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.     

MiR-1204 promotes ovarian squamous cell carcinoma growth by increasing glucose uptake

MiR-1204 has been recently identified as an oncogenic miRNA in breast cancer. Our study aims to investigate the role of miR-1204 in ovarian squamous cell carcinoma. Expression of miR-1204 and glucose transporter 1 in ovarian biopsies and plasma of both OC patients and healthy controls was detected by qRT-PCR. Correlations between patients’ clinicopathological data were analyzed by Chi-square test. MiR-1204 overexpression OC cell lines were established. Expression of GLUT-1 protein was detected by western blot. Glucose uptake was measured by glucose uptake assay. Cell proliferation was detected by CCK-8 assay. We found that miR-1204 expression was significantly correlated with tumor size. Expression levels of miR-1204 and GLUT-1 were significantly high in OC patients. Expression levels of miR-1204 were positively correlated with expression levels of GLUT-1 in OC patients. MiR-1204 overexpression significantly promoted GLUT-1 expression, glucose uptake and cell proliferation. MiR-1204 may promote ovarian squamous cell carcinoma growth by increasing glucose uptake.     

过表达miR-103促进猪前体脂肪细胞分化

为阐明miR-103在猪前体脂肪细胞分化过程中的调控作用,采用Real-time PCR检测猪前体脂肪细胞成脂分化过程中的miR-103表达谱,明确了其在分化过程中的表达趋势;使用miR-103的腺病毒超表达载体感染猪原代脂肪细胞,随后采用Real-time PCR和Western blotting分别检测成脂标记基因PPARγ、aP2的mRNA和蛋白表达量变化;油红O染色观察腺病毒miR-103侵染的前体脂肪细胞诱导分化第8天的成脂情况。结果显示,miR-103的表达量随着脂肪细胞分化而增加,在miR-103超表达的猪原代脂肪细胞的诱导分化过程中,成脂标记基因PPARγ、aP2的表达量与对照相比显著升高,分化第8天观察到明显的脂滴。说明miR-103能够促进猪前体脂肪细胞分化。     

LOXL2 promotes vasculogenic mimicry and tumour aggressiveness in hepatocellular carcinoma

Lysyl oxidase‐like 2 (LOXL2) has shown to promote metastasis and poor prognosis in hepatocellular carcinoma (HCC). Also, we have previously reported that vasculogenic mimicry (VM) is associated with invasion, metastasis and poor survival in HCC patients. In the present study, we investigated molecular function of LOXL2 in HCC and VM. We used the immunohistochemical and CD31/periodic acid‐Schiff double staining to detect the relationship between LOXL2 and VM formation. We performed the gain and loss of function studies and analysed the migratory, invasion and tube formation in HCC cell lines. We analysed the function of LOXL2 in VM formation and HCC metastasis both in vitro and in vivo. We have showed that LOXL2 was overexpression in HCC and was positively correlated with tumour grade, metastasis, VM formation and poor survival in 201 HCC patients. Secondly, our studies have showed that LOXL2 overexpression in HCC cells significantly promoted migration, invasion and tube formation. Finally, we found that LOXL2 may increase SNAIL expression, thereby enabling VM. Our study indicated that LOXL2 may promote VM formation and tumour metastasis by collaborating with SNAIL in HCC. What's more, the overexpression of LOXL2 indicated a poor prognosis in HCC patients.     

BRE over-expression promotes growth of hepatocellular carcinoma

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8 months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.     

NEDD4 promotes cell growth and motility in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death worldwide. In China, the situation is even worse as cancer incidence and mortality continue to increase rapidly. Although tremendous progress has been made toward HCC treatments, the benefits for liver cancer patients are still limited. Therefore, it is necessary to identify and develop novel therapeutic methods. Neuronally expressed developmentally downregulated 4 (NEDD4), an E3 ubiquitin ligase, plays a critical role in the development and progression of various types of human cancers. In our study, NEDD4 acts as an oncoprotein in both QGY7703 and SMMC7721 liver cancer cell lines. We found that depletion of NEDD4 by siRNA transfection led to inhibition of cell growth, invasion and migration, and promotion of apoptosis. In contrast, overexpression of NEDD4 via plasmid transfection resulted in facilitated cell proliferation, invasion and migration, and decreased apoptosis. Importantly, we observed that tumor suppressor LATS1, also a core component of Hippo pathway, was negatively regulated by NEDD4 in liver cancer cells. Our findings suggested that NEDD4 may be involved in the HCC progression via regulating LATS1 associated signaling pathway. Therefore, targeting NEDD4-LATS1 signaling could be a potential therapeutic option for HCC treatment.     

