Migration Of Human Hepatocellular Carcinoma Cells Research Articles

MicroRNA-639 is Down-Regulated in Hepatocellular Carcinoma Tumor Tissue and Inhibits Proliferation and Migration of Human Hepatocellular Carcinoma Cells Through the KAT7/Wnt/β-Catenin Pathway.

BackgroundThis study aimed to investigate the expression of microRNA-639 (miR-639) in tumor tissue from patients with hepatocellular carcinoma (HCC) and its effects on patient outcome, to identify the targets for miR-639 using bioinformatics and luciferase reporter analysis, and the effects of miR-639 in human HCC cells in vitro to identify the molecular pathways involved.Material/MethodsExpression levels of miR-639 were compared in tumor tissue and adjacent normal liver tissue from 50 patients with HCC, and Kaplan-Meier curves identified the association with overall survival (OS). miR-639 expression was measured in HCC cells cultured in vitro using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot. HCC cells were studied using the MTT assay, the colony formation assay, and the transwell assay. Bioinformatics and luciferase reporter analysis identified the role of the histone acetyltransferase gene, KAT7, in HCC.ResultsThe expression of miR-639 was significantly reduced in HCC tissues compared with normal adjacent liver tissues, and inhibited cell proliferation and epithelial-mesenchymal transition (EMT) of HCC cells. Bioinformatics and luciferase reporter analysis showed that miR-639 directly targeted KAT7 and inhibit its expression. KAT7 expression promoted cell proliferation, and migration of human HCC cells in vitro, and miR-639 inhibited cell proliferation and EMT by down-regulating the KAT7/Wnt/β-catenin signaling pathway.ConclusionsmiR-639 was down-regulated in HCC tumor tissue, and inhibited proliferation and migration of HCC cells by the down-regulation of KAT7/Wnt/β-catenin signaling and was associated with reduced OS. These findings supported the potential role of miR-639 as a tumor suppressor.

LncRNA RNCR3 Promotes the Progression of HCC by Activating the Akt/GSK3β Signaling Pathway.

Objective To explore the potential biological roles of long noncoding RNA (lncRNA) RNCR3 in human hepatocellular carcinoma (HCC). Methods First, the expression of RNCR3 was detected by qRT-PCR. Then, in vitro experiments were performed to investigate the effects of RNCR3 on the proliferation, cell cycle, migration, and invasion of HCC cells, while the effects of RNCR3 on HCC tumor growth and metastasis were investigated using in vivo experiments. Finally, western blot was used to study the activation of the Akt/GSK3β signaling pathway. Results RNCR3 was highly expressed in both HCC tissues and cells, and the expression of RNCR3 was closely related to tumor size, tumor number, TNM stage, and overall survival time. In vitro, RNCR3 served as an oncogene to promote cell proliferation, migration, and invasion, and in vivo, RNCR3 promoted the growth and metastasis of HCC tumors. In terms of mechanism, RNCR3 induced the phosphorylation of Akt (thr308 and ser473) and GSK3β (ser9) but decreased the expression of GSK3β, which activated the Akt/GSK3β signaling pathway. Conclusion The high expression of lncRNA RNCR3 in HCC can promote the proliferation, migration, invasion, growth, and metastasis of HCC by activating the NF-κB signaling pathway.

CRISPR/Cas9-mediated knockout of NSD1 suppresses the hepatocellular carcinoma development via the NSD1/H3/Wnt10b signaling pathway

