The Overexpression of IQGAP1 and β-Catenin Is Associated with Tumor Progression in Hepatocellular Carcinoma In Vitro and In Vivo

The IQ-domain GTPase-activating protein 1 (IQGAP1) is a multifunctional scaffold protein, which interacts with diverse proteins to regulate cell adhesion and cell migration. The abnormal expression of IQGAP1 widely exists in many cancers, but biological roles of IQGAP1 cooperation with its interacting proteins to involve in tumorigenesis remain to clarify. In this study, we have found that IQGAP1 interacts with β-catenin and regulates β-catenin expression in hepatocellular carcinoma (HCC) cells. The expression levels of IQGAP1 and β-catenin and their associations have a positive correlation with cell metastasis ability in several HCC cell lines. The up-regulation of IQGAP1 and β-catenin improves cell proliferation and migration ability of HCC cells, whereas the knockdown of IQGAP1 by small interfering RNA can decrease β-catenin expression, which results in the reduction of cell proliferation and migration ability in vitro. In addition, a significantly higher expression of IQGAP1 and β-catenin also usually exists in human HCC tissues, especially their overexpression is clinicopathologically associated with tumor malignancy. Generally the overexpression and interactions of IQGAP1 and β-catenin contribute to HCC progression by promoting cell proliferation and migration.     

Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2’s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development.     

The Suppressive Role and Aberrent Promoter Methylation of BTG3 in the Progression of Hepatocellular Carcinoma

Background

BTG3 (B-cell translocation gene 3) has been identified as a tumor suppressor and hypermethylation contributes to its down-regulation in some tumors, but its role in hepatocellular carcinoma (HCC) remain unknown. This study aimed to detect the expression and methylation status of BTG3 in HCC cell lines or tissues, and determine its function in HCC progression.

Methodology

The expression of BTG3 was detected in HCC cell lines and HCC tissue by real-time RT-PCR, Western blot or immunohistochemistry. The promoter methylation status of BTG3 was measured by using methylation-specific PCR in HCC cell lines. A series of assays were performed to evaluate the effect of BTG3 on proliferation, invasion and cell cycle transition in vitro.

Results

BTG3 expression was lower in HCC cell lines than in hepatocyte cell line LO2 (P<0.05). BTG3 was also down-regulated in HCC tissues. Its expression was positively correlated with differentiation and distant metastasis (P<0.05). Patients with lower BTG3 expression had shorter overall survival time (P=0.029). DNA methylation directed repression of BTG3 mRNA expression in HCC cell lines. BTG3 suppressed proliferation, invasion and induces G1/S cycle arrest of HCC cells in vitro.

Conclusion

Down-regulation of BTG3 due to the promoter hypermethylation is closely associated with proliferation, invasion and cell cycle arrest of HCC cells. It may be a novel prognostic biomarker for HCC patients.     

Overexpression of Endothelin 1 Triggers Hepatocarcinogenesis in Zebrafish and Promotes Cell Proliferation and Migration through the AKT Pathway

Hepatocarcinogenesis commonly involves the gradual progression from hepatitis to fibrosis and cirrhosis, and ultimately to hepatocellular carcinoma (HCC). Endothelin 1 (Edn1) has been identified as a gene that is significantly up-regulated in HBx-induced HCC in mice. In this study, we further investigated the role of edn1 in hepatocarcinogenesis using a transgenic zebrafish model and a cell culture system. Liver-specific edn1 expression caused steatosis, fibrosis, glycogen accumulation, bile duct dilation, hyperplasia, and HCC in zebrafish. Overexpression of EDN1 in 293T cells enhanced cell proliferation and cell migration in in vitro and xenotransplantation assays and was accompanied with up-regulation of several cell cycle/proliferation- and migration-specific genes. Furthermore, expression of the unfolded protein response (UPR) pathway-related mediators, such as spliced XBP1, ATF6, IRE1, and PERK, was also up-regulated at both the RNA and protein levels. In the presence of an EDN1 inhibitor or an AKT inhibitor, these increases were diminished and the EDN1-induced migration ability also was disappeared, suggesting that the EDN1 effects act through activation of the AKT pathway to enhance the UPR and subsequently activate the expression of downstream genes. Additionally, p-AKT is enhanced in the edn1 transgenic fish compared to the GFP-mCherry control. The micro RNA miR-1 was found to inhibit the expression of EDN1. We also observed an inverse correlation between EDN1 and miR-1 expression in HCC patients. In conclusion, our data suggest that EDN1 plays an important role in HCC progression by activating the PI3K/AKT pathway and is regulated by miR-1.     

