compared with normal liver cell lines (HL‐7702). Overexpression of HOXA11‐AS promoted HCC proliferation and invasion and induced the epithelial‐mesenchymal transition (EMT) and knockdown of HOXA11‐AS suppressed the HCC cell proliferation and invasion. However, we showed that miR‐214‐3p expression was down‐regulated in the HCC tissues and cell lines. Ectopic expression of miR‐214‐3p suppressed HCC cell proliferation and invasion. Furthermore, we indicated that overexpression of HOXA11‐AS decreased the miR‐214‐3p expression and the expression of miR‐214‐3p was negatively related with the HOXA11‐AS expression in HCC samples. Ectopic expression of HOXA11‐AS increased HCC proliferation and invasion and induced EMT through inhibiting miR‐214‐3p expression. These data suggested that HOXA11‐AS/miR‐214‐3p axis was responsible for development of HCC.

POU5F1B promotes hepatocellular carcinoma proliferation by activating AKT

Hepatocellular carcinoma (HCC) is one of the most common tumors, so far, there still aren’t good therapeutic methods. Looking for new targets makes a top priority. In this study, we found a OCT4 pseudogene, POU5F1B was significantly upregulated in HCC cells and tissues, patients with high POU5F1B had shorter survival time compared to patients with low POU5F1B expression. POU5F1B overexpression promoted HCC proliferation, while its knockdown inhibited HCC proliferation determined by MTT assay, soft agar growth assay, BrdU incorporation assay, and cell cycle assay. Mechanism analysis suggested POU5F1B could activate AKT, AKT inhibition in HCC cells with POU5F1B overexpression significantly inhibited cell proliferation compared to cells only with POU5F1B overexpression, suggesting POU5F1B promoted HCC proliferation by activating AKT. Finally, we analyzed the relationship between POU5F1B expression and AKT activity in HCC tissues, and found POU5F1B expression was positive correlated with activated AKT. Taken together, our findings suggested POU5F1B promoted HCC proliferation by activating AKT, and provided a new target for HCC therapy.

PRMT5 promotes cell proliferation by inhibiting BTG2 expression via the ERK signaling pathway in hepatocellular carcinoma.

Increasing evidence suggests that PRMT5, a protein arginine methyltransferase, has roles in cell growth regulation and cancer development. However, the role of PRMT5 in hepatocellular carcinoma (HCC) progression remains unclear. Here, we showed that PRMT5 expression was frequently upregulated in HCC tissues, and its expression was inversely correlated with overall survival in HCC patients. PRMT5 knockdown markedly inhibited in vitro HCC proliferation and in vivo tumorigenesis. We revealed that the mechanism of PRMT5‐induced proliferation was partially mediated by BTG downregulation, leading to cell cycle arrest during the G1 phase in HCC cells. Ectopic BTG2 overexpression decreased HCC growth, caused cell cycle arrest at the G1 phase, and downregulated Cyclin D1 and Cyclin E1 protein expression. Furthermore, we found that PRMT5‐induced ERK phosphorylation regulated BTG2 expression in HCC cells, whereas pretreatment with a selective ERK1/2 inhibitor (PD184352) significantly reversed the effect of PRMT5 on BTG2 expression. Our results indicated that PRMT5 promotes HCC proliferation by downregulating BTG2 expression via the ERK pathway.

MiR-182-5p promotes hepatocellular carcinoma progression by repressing FOXO3a

BackgroundHigh frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma (HCC). microRNA (miR)-182-5p emerged as a high-priority miRNA in HCC and was found to be related to HCC metastasis. Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown.MethodsReal-time PCR (RT-PCR) and in situ hybridization (ISH) were conducted to assess the expression of miR-182-5p in HCC cells and tissues. Cell Counting Kit-8 (CCK-8), transwell assays were performed to detected cells proliferation and migration ability. Flow cytometry assays were used to detect cell apoptosis rate, and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis. The target of miR-182-5p was validated with a dual-luciferase reporter assay and western blotting. Immunohistochemistry, immumoblotting, and immunoprecipitation were performed to test relative protein expression.ResultsWe showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery. miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo. miR-182-5p directly targets 3′-UTR of FOXO3a and repressed FOXO3a expression, activating AKT/FOXO3a pathway to promote HCC proliferation. Notably, miR-182-5p activated Wnt/β-catenin signaling by inhibiting the degradation of β-catenin and enhancing the interaction between β-catenin and TCF4 which was mediated by repressed FOXO3a.ConclusionsConsistently, miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery, and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.