BackgroundThe NSD family of histone lysine methyltransferases have emerged as important biomarkers that participate in a variety of malignancies. Recent evidence has indicated that somatic dysregulation of the nuclear receptor binding SET domain-containing protein 1 (NSD1) is associated with the tumorigenesis in HCC, suggesting that NSD1 may serve as a prognostic target for this malignant tumor. However, its mechanism in human hepatocellular carcinoma (HCC), the major primary malignant tumor in the human liver, remains unclear. Hence, we investigated how NSD1 regulated HCC progression via regulation of the Wnt/β-catenin signaling pathway.MethodsReverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was performed to identify the expression of NSD1 in HCC cells and clinically obtained tissues. The relationship between NSD1 expression and prognosis was analyzed by Kaplan-Meier survival curve. Further, a NSD1 knockout cell line was constructed by CRISPR/Cas9 genomic editing system, which was investigated in a battery of assays such as HCC cell proliferation, migration and invasion, followed by the investigation into NSD1 regulation on histone H3, Wnt10b and Wnt/β-catenin signaling pathway via ChIP. Finally, a nude mouse xenograft model was conducted in order to assess tumorigenesis affected by NSD1 knockout in vivo.ResultsNSD1 was overexpressed in HCC tissues and cell lines in association with poor prognosis. Knockout of NSD1 inhibited the proliferation, migration and invasion abilities of HCC cells. CRISPR/Cas9-mediated knockout of NSD1 promoted methylation of H3K27me3 and reduced methylation of H3K36me2, which inhibited Wnt10b expression. The results thereby indicated an inactivation of the Wnt/β-catenin signaling pathway suppressed cell proliferation, migration and invasion in HCC. Moreover, these in vitro findings were reproduced in vivo on tumor xenograft in nude mice.ConclusionIn conclusion, the study provides evidence that CRISPR/Cas9-mediated NSD1 knockout suppresses HCC cell proliferation and migration via the NSD1/H3/Wnt10b signaling pathway, suggesting that NSD1, H3 and Wnt10b may serve as potential targets for HCC.

Olive oil polyphenols suppress the TGF-α-induced migration of hepatocellular carcinoma cells.

Oleuropein and 3-hydroxytyrosol (3-HT) are natural polyphenols present in olive oil that are known to exhibit potent anti-oxidant activities and exert protective effects against a number of human diseases. In the liver, olive oil polyphenols have been demonstrated to prevent hepatocellular carcinoma (HCC) cell growth. However, little is known about their effects against HCC cell migration. Therefore, the present study investigated whether or not oleuropein and 3-HT were involved in the suppression of transforming growth factor-α (TGF-α)-induced migration of human HCC cells using human HCC-derived HuH7 cells. The TGF-α-induced migration of HuH7 cells was significantly and dose-dependently suppressed by oleuropein and 3-HT. This study group demonstrated previously that the TGF-α-induced activation of AKT and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) were involved in HuH7 cell migration. In addition to these protein kinases, the present study examined the involvement of TGF-α-induced activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and Rho kinase in HuH7 cell migration. TGF-α-induced HuH7 cell migration was decreased by SB203580, a p38 MAPK inhibitor, and Y27632, a Rho kinase inhibitor. However, PD98059, an inhibitor of the upstream kinase activating ERK, did not suppress the TGF-α-induced migration of HuH7 cells. Although AKT, SAPK/JNK, p38 MAPK and Rho kinase pathways were suggested to be involved in the TGF-α-induced migration of HuH7 cells, 10-30 µM 3-HT did not exhibit any suppressive effect on the TGF-α-stimulated activities of these kinases. The results of the present study suggest that olive oil polyphenols suppressed the TGF-α-induced migration of HCC cells.

MiR-221 Promotes Hepatocellular Carcinoma Cells Migration via Targeting PHF2.

MicroRNAs (MiRNAs), which regulate the gene expression leading to translational inhibition or mRNA degradation, are involved in carcinogenesis and tumor progression. Previous studies have demonstrated that miR-221 was one of the most consistent overexpressed miRNAs in several types of cancer. However, the role of miR-221 in human liver cancer progression is not yet fully elucidated. Levels of miR-221 and plant homeodomain finger 2 (PHF2) expressions in human hepatocellular carcinoma (HCC) tissues and cell lines were detected using western blotting and quantitative real-time PCR (qRT-PCR). Cell migration was studied using the transwell assays. A dual-luciferase reporter system was used to validate the target gene of miR-221. The results indicated that miR-221 promoted HCC cell migration. By performing subsequent systematic bioinformatic analyses, we found PHF2 was the target gene of miR-221 and the direct binding relationship was further validated by dual-luciferase reporter assay. In addition, lower expression of PHF2 promoted HCC cell migration and linked to worse overall survival in HCC patients. Finally, the negative correlation between miR-221 and PHF2 expression levels in HCC specimens was further confirmed. Taken together, our findings implied that miR-221 could be a potential candidate for the therapeutics of HCC metastasis.