MicroRNA-1271 functions as a potential tumor suppressor in hepatitis B virus–associated hepatocellular carcinoma through the AMPK signaling pathway by binding to CCNA1

Hepatocellular carcinoma (HCC) is mainly associated with hepatitis B virus (HBV) infection and characterized by metastasizing and infiltrating adjacent and distant tissues. Notably, microRNA-1271 (miR-1271) is a tumor suppressor in various cancers. Therefore, we evaluate the ability of miR-1271 to influence cell proliferation, migration, invasion, and apoptosis in HBV-associated HCC through the Adenosine monophosphate–activated protein kinase (AMPK) signaling pathway via targeting CCNA1. HBV-associated HCC and adjacent normal tissues were collected to identify the expression of miR-1271 and CCNA1. To verify the relationship between miR-1271 and CCNA1, we used bioinformatics prediction and the dual-luciferase reporter gene assay. The effects of miR-1271 on HBV-associated HCC cell behaviors were investigated by treatment of the miR-1271 mimic, the miR-1271 inhibitor, or small interfering RNA against CCNA1. The HBV-DNA quantitative assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid assay, scratch test, transwell assay, and flow cytometry were used to detect HBV-DNA replication, cell proliferation, invasion, migration, and apoptosis. MiR-1271 showed a low expression, whereas CCNA1 showed a high expression in HBV-associated HCC tissues. We identified that miR-1271 targeted and negatively regulated CCNA1. Upregulated miR-1271 and downregulated CCNA1 inhibited the HBV-associated HCC cell HBV-DNA replication, proliferation, migration, and invasion, while accelerating apoptosis by activating the AMPK signaling pathway. MiR-1271 promotes the activation of the AMPK signaling pathway by binding to CCNA1, whereby miR-1271 suppresses HBV-associated HCC progression. This study points to a potential therapeutic approach of downregulation of miR-1271 in HBV-associated HCC treatment.     

RANKL Promotes Migration and Invasion of Hepatocellular Carcinoma Cells via NF-κB-Mediated Epithelial-Mesenchymal Transition

Background

Metastasis accounts for the most deaths in patients with hepatocellular carcinoma (HCC). Receptor activator of nuclear factor kappa B ligand (RANKL) is associated with cancer metastasis, while its role in HCC remains largely unknown.

Methods

Immunohistochemistry was performed to determine the expression of RANK in HCC tissue (n = 398). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to examine the expression of RANK, E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist and MMPs in HCC cells. Wound healing and Transwell assays were used to evaluate cell migration and invasion ability.

Results

We found that expression of RANK, the receptor of RANKL, was significantly higher in HCC tumor tissues than in peritumor liver tissues (p<0.001). Constitutive expression of RANK was detected in HCC cell lines, which can be up-regulated when HCC cells were stimulated with RANKL. Notably, in vitro experiments showed that activation of RANKL-RANK axis significantly promoted migration and invasion ability of HCC cells. In addition, RANKL stimulation increased the expression levels of N-cadherin, Snail, and Twist, while decreased the expression of E-cadherin, with concomitant activation of NF-κB signaling pathway. Moreover, administration of the NF-κB inhibitor attenuated RANKL-induced migration, invasion and epithelial-mesenchymal transition of HCC cells.

Conclusions

RANKL could potentiate migration and invasion ability of RANK-positive HCC cells through NF-κB pathway-mediated epithelial-mesenchymal transition, which means that RANKL-RANK axis could be a potential target for HCC therapy.     

Decreased expression of SERPINB1 correlates with tumor invasion and poor prognosis in hepatocellular carcinoma

