MiR-103 regulates hepatocellular carcinoma growth by targeting AKAP12

AKAP12/Gravin (A kinase anchor protein 12) belongs to the group of A-kinase scaffold proteins and functions as a tumor suppressor in some human primary cancers. While AKAP12 is found consistently downregulated in hepatocellular carcinoma (HCC), its involvement in hepatocarcinogenesis has not been fully elucidated. We identified targeting sites for miR-103 in the 3′-untranslated region (3′-UTR) of AKAP12 by bioinformatic analysis and confirm their function by a luciferase reporter gene assay. We reveal miR-103 expression to be inversely correlated with AKAP12 in HCC tissue samples and show that overexpressed miR-103 promotes cell proliferation and inhibits apoptosis by downregulating AKAP12 expression in HCC cell lines. On the other hand, repression of miR-103 suppresses proliferation and promotes apoptosis in HCC cells by increasing AKAP12. In xenografted HCC tumors, overexpression of AKAP12 suppresses tumor growth whereas overexpression of miR-103 enhances tumor growth while repressing AKAP12. Since the activation of telomerase is crucial for cells to gain immortality and proliferation ability, we investigated whether AKAP12 expression affected telomerase activity in HCC cells. Both AKAP12 overexpression and protein kinase Cα (PKCα) inhibition prevent nuclear translocation and phosphorylation of TERT and reduce telomerase activity in HCC cells. These findings indicate that miR-103 potentially acts as an oncogene in HCC by inhibiting AKAP12 expression and raise the possibility that miR-103 increases telomerase activity by increasing PKCα activity. Thus, miR-103 may represent a new potential diagnostic and therapeutic target for HCC treatment.     

Overexpressed homeobox B9 regulates oncogenic activities by transforming growth factor-β1 in gliomas

Glioma is the leading cause of deaths related to tumors in the central nervous system. The mechanisms of gliomagenesis remain elusive to date. Homeobox B9 (HOXB9) has a crucial function in the regulation of gene expression and cell survival, but its functions in glioma formation and development have yet to be elucidated. This study showed that HOXB9 expression in glioma tissues was significantly higher than that in nontumor tissues. Higher HOXB9 expression was also significantly associated with advanced clinical stage in glioma patients. HOXB9 overexpression stimulated the proliferation, migration, and sphere formation of glioma cells, whereas HOXB9 knockdown elicited an opposite effect. HOXB9 overexpression also increased the tumorigenicity of glioma cells in vivo. Moreover, the activation of transforming growth factor-β1 contributed to HOXB9-induced oncogenic activities. HOXB9 could be used as a predictable biomarker to be detected in different pathological and histological subtypes in glioma for diagnosis or prognosis.     

PSG9 promotes angiogenesis by stimulating VEGFA production and is associated with poor prognosis in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is a common malignant solid tumor characterized by rich vascularization. Pregnancy-specific glycoprotein 9(PSG9) is a member of the carcinoembryonic antigen(CEA)/PSG family and is produced at high levels during pregnancy. We previously identified PSG9 as an HCC-related protein. However, the expression of PSG9 and its regulation during HCC carcinogenesis remain poorly explored. In the present study, we first found that the levels of PSG9 protein were significantly increased in the plasma of HCC patients. PSG9 overexpression also increased the proliferation ability of an HCC cell line. High expression of PSG9 was associated with angiogenesis by accelerating VEGFA expression. In addition, Cox's proportional hazards model analysis revealed that the plasma level of PSG9 was an independent prognostic factor for overall survival. We propose that PSG9 is a novel indicator of prognosis in patients with HCC and could serve as a novel therapeutic target for HCC. Furthermore,our results indicate that PSG9 protein may facilitate the development of HCC by fostering angiogenesis via promoting VEGFA production in cancer cells.     

PNO1 regulates autophagy and apoptosis of hepatocellular carcinoma via the MAPK signaling pathway

Some studies have reported that activated ribosomes are positively associated with malignant tumors, especially in hepatocellular carcinoma (HCC). The RNA-binding protein PNO1 is a critical ribosome rarely reported in human tumors. This study aimed to explore the molecular mechanisms of PNO1 in HCC. Using 150 formalin-fixed and paraffin-embedded samples and 8 fresh samples, we found high PNO1 expression in HCC tumor tissues through Western blotting and RT-PCR. Moreover, the higher PNO1 expression was associated with poor HCC prognosis patients. In vitro and in vivo experiments indicated that PNO1 overexpression promoted the proliferation and depressed the apoptosis of HCC cells. High PNO1 expression also increased the autophagy of HCC cells. The molecular mechanisms underlying PNO1 were examined by RNA-seq analysis and a series of functional experiments. Results showed that PNO1 promoted HCC progression through the MAPK signaling pathway. Therefore, PNO1 was overexpressed in HCC, promoted autophagy, and inhibited the apoptosis of HCC cells through the MAPK signaling pathway.Subject terms: Cancer genomics, Cancer genomics     