C1QTNF1-AS1 regulates the occurrence and development of hepatocellular carcinoma by regulating miR-221-3p/SOCS3.

The aim of our study was to explore how C1QTNF1-AS1 regulated miR-221-3p/SOCS3 axis in human hepatocellular carcinoma (HCC). Differentially expressed lncRNAs and genes were examined via RNA-seq. GO analysis and KEGG pathway enrichment analysis were carried out based on the function of dys-regulated mRNAs. RT-qPCR was employed to detect the relative mRNA expression level of C1QTNF1-AS1, miR-221-3p, SOCS3 and key genes in the JAK/STAT signaling pathway in HCC tissues and cells, and western blot analysis was conducted to detect the relative protein expression levels of SOCS3 and key proteins in the JAK/STAT signaling pathway in HCC tissues and cells. MTT assay, transwell assay and flow cytometry were utilized to assess HCC cell proliferation, invasion, migration and apoptosis. Dual luciferase reporter gene assay was used to verify the targeted relationship between C1QTNF1-AS1 and miR-221-3p, as well as between miR-221-3p and SOCS3. A tumorigenicity assay in nude mice was conducted to investigate the effects of C1QTNF1-AS1 on HCC tumor growth in vivo. C1QTNF1-AS1 and SOCS3 were down-regulated, while miR-221-3p was up-regulated in HCC tissues and cells. In HepG2 and Huh7 cells, overexpression of C1QTNF1-AS1 or SOCS3, as well as silence of miR-221-3p inhibited HCC cell proliferation, migration, and invasion and promoted HCC cell apoptosis. The results of the dual luciferase reporter gene assay indicated that miR-221-3p could directly target both C1QTNF1-AS1 and SOCS3. In addition, up-regulation of C1QTNF1-AS1 suppressed HCC tumor growth in vivo. Overexpression of C1QTNF1-AS1 down-regulated miR-221-3p and subsequently up-regulated SOCS3, thereby inhibiting HCC cell proliferation, migration and invasion and promoting apoptosis through the JAK/STAT signaling pathway.

MiRNA-365B Promotes Hepatocellular Carcinoma Cell Migration and Invasion By Downregulating SGTB

Aim: miR-365b, a miRNA at chromosomal breakpoint, was often amplified and upregulated in human hepatocellular carcinoma (HCC). However, the role of miR-365b dysregulation remains unclear. Materials & methods: miR-365b function assays and its target gene analyses were performed. Results: We revealed that miR-365b promoted HCC cell motility and spreading. Furthermore, SGTB was found to be a downstream target of miR-365b, and knockdown of the SGTB gene could mimic the effect of miR-365b in hastening HCC cell migration and invasion. Conclusion: These results imply that miR-365b plays a tumor-promoting role in HCC by suppressing SGTB expression, offering novel potential targets for the treatment of HCC.

MicroRNA‑181c suppresses growth and metastasis of hepatocellular carcinoma by modulating NCAPG.