Far Upstream Element-Binding Protein 1 Facilitates Hepatocellular Carcinoma Invasion and Metastasis

Objective: To identify the role of FUBP1 in hepatocellular carcinoma (HCC) progression, invasion and metastasis. Methods: The expression and clinical significance of FUBP1 in HCC was analyzed by university of California santa cruz (UCSC) Xena online bioinformatics analysis tool and TMA from 451 HCC patients who underwent surgery that removed the primary tumor. CD45-EpCam circulating tumor cell was analyzed by CellSearchTM system. Gain-of-function and loss-of-function studies for FUBP1 were performed by implementing lentivirus infection system. HCC cell proliferation, migration and long term survival were measured by CCK-8, transwell assay and clone formation test respectively. The effect of tumor cell growth in vivo was measured by using tumor xenograft mice. Results: mRNA expression level of FUBP1 is higher in HCC tumors compared to adjacent normal liver tissue. Statistical analysis shows that higher expression of FUBP1 is related with hepatitis B virus (HBV) infection background and often with microvascular invasion. Kaplan-Meier analysis revealed significantly shorter median TTR (Time to Recurrence) and overall survival (OS) in patients with high FUBP1 compared with those patients with low FUBP1. The 2-year and 5-year recurrence rate is higher, corresponding with lower OS, in patients with high FUBP1 expression than low FUBP1. Univariate and multivariate analysis revealed that FUBP expression level was an independent indicator for both TTR and OS. In addition, FUBP1 expression in tumors positively correlated with the percentage of CD45-EpCam circulating tumor cell in blood before operation. Overexpression of FUBP1 enhances HCC proliferation, invasion and metastasis. By using shRNAs to deplete FUBP1 expression, HCC cells displayed decreased cell proliferation, soft-agar growth and invasion. In contrast, FUBP1 overexpression promotes primary tumor growth and lung metastasis. qPCR and Western Blot confirmed that the FUBP1 overexpression induces upregulation of N-cadherin and Vimentin whereas inhibiting E-cadherin, important epithelial mesenchymal transition (EMT) markers. By performing gene expression profiling, we identified that FUBP1 activates the TGF-β/smad pathway through promoting the expression of THBS1 (Thrombospondin-1, TSP-1). LSKL (Peptide antagonist of Thrombospondin-1) could inhibit HCC proliferation and invasion in vitro and vivo through blocking activation of TGF-β/smad pathway mediated by THBS1. Conclusions: High level of FUBP1 in HCC predicts poor prognosis after surgery. Overexpression of FUBP1 promotes HCC proliferation, invasion and metastasis, at least partially through enriching the generation of CTCs by activates TGF-I²/smad pathway and enhancing EMT in vitro and vivo. LSKL could inhibit HCC proliferation and invasion in vitro and vivo through blocking activation of TGF-β/smad pathway mediated by THBS1. Funding: National Science and Technology Major Project (2013ZX10002011-004, 2018ZX10203204-006-002). National Key Research and Development Program (2016YFC0902400), National Natural Science Foundation of China (81572823, 81572884). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: This study was approved by the Clinical Trail Ethics Committee of Zhongshan Hospital and all other four clinical centers. Approval for research protocol and use of human subjects was obtained from the research ethics committee of Zhongshan Hospital. Informed consent was obtained from each patient.

Overexpression of eEF1A1 regulates G1-phase progression to promote HCC proliferation through the STAT1-cyclin D1 pathway

Hepatocellular carcinoma (HCC) is a common cancer worldwide with an aggressive and highly proliferative activity. Studies had confirmed that HCC cell proliferation is associated with the cell cycle's G1 phase, but the detailed molecular mechanism has not been thoroughly elucidated to date. Eukaryotic translation elongation factor 1A1 (eEF1A1) is an evolutionarily conserved elongation factor protein and is involved in tumor cell proliferation. However, which phase of the cell cycle is regulated by eEF1A1 to influence cell proliferation in HCC and its detailed molecular mechanism remain unclear. In this study, we observed that eEF1A1 influences HCC cell proliferation by regulating the cell cycle's G1 phase. In addition, eEF1A1 influences G1 phase by regulating cyclin D1 expression, promoting HCC cell proliferation both in vitro and in vivo. Moreover, our results indicated that eEF1A1 regulates cyclin D1 expression through STAT1 signaling. STAT1 increases the transcriptional activity of cyclin D1 by binding to the cyclin D1 promoter. Taken together, these findings enabled us to identify a novel mechanism by which eEF1A1 regulates the cell cycle's G1 phase to promote tumor proliferation by regulating cyclin D1 expression through STAT1 signaling in HCC.