Mutually exclusive antiproliferative effect of cell line-specific HOX inhibition in epithelial ovarian cancer cell lines: SKOV-3 vs RMUG-S

We aimed to discover cell line-specific overexpressed HOX genes responsible for chemoresistance and to identify the mechanisms behind HOX-induced cell line-specific chemoresistance in EOC. Ten HOX genes and eight EOC cell lines were tested for any cell line-specific overexpression that presents a mutually exclusive pattern. Cell viability was evaluated after treatment with cisplatin and/or siRNA for cell line-specific overexpressed HOX genes. Immunohistochemical (IHC) staining for HOXB9 was performed in 84 human EOC tissues. HOXA10 and HOXB9 were identified as cell line-specific overexpressed HOX genes for SKOV-3 and RMUG-S, respectively. Inhibiting the expression of cell line-specific HOX genes, but not of other HOX genes, significantly decreased cell viability. In SKOV-3 cells, cell viability decreased to 46.5% after initial 10 µM cisplatin treatment; however, there was no further decrease upon additional treatment with HOXA10 siRNA. In contrast, cell viability did not significantly decrease upon cisplatin treatment in RMUG-S cells, but decreased to 65.5% after additional treatment with HOXB9 siRNA. In both cell lines, inhibiting cell line-specific HOX expression enhanced apoptosis but suppressed the expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, MMP9, and Oct4. IHC analysis showed that platinum-resistant cancer tissues more frequently had high HOXB9 expression than platinum-sensitive cancer tissues. HOXB9, which is overexpressed in RMUG-S but not in SKOV-3 cells, appeared to be associated with cell line-specific platinum resistance in RMUG-S. Inhibiting HOXB9 overexpression in RMUG-S cells may effectively eliminate platinum-resistant ovarian cancer cells by facilitating apoptosis and inhibiting EMT.     

Downregulation of proapoptotic proteins Bax and Bcl-X(S) in p53 overexpressing hepatocellular carcinomas

As the occurrence of structural p53 mutations in hepatocellular carcinoma (HCC) in Thailand was previously reported to be much lower than that found in other high-incidence HCC areas, we analyzed 16 HCC samples from Thailand to determine the expression and functionality of p53 protein. We observed the overexpression of p53 protein in 69% of HCC, despite the prevalence of the wild-type p53 gene. However, the overexpressed p53 protein was nonfunctional as suggested by its inability to modulate the expressions of several p53 effector proteins (p21 and Bcl-2 family proteins). In addition, we observed significant underexpression of two proapoptotic proteins, Bax and Bcl-X(S), in 81% (P = 0.02) and 64% (P = 0.03) of HCC, respectively. Consequently, the ratios of proapoptotic to antiapoptotic BCL-2 family proteins were reduced in 88% of the HCC tumor tissues when compared to normal tissues, such that the rheostat between BCL-2 family proteins is strongly skewed toward enhanced cell survival in the tumor cells.     

Characterization of bridging integrator 1 (BIN1) as a potential tumor suppressor and prognostic marker in hepatocellular carcinoma

It has been shown that bridging integrator 1 (BIN1) can interact with c-myelocytomatosis (c-Myc) oncoprotein in cancer. However, the role of BIN1 in hepatocellular carcinoma (HCC) is not clear. In the present study, we investigated the expression and prognostic role of BIN1 in primary HCC and evaluated the function of BIN1 in hepatocarcinogenesis. Using real-time polymerase chain reaction and Western blot analysis, we found significantly decreased expression of BIN1 in primary HCC tumor tissues (n = 42) compared with adjacent normal tissues and in HCC cell lines. Immunohistochemistry analysis also found decreased BIN1 expression in HCC tumor tissues (n = 117). In clinicopathological analysis, loss of BIN1 expression correlated significantly (P < 0.05) with differentiation scores and tumor size. Importantly, decreased expression of BIN1 in tumors was found to be closely associated with a poor prognosis, and we conclude that BIN1 was an independent prognostic factor in a multivariate analysis. In mechanistic studies, restoring BIN1 expression in BIN1-null HCC cells significantly inhibited cell proliferation and colony formation and induced apoptosis of HCC cells. Furthermore, we found that BIN1 overexpression could significantly suppress the motility and invasion of HCC cells in vitro. Our results indicate that BIN1 may function as a potential tumor suppressor and serve as a novel prognostic marker in HCC patients. The BIN1 molecule might play an important role in tumor growth, cell motility and invasion. Modulation of BIN1 expression may lead to clinical applications of this critical molecule in the control of hepatocellular carcinoma as well as in early and effective diagnosis of this aggressive tumor.     