Purpose: Numerous studies have shown that the expression of microRNA-181c (miR-181c) is inhibited in various cancers, which suggests that it has a cancer suppressive effect. In the current study, we evaluated the regulation and characteristics of miR-181c in human hepatocellular carcinoma (HCC).Materials and methods: Samples of tumor tissues and adjacent non-tumor tissues were collected from 52 patients with HCC, and expression levels of miR-181c in these samples were investigated via quantitative real-time polymerase chain reaction. HCC cell migration and invasion were investigated via wound healing assays and transwell assays. HCC cell apoptosis rates were assessed via flow cytometry, and HCC proliferation was assessed via 5-ethynyl-20-deoxyuridine assays. In vivo tumors were initiated by subcutaneously inoculating HCC cells into nude mice. And various biomarkers were investigated via western blotting.Results: In microarray datasets and tumor tissues, significant downregulation of miR-181c was apparent compared with non-tumorous adjacent tissues. Expression of miR-181c in HCC cells was also significantly lower than it was in normal human liver cells. miR-181c regulated the migration, invasion, apoptosis, and proliferation of HCC cell lines in vitro, and tumor development in vivo. Observations also suggest that miR-181c regulates NCAPG in HCC cells, and its expression affects cellular invasion, migration, proliferation, and apoptosis. There was a negative correlation between miR-181c expression and NCAPG in HCC tissue samples.Conclusion: miR-181c exhibits tumor-suppression via the regulation of NCAPG levels.

LncRNA AY promotes hepatocellular carcinoma metastasis by stimulating ITGAV transcription.

Rationale: Tumor metastasis is the main cause for cancer-related death. However, the driving molecules of metastasis remain largely unknown. Here, we aim to identify long non-coding RNAs (lncRNAs) critical for human hepatocellular carcinoma (HCC) metastasis.Methods: Microarrays were used to screen a comprehensive set of lncRNAs with differential expression profiles in sulfatide-treated cells. Mass spectrometry, protein arrays, and RNA pull-down experiments were used to identify proteins that interacted with lncRNA. Epigenetic analysis was used to study lncRNA-mediated regulation mechanisms.Results: We identified lncRNA AY927503 (AY) as a metastasis-associated molecule that was highly expressed in human hepatocellular carcinoma (HCC) and correlated with metastatic events and poor prognosis in patients with HCC. AY promoted HCC cell migration, stemness, 5-fluorouracil resistance, and metastasis in mice. However, knockdown of integrin αV (ITGAV) abolished AY-stimulated migration, cell viability in HCC cells or tube formation. AY strongly promoted ITGAV transcription and αVβ3 expression by interacting with the ITGAV promoter specifically and stimulating its activity. AY was identified to interact with histone 1FX (H1FX), but deletion of the central domain of AY (AY∆371-522) abolished H1FX binding and ITGAV promoter stimulation. AY significantly enriched H3K4Me3 and acH3K9/14 but reduced H3K27Me3 and H1FX occupancy on the ITGAV promoter, which remodeled chromatin structures for RNA polymerase II recruitment. Knockdown of H1FX abrogated ITGAV transcription stimulated by AY.Conclusions: Our findings suggested that lncRNA AY promoted HCC metastasis via induction of chromatin modification for ITGAV transcription as a pioneer factor and was a potential molecular signature for metastasis or poor prognosis in patients with HCC.

LncRNA CDKN2BAS predicts poor prognosis in patients with hepatocellular carcinoma and promotes metastasis via the miR-153-5p/ARHGAP18 signaling axis.

Background: Growing evidence shows that long noncoding RNAs (lncRNAs) play a crucial role in cancer progression. However, whether lncRNA CDKN2BAS is involved in human hepatocellular carcinoma (HCC) metastasis remains unclear.Methods: Human lncRNA microarray analysis was performed to detect differential expression levels of lncRNAs in metastatic HCC tissues. Effects of CDKN2BAS on cell proliferation, migration, and apoptosis were determined by MTT assay, colony formation assay, migration assay, scratch assay, and flow cytometry. The xenograft experiment was used to confirm the effect of CDKN2BAS on HCC in vivo. qRT-PCR and Western blot were performed to determine the expression levels of mRNAs and proteins. Luciferase reporter assay was used to identify the specific target relationships.Results: CDKN2BAS was remarkably up-regulated in metastatic HCC tissues compared with the adjacent non-tumor tissues. CDKN2BAS promotes HCC cell growth and migration in vitro and in vivo. Additionally, CDKN2BAS upregulated the expression of Rho GTPase activating protein 18 (ARHGAP18) by sponging microRNA-153-5p (miR-153-5p), and thus promoted HCC cell migration. Besides, CDKN2BAS downregulated the expression of Krüppel-like factor 13 (KLF13) and activated MEK-ERK1/2 signaling, thus reducing apoptosis in HCC cells.Conclusions: Our study revealed that lncRNA CDKN2BAS promotes HCC metastasis by regulating the miR-153-5p/ARHGAP18 signaling.