Hepatitis B virus X protein inhibits apoptosis by modulating endoplasmic reticulum stress response.

Chronic Hepatitis B virus (HBV) infection is a major risk of hepatocellular carcinoma (HCC) worldwide. Hepatitis B virus X protein (HBx) is encoded by one of the four open reading frames of HBV, and is well known as an important coactivator for HBV replication and HBV-associated hepatocellular carcinogenesis. However, its role in keeping cells from apoptosis to promote HCC proliferation remains controversial. Here, we used HBx expressing HCC cells as a model, to investigate the mechanism of HBx-mediated cellular response to endoplasmic reticulum (ER) stress. We found that HBx protein was localized in ER lumen and interacted with GRP78 directly. This interaction resulted in suppression of eIF2α phosphorylation, inhibited expression of ATF4/CHOP/Bcl-2, and reduced cleavage of poly ADP-ribose polymerase (PARP) and level of γH2AX, thus preventing HCC cells from cell death and negatively regulating DNA repair. This study reveals a novel mechanism of the HBx-mediated oncogenesis and provides a basis for potential HBx-targeted therapeutic intervention of HCC.

Abstract 1390: Deregulation of RNA N6-adenosine methylation contribute to liver carcinogenesis

Abstract Primary liver cancer, particularly the most common type hepatocellular carcinoma (HCC), is the third leading cause of cancer death worldwide. Case is even worse in Asia due to extremely high prevalence of HBV/HCV infection, which enhances the risk of HCC. However, the exact mechanism of how HCC develops is still not clearly defined. Previous studies have shown strong evidence for epigenetic alterations in human carcinogenesis, which mainly focus on abnormal DNA methylation, histone modifications, and chromatin remodeling. Recent studies suggest that diverse chemical modifications on RNAs, also known as “epi-transcriptome”, constitute another layer of gene expression regulation. N-6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA, which has been found to functionally influence mRNA stability, alternative splicing, and translation efficiency. However, the roles of m6A deregulation in human carcinogenesis remain unclear. Through whole-transcriptome sequencing, we identified that METTL3, the major component of m6A methyltransferase, was significantly up-regulated in human HCC. Up-regulation of METTL3 negatively associated with poor patient overall and disease free survival. Functionally knockdown/knockout of METTL3 dramatically inhibited HCC proliferation, migration and colony formation abilities in vitro and significantly suppressed in vivo tumorigenicity. In contrast, overexpression of METTL3 using CRISPR/dCas9 SAM system modestly but significantly enhanced HCC proliferation, migration, anchorage-independent growth in vitro and tumor formation in vivo. In order to find the downstream mechanisms which might attributed to METTL3 oncogenic function we performed RNA-Seq on two independent METTL3 knockdown cell lines to interrogated their transcriptome changes. Based on RNA-Seq data, we identified a prominent tumor suppressor SOCS2 as a potential down-stream target of METTL3. With m6A-RNA immunoprecipitation (RIP) assay, we confirmed that SOCS2 mRNA was subject to m6A modification in cancer cells. Inactivation of METTL3 by RNAi or treatment of methylation inhibitor (DAA) impaired m6A mediated mRNA degradation and thereby stabilized SOSC2 mRNA. We further confirm SOCS2 tumor suppressor function in HCC. Downregulation of SOCS2 was associated with poor patient overall survival and disease free survival of HCC patients. Knockdown of SOCS2 significantly promoted HCC proliferation, migration in HCC cells. In conclusion, our findings suggested that deregulation of METTL3 and its associated m6A modification could contribute to human carcinogenesis by imposing an epigenetic control on the stability and expressions of critical tumor suppressor genes. Citation Format: Mengnuo Chen, Larry L. Wei, Cheuk Ting Law, Felice HC Tsang, Iris MJ Xu, Joyce Lee MF Lee, Carmen Wong CL Wong, Irene OL Ng, Chun-Ming Wong. Deregulation of RNA N6-adenosine methylation contribute to liver carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1390. doi:10.1158/1538-7445.AM2017-1390