SERPINB1 (serine protease inhibitor, clade B, member1) is a member of the SERPINB family. Recent studies suggested that SERPINB1 may suppress the migration and invasion of lung and breast cancers. In this study, we investigated a possible involvement of SERPINB1 in the regulation of hepatocellular carcinoma metastasis (HCC). The expression of SERPINB1 was evaluated using western blot analysis in 8 paired fresh HCC specimens and immunohistochemistrical assay on 67 paraffin-embedded HCC slices. SERPINB1 was downregulated in HCC specimens and correlatively related with two clinicopathologic features of HCC, metastasis (P = 0.000) and vein invasion (P = 0.006). Univariate and multivariate survival analyses showed a lower level of SERPINB1 expression is associated with poor prognosis and clinical outcome (P = 0.001). In addition, small interfering RNA targeting SERPINB1 was used to knock down the expression of SERPINB1 in Huh7 and BEL-7404 cells. We showed that interference of SERPINB1 promoted migration and invasion of HCC cells, while cell proliferation was not affected. Finally, we observed an apparent increase in the level of active matrix metalloproteinase-2 (MMP2) after SERPINB1 knockdown, implying that SERPINB1 might participate in the regulation of HCC metastasis through modulating the activation of matrix metalloproteinases. Overall, our results suggested an inhibitory role of SERPINB1 in the migration and invasion of HCC, implying that SERPINB1 might be a potential prognostic indicator of HCC metastasis.     

PAK1 promotes proliferation,migration and invasion of hepatocellular carcinoma by facilitating EMT via directly up-regulating Snail

BackgroundHepatocellular carcinoma (HCC) is a primary cause of cancer mortality. PAK1 plays key roles in many types of cancers. However, the role of PAK1 in HCC is not clear.MethodsqRT-PCR and Western blotting were used to determine expressions of PAK1, Snail and epithelial mesenchymal transition (EMT)-related proteins. Luciferase reporter assay was used to measure the interaction between PAK1 and Snail. Wound healing, transwell, colony formation assays and flow cytometry were used to assess cell migration, invasion, proliferation and apoptosis. Mouse tumor xenograft model was used to determine the effect of PAK1 on tumor growth in vivo.ResultsPAK1 and Snail were up-regulated in HCC cells. PAK1 knockdown suppressed cell proliferation, migration and invasion, and increased apoptosis of HCC cells. PAK1 knockdown also inhibited tumor growth in vivo. Mechanistically, PAK1 promoted EMT by targeting Snail. Knockdown of PAK1 could up-regulate pro-apoptotic proteins but down-regulate proliferation-related proteins via suppressing β-catenin signaling pathway.ConclusionPAK1 promotes EMT process by increasing Snail, and facilitates progression of HCC by activating β-catenin pathway.     

Downregulation of circDYNC1H1 exhibits inhibitor effect on cell proliferation and migration in hepatocellular carcinoma through miR-140-5p

Circular RNAs have been found to be aberrantly expressed in tumors and their significance in tumorigenesis has been focused on. The role of circDYNC1H1 in hepatocellular carcinoma (HCC) pathogenesis and its relationship with miR-140-5p were explored. The expression of circDYNC1H1, miR-140-5p, and SULT2B1 in HCC tissues and cells was measured, and Pearson's analysis was used to analyze their expression correlation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assays were performed to determine cell proliferation and migration. Binding between circDYNC1H1 and miR-140-5p was evaluated with RNA pull-down assay. A luciferase reporter assay was conducted to assess the interaction between circDYNC1H1 and miR-140-5p and between miR-140-5p and SULT2B1. circDYNC1H1 was highly expressed in HCC tissues (n = 20), and it was negatively associated with the expression of miR-140-5p but positively correlated with SULT2B1 messenger RNA expression. circDYNC1H1 was upregulated in cell lines of HCC. Interference of circDYNC1H1 suppressed cell proliferation and migration of HCC. circDYNC1H1 acted as a sponge of miR-140-5p. miR-140-5p controlled SULT2B1 expression by targeting its 3′-untranslated region. circDYNC1H1 enhanced SULT2B1 expression via sponging miR-140-5p. Downregulation of circDYNC1H1 disturbed cell proliferation and migration of HCC through miR-140-5p/SULT2B1 pathway. Silencing of circDYNC1H1 delayed tumor growth in HCC mouse model. Acting like a sponge of miR-140-5p, silenced circDYNC1H1 downregulated SULT2B1 to restrain HCC cell proliferation and migration, which is adverse to HCC growth and progression.     