TSG101 promotes the proliferation, migration and invasion of hepatocellular carcinoma cells by regulating the PEG10.

The tumour susceptibility gene 101 (TSG101) is reported to play important roles in the development and progression of several human cancers. However, its potential roles and underlined mechanisms in human hepatocellular carcinoma (HCC) are still needed to be further clarified. In the present study, we reported that knock down of TSG101 suppressed the proliferation, migration and invasion of HCC cells, while overexpression of TSG101 facilitated them. Molecularly, the results revealed that knock down of TSG101 significantly decreased the cell cycle related regulatory factor p53 and p21. In another point, knock down of TSG101 also obviously decreased the level of metallopeptidase inhibitor TIMP1 (Tissue inhibitors of metalloproteinases 1), which results in inhibition of MMP2, MMP7 and MMP9. In contrast, overexpression of TSG101 had opposite effects. The iTRAQ proteomics analysis identified that oncogenic protein PEG10 (Paternally expressed gene 10) might be a potential downstream target of TSG101. Further investigation showed that TSG101 interacted with PEG10 and protected it from proteasomal degradation thereby regulating the expression of p53, p21 and MMPs. Finally, we found that both TSG101 and PEG10 proteins are up‐regulated and presented a direct correlation in HCC patients. In conclusion, these results suggest that TSG101 is up‐regulated in human HCC patients, which may accelerate the proliferation, migration and invasion of HCC cells through regulating PEG10.

Involvement of H19/miR-326 axis in hepatocellular carcinoma development through modulating TWIST1.

Overexpression of long noncoding RNA (lncRNA) H19 has been observed in various cancers, which indicates that H19 exert important roles in the progression of carcinogenesis. MiR-326 has been reported to play tumor suppressive roles in multiple tumors. Recently, the competing endogenous RNA (ceRNA) hypothesis has implied that lncRNAs might function as molecular sponges for microRNAs in various cancers. However, the roles of H19/miR-326 in human hepatocellular carcinoma (HCC) still remain unclear. The aim of our study was to determine H19/miR-326 expression in HCC cells and investigate their roles in HCC development. We found that H19 was significantly elevated and miR-326 was decreased in HCC cells including Hep3B, HepG2, MHCC-97L, SK-hep1, Hun7, SMCC-7721 compared with LO2 cells, respectively. In the subsequent experiments, we observed that inhibition of H19 can repress HCC cell growth, migration, and invasion in vitro. H19 downregulation can increase miR-326 expression in HCC cells. Meanwhile, miR-326 mimics can also inhibit HCC progression, whereas miR-326 inhibitors exhibited a reverse phenomenon by modulating H19 expression. In addition, a negative association between H19 and miR-326 was predicted and confirmed. Furthermore, the transcription factor TWIST1 has been recognized as a significant regulator in tumor progression. Here, by performing bioinformatics analysis, TWIST1 was identified as a downstream target of miR-326. The findings of our study implied that lncRNA H19 can serve as a ceRNA to sponge miR-326 and modulate TWIST1 levels in HCC pathogenesis. Taken these together, these findings indicated that H19/miR-326/TWIST1 axis was involved in HCC development and can indicate a novel HCC target.

T-box Transcription Factor Tbx3 Contributes to Human Hepatocellular Carcinoma Cell Migration and Invasion by Repressing E-Cadherin Expression.