Cyclin-dependent kinase inhibitor 3 is overexpressed in hepatocellular carcinoma and promotes tumor cell proliferation

Cyclin-dependent kinase inhibitor 3 (CDKN3) belongs to the protein phosphatases family and has a dual function in cell cycling. The function of this gene has been studied in several kinds of cancers, but its role in human hepatocellular carcinoma (HCC) remains to be elucidated. In this study, we found that CDKN3 was frequently overexpressed in both HCC cell lines and clinical samples, and this overexpression was correlated with poor tumor differentiation and advanced tumor stage. Functional studies showed that overexpression of CDKN3 could promote cell proliferation by stimulating G1-S transition but has no impact on cell apoptosis and invasion. Microarray-based co-expression analysis identified a total of 61 genes co-expressed with CDKN3, with most of them involved in cell proliferation, and BIRC5 was located at the center of CDKN3 co-expression network. These results suggest that CDKN3 acts as an oncogene in human hepatocellular carcinoma and antagonism of CDKN3 may be of interest for the treatment of HCC.     

miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells

MicroRNAs (miRNAs) are small endogenous conserved RNAs regulating genes expression through base pairing with the 3′-untranslated region (3′-UTR) of target messenger RNAs. MiR-214-5p is a newly identified miRNA with its biological role largely unknown. In this study, we explored miR-214-5p expression status in 78 paired tumor and nontumor tissues obtained from patients with hepatocellular carcinoma (HCC) by RT-qPCR. The effects of miR-214-5p expression on HCC cell proliferation, cell cycle progression, and cell migration were measured by CCK-8 assay, flow cytometry, and wound-healing assay. A dual-luciferase activity assay was performed to identify whether KLF5 was a target of miR-214-5p. Kaplan-Meier curve and log-rank test were used to investigate the effects of miR-214-5p and KLF5 on overall survival and disease-free survival of patients with HCC. We found miR-214-5p expression was sharply reduced in HCC tissues and cell lines compared with the normal tissues and cell lines. Functional assay revealed that miR-214-5p overexpression could downregulate cell proliferation, cell migration, and arrested cell cycle at G0/G1 phase. Further, we validated Krüppel-like factor 5 (KLF5) as a direct target of miR-214-5p, and was upregulated in HCC and inversely correlated with the expression of miR-214-5p. Moreover, we found the low expression of miR-214-5p and high expression of KLF5 were correlated with tumor size, tumor stage, and poorer 5-year overall survival and disease-free survival of patients with HCC. In conclusion, our results suggested miR-214-5p functions as a tumor suppressor through targeting KLF5 in HCC. Also, miR-214-5p and KLF5 were identified as potential prognostic markers and might be therapeutic targets in HCC.     

miR-101 Inhibiting Cell Proliferation,Migration and Invasion in Hepatocellular Carcinoma through Downregulating Girdin

miR-101 is considered to play an important role in hepato-cellular carcinoma (HCC), but the underlying molecular mechanism remains to be elucidated. Here, we aimed to confirm whether Girdin is a target gene of miR-101 and determine the tumor suppressor of miR-101 through Girdin pathway. In our previous studies, we firstly found Girdin protein was overexpressed in HCC tissues, and it closely correlated to tumor size, T stage, TNM stage and Edmondson-Steiner stage of HCC patients. After specific small interfering RNA of Girdin was transfected into HepG2 and Huh7.5.1 cells, the proliferation and invasion ability of tumor cells were significantly inhibited. In this study, we further explored the detailed molecular mechanism of Girdin in HCC. Interestingly, we found that miR-101 significantly low-expressed in HCC tissues compared with that in matched normal tissues while Girdin had a relative higher expression, and miR-101 was inversely correlated with Girdin expression. In addition, after miR-101 transfection, the proliferation, migration and invasion abilities of HepG2 cells were weakened. Furthermore, we confirmed that Girdin is a direct target gene of miR-101. Finally we confirmed Talen-mediated Girdin knockout markedly suppressed cell proliferation, migration and invasion in HCC while down-regulation of miR-101 significantly restored the inhibitory effect. Our findings suggested that miR-101/Girdin axis could be a potential application of HCC treatment.     