Underexpression of miR-34a in Hepatocellular Carcinoma and Its Contribution towards Enhancement of Proliferating Inhibitory Effects of Agents Targeting c-MET

Aberrant expression of microRNA-34a (miR-34a) has been reported to be involved in the tumorigenesis and progression of various classes of malignancies. However, its role in hepatocellular carcinoma (HCC) has not been completely clarified. In the current study, we have investigated the clinical significance and the in vitro contribution of miR-34a on biological functions of human HCCs. miR-34a expression in eighty-three cases of HCC formalin-fixed paraffin-embedded (FFPE) tissues decreased significantly compared to that in the adjacent liver tissues (P<0.01), as detected by real-time quantitative RT-PCR (RT-qPCR). miR-34a expression in the groups of TNM stage I and II, without metastasis and without portal vein tumor embolus, was significantly higher than that of their corresponding groups (P<0.05). In functional experiments, miR-34a mimic suppressed cell growth, migration and invasion, meanwhile it increased cellular apoptosis and caspase activity in HCC cells. miR-34a mimic also reduced phospho-ERK1/2 and phospho-stat5 signaling. In addition, miR-34a mimic enhanced the effect of cell proliferation inhibition and caspase activity induction of agents targeting c-MET (siRNAs and small molecular inhibitor su11274). In conclusion, miR-34a may act as a tumor suppressor miRNA of HCC. The strategies to increase miR-34a level might be a critical targeted therapy for HCC in future.     

mTOR/miR-145-regulated exosomal GOLM1 promotes hepatocellular carcinoma through augmented GSK-3β/MMPs

Golgi membrane protein 1 (GOLM1/GP73) is a serum marker of hepatocellular carcinoma (HCC). We have previously shown that mTOR promoted tumorigenesis of HCC through stimulating GOLM1 expression. In this study, we demonstrated that the mammalian target of rapamycin (mTOR) was a negative regulator of microRNA-145 (miR-145) expression. miR-145 inhibited GOLM1 expression by targeting a coding sequence of GOLM1 gene. GOLM1 and miR-145 were inversely correlated in human HCC tissues. GOLM1-enriched exosomes activated the glycogen synthase kinase-3β/matrix metalloproteinases (GSK-3β/MMPs) signaling axis of recipient cells and accelerated cell proliferation and migration. In contrast, miR-145 suppressed tumorigenesis and metastasis. We suggest that mTOR/miR-145/GOLM1 signaling pathway should be targeted for HCC treatment.     

Knockdown of MACC1 expression suppressed hepatocellular carcinoma cell migration and invasion and inhibited expression of MMP2 and MMP9

Expression of MACC1 (metastasis-associated in colon cancer-1) protein is associated with metastasis of various human cancers. This study analyzed MACC1 protein expression in hepatocellular carcinoma (HCC) tissue specimens and then investigated the effects of MACC1 knockdown on HCC cell migration and invasion, and gene expression levels. Sixty pairs of HCC and adjacent normal liver tissues from HCC patients were analyzed for MACC1 expression immunohistochemically. The HCC cell lines Hep3B, Huh7, MHCC97H, SMMC-7721, Bel-7402, and HepG2 and the normal liver cell line LO2 were used to assess expressions of MACC1 mRNA and MACC1 protein using qRT-PCR and western blot, respectively. MACC1 short hairpin RNA (shRNA) was used to knockdown MACC1 protein expression in Huh7 cells. Changes in the tumor phenotype of these cells were analyzed with wound healing assay and invasion assays, and differences in gene expression were evaluated via western blot. Immunofluorescence was used to locate MACC1 protein in the above cell lines. MACC1 was highly expressed in HCC tissues and the nuclear expression of MACC1 protein was associated with poor tumor differentiation and intrahepatic metastasis or portal invasion. Moreover, MACC1 mRNA and MACC1 protein was also expressed in HCC cell lines. Immunostaining showed that MACC1 protein was localized in both nuclei and cytoplasm of HCC cell lines and the nuclear localization of MACC1 protein was associated with increased aggressiveness of HCC in cell lines. Knockdown of MACC1 expression using MACC1-shRNA reduced Huh7 cell migration and invasion abilities, which was associated with downregulation of MMP2, MMP9, and c-Met proteins in Huh7 cells. Localization of MACC1 protein to the nucleus may predict HCC progression. Knockdown of MACC1 expression using MACC1 shRNA warrants further evaluation as a novel therapeutic strategy for control of HCC.     