Tbx3, a member of the T-box family of transcription factors, contributes directly to tumor formation, migration, and invasion. However, the role of Tbx3 in the metastasis of HCC remains unclear. In the present study, Tbx3 expression was detected in HCC tissues and cells by Western blot, and Tbx3 expression was regulated by use of siRNAs or lentivirus-mediated vectors. Here we found that Tbx3 protein expression increased in HCC tissues and cell lines. Tbx3 expression was positively associated with multiple tumor nodes, venous infiltration, and advanced TNM tumor stage. Survival analysis demonstrated that Tbx3 expression was an independent prognostic factor for HCC patients. In vitro assays further validated that Tbx3 indeed prompted HCC cell migration and invasion. In addition, Tbx3 expression was negatively related with E-cadherin expression in HCC tissues. Mechanically, Tbx3 inhibited the expression of E-cadherin, and then facilitated epithelial–mesenchymal transition (EMT) of HCC cells. Furthermore, the effect of Tbx3 knockdown on HCC cells was attenuated by E-cadherin knockdown. In conclusion, Tbx3 may be a novel prognostic factor, and it contributes to HCC cell migration, invasion, and EMT by repressing E-cadherin expression. Thus, Tbx3 may be recommended as a therapeutic target for HCC patients.

RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2.

METTL3 is frequently up-regulated in human HCC and contributes to HCC progression. METTL3 represses SOCS2 expression in HCC through an m6A-YTHDF2-dependent mechanism. Our findings suggest an important mechanism of epigenetic alteration in liver carcinogenesis. (Hepatology 2018;67:2254-2270).

Teroxirone suppresses growth and motility of human hepatocellular carcinoma cells

AimsThe prevalent human hepatocellular carcinoma (HCC) is a leading cause of global cancer-related mortality. The small molecular weight triepoxide derivative, 1,3,5-triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl) (teroxirone), has been proved effective against the proliferation of lung cancer cells. The purpose is to further examine if teroxirone regulate growth and metastatic potential of HCC cells with aims at disclosing more of the reaction mechanisms. Main methodsMeasurements of cell viability and flow cytometry were conducted to test sensitivities of teroxirone against HCC cells. The signaling pathway leading to apoptotic death was unraveled by Western blotting analysis. The metastatic progression was evaluated by cell-based phenotype assay that included migration, invasion, gelatin zymography and wound assay. The in vivo drug efficiency was done in immune-deficient mice with the established xenograft tumors. Key findingsTeroxirone inhibited growth of HCC cells, but not hepatic cells. The drug induced apoptosis in HCC cells bearing mutant p53. Pretreatment of caspase-3 inhibitor restored cell viabilities by suppressing extrinsic pathway-mediated cell death. More experiments suggested that sub-apoptotic concentrations of teroxirone mitigated migration, invasion and wound healing of HCC cells. The drug reduced growth of the xenograft tumors as established in animal models by activating apoptotic death. SignificanceThe findings asserted that teroxirone is an eligible addition to the existing options as an anticancer agent to eliminate HCC.

CFIm25 inhibits hepatocellular carcinoma metastasis by suppressing the p38 and JNK/c-Jun signaling pathways.

Alternative polyadenylation (APA), a post-transcriptional modification, has been implicated in many diseases, but especially in tumor proliferation. CFIm25, the 25 kDa subunit of human cleavage factor Im (CFIm), is a key factor in APA. We show that CFIm25 expression is reduced in human hepatocellular carcinoma (HCC), and its expression correlates with metastasis. Kaplan-Meier analysis indicated that CFIm25 is related to overall survival in HCC. Moreover, CFIm25 expression is negatively related to the metastatic potential of HCC cell lines. CFIm25 knockdown promotes cell invasion and migration in vitro, while overexpression of CFIm25 inhibits cell invasion and migration in vitro and inhibits intrahepatic and lung metastasis in vivo. Additional studies showed that CFIm25 disrupts epithelial-mesenchymal transition by increasing E-cadherin, that it inhibits HCC cell migration and invasion by blocking the p38 and JNK/c-Jun signaling pathways, and that CFIm25 knockdown increases the transcriptional activity of activating protein-1 (AP-1). These findings indicate that therapy directed at increasing CFIm25 expression is a potential HCC treatment.

Methionine Sulfoxide Reductase B1 Regulates Hepatocellular Carcinoma Cell Proliferation and Invasion via the Mitogen-Activated Protein Kinase Pathway and Epithelial-Mesenchymal Transition.