VEGF is essential for the growth and migration of human hepatocellular carcinoma cells

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF induces new vessel formation and tumor growth by inducing mitogenesis and chemotaxis of normal endothelial cells and increasing vascular permeability. However, little is known about VEGF function in the proliferation, survival or migration of hepatocellular carcinoma cells (HCC). In the present study, we have found that VEGF receptors are expressed in HCC line BEL7402 and human HCC specimens. Importantly, VEGF receptor expression correlates with the development of the carcinoma. By using a comprehensive approaches including TUNEL assay, transwell and wound healing assays, migration and invasion assays, adhesion assay, western blot and quantitative RT-PCR, we have shown that knockdown of VEGF165 expression by shRNA inhibits the proliferation, migration, survival and adhesion ability of BEL7402. Knockdown of VEGF165 decreased the expression of NF-κB p65 and PKCα while increased the expression of p53 signaling molecules, suggesting that VEGF functions in HCC proliferation and migration are mediated by P65, PKCα and/or p53.     

Dyskerin overexpression in human hepatocellular carcinoma is associated with advanced clinical stage and poor patient prognosis

Background

Dyskerin (encoded by the DKC1 gene) is an essential nucleolar protein involved in cell proliferation, where it is required for the pseudo-uridylation of ribosomal RNA (rRNA) molecules and the stabilization of the telomerase RNA component. Dyskerin expression has been reported to predict poor survival in some cancer patients. The aim of the present study was to analyze the expression of dyskerin in hepatocellular carcinoma (HCC) and to determine its correlation with clinicopathologic features, including the survival of patients with HCC.

Methodology/Principal Findings

Dyskerin protein expression was detected by immunohistochemistry in paraffin sections of 252 HCC cases and 80 noncancerous liver tissues. The correlation was analyzed between dyskerin expression levels and clinicopathologic variables and prognosis. Dyskerin protein was significantly overexpressed in HCC tissues when compared to noncancerous liver tissue. Dyskerin overexpression was positively correlated with the hepatitis B surface antigen status, serum alpha-fetoprotein, and advanced clinical stage in HCC patients. A survival analysis indicated that HCC patients with higher dyskerin expression had a significantly shorter overall survival and 5-year survival time when compared to those with low expression. A multivariate analysis suggested that dyskerin overexpression was an independent factor for prognosis (hazard risk, 2.912; P = 0.007). Expression of DKC1 mRNA was measured by quantitative RT-PCR in 80 HCC and 50 non-cancerous tissues. The relationship between DKC1, TERT, MKI67, and MYC mRNA expression in HCC tissues was also evaluated. DKC1 mRNA was significantly overexpressed in HCC tissues and showed a significant correlation with MKI67 and MYC mRNA but a weak correlation with TERT mRNA.

Conclusions/Significance

Dyskerin overexpression in HCC patients was correlated with MYC and MKI67 expression and showed a possible involvement in the tumorigenic process. Dyskerin overexpression may be an unfavorable prognostic factor in patients with HCC.     

HOXB1 Is a Tumor Suppressor Gene Regulated by miR-3175 in Glioma

The HOXB1 gene plays a critical role as an oncogene in diverse tumors. However, the functional role of HOXB1 and the mechanism regulating HOXB1 expression in glioma are not fully understood. A preliminary bioinformatics analysis showed that HOXB1 is ectopically expressed in glioma, and that HOXB1 is a possible target of miR-3175. In this study, we investigated the function of HOXB1 and the relationship between HOXB1 and miR-3175 in glioma. We show that HOXB1 expression is significantly downregulated in glioma tissues and cell lines, and that its expression may be closely associated with the degree of malignancy. Reduced HOXB1 expression promoted the proliferation and invasion of glioma cells, and inhibited their apoptosis in vitro, and the downregulation of HOXB1 was also associated with worse survival in glioma patients. More importantly, HOXB1 was shown experimentally to be a direct target of miR-3175 in this study. The downregulated expression of miR-3175 inhibited cell proliferation and invasion, and promoted apoptosis in glioma. The oncogenicity induced by low HOXB1 expression was prevented by an miR-3175 inhibitor in glioma cells. Our results suggest that HOXB1 functions as a tumor suppressor, regulated by miR-3175 in glioma. These results clarify the pathogenesis of glioma and offer a potential target for its treatment.