Dulcitol suppresses proliferation and migration of hepatocellular carcinoma via regulating SIRT1/p53 pathway

BackgroundHepatocellular carcinoma (HCC) spreads further with continuance and increasing incidence due to its high-grade malignancy and metastasis. More effectual strategies on blocking proliferation and metastasis of cancer cells should be studied in HCC. Dulcitol, a natural product extracted from euonymus alatus, was reported that it could induce apoptosis of C6 glioma cells. However, the underlying mechanism of Dulcitol on HCC remains unclea.PurposeIn this study, we aimed to reveal the effect and potential mechanisms of Dulcitol on hepatocellular carcinoma in vitro and in vivo.Study design and methods The cell proliferation and apoptosis were evaluated by MTT, Ki-67 and Hoechst 33258/PI double staining. The migratory and invasive abilities of HepG2 cells were measured by wound-healing and transwell assays. Pathological changes of tumor tissue were observed by HE staining and IHC methods. The expression levels of protein were detected using Western Blot analysis.ResultsThe results showed that Dulcitol inhibited HepG2 cells proliferation by down-regulating the protein expression of SIRT1, Bcl-2, along with up-regulating p53, acetylated-p53 (K382), cleaved-caspase9, cleaved-caspase3, Bax, and cytochrome c in a dose-dependent manner. Furthermore, Dulcitol surpressed the migration and invasion of HepG2 cells through decreasing the levels of MMP-2, uPA and MMP-9 and increasing E-cadherin associated with tumor invasion. In vivo, Dulcitol distinctly inhibited the growth of HepG2 cancer xenograft tumors via inhibiting SIRT1/p53 pathway.ConclusionsOur findings suggested that Dulcitol acted as a SIRT1 inhibitor, inducing apoptosis and inhibiting proliferation, migration and invasion of HepG2 cells and its modulatory mechanism seemed to be associated with regulation of MMPs, SIRT1/p53 pathways.     

Retracted: In vivo and in vitro effects of microRNA-221 on hepatocellular carcinoma development and progression through the JAK–STAT3 signaling pathway by targeting SOCS3

Hepatocellular carcinoma (HCC), as the third leading cancer-caused deaths, prevails with high mortality, and affects more than half a million individuals per year worldwide. A former study revealed that microRNA-221 (miR-221) was involved in cell proliferation of liver cancer and HCC development. The current study aims to evaluate whether miR-221 targeting SOCS3 affects HCC through JAK–STAT3 signaling pathway. A series of miR-221 mimic, miR-221 inhibitor, siRNA against SOCS3, and SOCS3 plasmids were introduced to SMMC7721 cells with the highest miR-221 expression assessed. The expression of JAK–STAT3 signaling pathway–related genes and proteins was determined by Western blot analysis. Cell apoptosis, viability, migration, and invasion were evaluated by means of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, and transwell assays, respectively. HCC xenograft in nude mice was performed to measure HCC tumor growth. miR-221 was found to be highly expressed but SOCS3 was poorly expressed in HCC tissues. miR-221 expression was correlated with lymph node metastasis (LNM) and tumor node metastasis (TNM) of HCC, and SOCS3 expression was correlated with LNM, differentiation and TNM of HCC. SOCS3 is a target gene of miR-221. MiR-221 mimic or si-SOCS3 exposure was found to induce cell viability, migration, and invasion, and reduce apoptosis. MiR-221 inhibitor was observed to have inhibitory effects on HCC cell proliferation, migration, and invasion. Moreover, the expression of JAK–STAT3 signaling pathway was suppressed by miR-221 inhibitor. Downregulated miR-221 expression could promote its target gene SOCS3 to inhibit the proliferation, invasion and migration of HCC cells by repressing JAK–STAT3 signaling pathway.     

lncRNA RMRP knockdown suppress hepatocellular carcinoma biological activities via regulation miRNA-206/TACR1

Long noncoding RNA, RNA component of mitochondrial RNA processing endoribonuclease (RMRP) plays an important role in cancer development and is closely correlated with prognosis in cancer patients. However, whether RMRP affects prognosis in patients with hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to investigate the expression level of RMRP in HCC and its correlation with prognosis in patients with HCC and explain the effects and associated mechanisms by conducting an in vitro study. The high expression level of RMRP was correlated with poor prognosis in patients with HCC. Using in vitro analysis, RMRP knockdown suppressed HCC cell proliferation, invasion, and migration (P < .05). miRNA-206 overexpression had similar effects in HCC cell lines (Bel-7402 and Huh-7). Using Western blot analysis and cellular immunofluorescence detection, RMRP downregulation significantly suppressed TACR1/Erk1/2 pathway, while miRNA-206 was significantly upregulated (P < .05). RMRP downregulation inhibits HCC-related biological activities by the regulation of miRNA-206/TACR1.