Methionine sulfoxide reductase B1 (MsrB1) is a member of the selenoprotein family, which contributes to the reduction of methionine sulfoxides produced from reactive oxygen species (ROS) by redox processes in energy pathways. However, few studies have examined the role of MsrB1 in human hepatocellular carcinoma (HCC). We observed that MsrB1 is highly expressed in HCC tissues and that its expression correlated with the prognoses of patients with HCC after hepatectomy. In vitro, knockdown of MsrB1 inhibits HCC cell growth by MTT and EdU proliferation assay, and MsrB1 interference enhances H2O2/trx-induced apoptosis. We observed that phosphorylation of the key proteins of the MAPK pathway, namely, ERK, MEK, and p53, was inhibited, but PARP and caspase 3 were increased, thus infecting mitochondrial integrity. In vivo, MsrB1 knockdown effectively inhibited tumor growth. Furthermore, MsrB1 knockdown reduced HCC cell migration and invasion in a transwell assay through inhibition of cytoskeletal rearrangement and spread. This change was linked to epithelial-mesenchymal transition (EMT) inhibition resulting from increases in E-cadherin expression and decreases in expression in TGF-β1, Slug, MMP-2/9, and so on. MsrB1 regulates HCC cell proliferation and migration by modulating the MAPK pathway and EMT. Thus, MsrB1 may be a novel therapeutic target with respect to the treatment of HCC.

Altered fatty acid‐binding protein 4 (FABP4) expression and function in human and animal models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality. Risk factors for developing HCC include viral hepatitis, alcohol and obesity. Fatty acid-binding proteins (FABPs) bind long-chain free fatty acids (FFAs) and are expressed in a tissue-specific pattern; FABP1 being the predominant hepatic form, and FABP4 the predominant adipocyte form. The aims of this study were to investigate the expression and function of FABPs1-9 in human and animal models of obesity-related HCC. FABP1-9 expression was determined in a mouse model of obesity-promoted HCC. Based on these data, expression and function of FABP4 was determined in human HCC cells (HepG2 and HuH7) in vitro. Serum from patients with different underlying hepatic pathologies was analysed for circulating FABP4 levels. Livers from obese mice, independent of tumour status, exhibited increased FABP4 mRNA and protein expression concomitant with elevated serum FABP4. In vitro, FABP4 expression was induced in human HCC cells by FFA treatment, and led to FABP4 release into culture medium. Treatment of HCC cells with exogenous FABP4 significantly increased proliferation and migration of human HCC cells. Patient serum analysis demonstrated significantly increased FABP4 in those with underlying liver disease, particularly non-alcoholic fatty liver disease (NAFLD) and HCC. These data suggest FABP4, an FABP not normally expressed in the liver, can be synthesized and secreted by hepatocytes and HCC cells, and that FABP4 may play a role in regulating tumour progression in the underlying setting of obesity.

Expression and purification of an FGF9 fusion protein in E. coli, and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration.

Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6×His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity. Both purified forms of rhFGF9, with and without fusion tags, significantly stimulated the proliferation of NIH3T3 cells. The FGF9 subfamily, including FGF9, FGF16, and FGF20, in addition to rhFGF16, rhFGF9, and rhFGF20, were shown to stimulate the proliferation and migration of HuH7 human hepatocellular carcinoma (HCC) cells. Mechanistic studies revealed that the stimulation of HuH7 cell proliferation and migration with rhFGF9 and rhFGF20 were associated with the activation of the extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) pathways and matrix metalloproteinase-26 (MMP26). Inhibition of the ERK and NF-κB pathways blocked cell migration, and NF-κB was demonstrated to be regulated by ERK. Therefore, the present study demonstrates a simple method for the preparation of biologically active rhFGF9 protein. Furthermore, the results indicate that exogenous rhFGF9- and rhFGF20-activated ERK/NF-κB signal transduction pathways play important roles in the regulation of HCC cell proliferation and migration, and this discovery helps to find the potential for new solutions of the treatment of liver cancer.

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.