Hepatoma‐derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells

We aimed to elucidate the effects of hepatoma‐derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real‐time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP‐2 and MMP‐9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF‐siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF‐siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

Aberrant porphyrin metabolism in hepatocellular carcinoma

In 15 patients with hepatocellular carcinoma (HCC) and 14 patients with liver cirrhosis (LC), urinary excretions of delta-aminolevulinic acid (ALA), porphobilinogen (PBG), uroporphyrin (UP), coproporphyrin (CP), and erythrocyte contents of CP and protoporphyrin (PP) were examined. In patients with HCC, urinary excretions of ALA and PBG and erythrocyte contents of CP and PP were not increased, but urinary excretions of UP and CP were significantly increased more than those of LC patients. Urinary excretions of UP and CP had no correlations with liver function tests and excretion of UP correlated slightly with blood hemoglobin level. After administration of ALA intravenously, urinary excretions of UP and CP were clearly increased in patients with HCC compared to normal controls. A Red fluorescent area was present at the cancerous area but not in the noncancerous cirrhotic area in a patient with HCC. These results suggest that aberrant porphyrin metabolism occurred in patients with HCC compared to other liver diseases.     

Thymidine phosphorylase promotes angiogenesis and tumour growth in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer, and thymidine phosphorylase (TP) is a regulator of angiogenesis. To investigate the biological activities of TP in ICC, we established human cholangiocarcinoma RBE cell lines overexpressing TP or silencing TP. Overexpression of TP enhanced viability, suppressed apoptosis and increased tube formation in human umbilical vein endothelial cells, while downregulation of TP reversed these effects. Moreover, an orthotopic xenograft mouse model of ICC was built to further explore TP's function in ICC in vivo. Histological analysis using H&E, TUNEL and Ki67 staining showed that TP promoted tumour growth and inhibited cell apoptosis. Immunostaining for CD31 revealed an elevation in microvessel density in the presence of TP. Besides, upregulation of TP increased the expression of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8 and tumour necrosis factor alpha. In contrast, TP knockdown inhibited tumour growth, suppressed microvessel formation and decreased the expression of angiogenesis-related proteins. Therefore, we suggest that TP promotes angiogenesis and tumour growth in ICC, which can be a potent therapeutic target for ICC treatment.     

RAD001 (everolimus) inhibits tumour growth in xenograft models of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and highly resistant to available chemotherapies. Mammalian target of rapamycin (mTOR) functions to regulate protein translation, angiogenesis and cell cycle progression in many cancers including HCC. In the present study, subcutaneous patient-derived HCC xenografts were used to study the effects of an mTOR inhibitor, RAD001 (everolimus), on tumour growth, apoptosis and angiogenesis. We report that oral administration of RAD001 to mice bearing patient-derived HCC xenografts resulted in a dose-dependent inhibition of tumour growth. RAD001-induced growth suppression was associated with inactivation of downstream targets of mTOR, reduction in VEGF expression and microvessel density, inhibition of cell proliferation, up-regulation of p27^Kip1 and down-regulation of p21^Cip1/Waf1, Cdk-6, Cdk-2, Cdk-4, cdc-25C, cyclin B1 and c-Myc. Our data indicate that the mTOR pathway plays an important role in angiogenesis, cell cycle progression and proliferation of liver cancer cells. Our study provides a strong rationale for clinical investigation of mTOR inhibitor RAD001 in patients with HCC.     

Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis

Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor-C (VEGF-C) is a recently described lymphangiogenic factor. Increased expression of VEGF-C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF-C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF-C expression, driven by the rat insulin promoter (Rip), is targeted to beta-cells of the endocrine pancreas. In contrast to wild-type mice, which lack peri-insular lymphatics, RipVEGF-C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic beta-cell tumours that are neither lymphangiogenic nor metastatic. Double-transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of beta-cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.     

CMTM5 is downregulated and suppresses tumour growth in hepatocellular carcinoma through regulating PI3K-AKT signalling

Background

Human chemokine like factor (CKLF)-like MAL and related proteins for vesicle trafficking transmembrane, domain-containing member 5 (CMTM5) has been shown to involved and may function as a tumour suppressor in tumorigenesis. The current study aimed to investigate the expression and function of CMTM5 in human hepatocellular carcinoma (HCC).

Methods

CMTM5 expression was examined by immunohistochemistry, and its clinical significance was analysed in 76 HCC specimens. The role and molecular mechanisms of CMTM5 in cell proliferation, apoptosis and invasion were examined in vitro and in vivo.

Results

CMTM5 expression was significantly downregulated in HCC tissues as well as cell lines. The expression of CMTM5 was absent in 77.6% of HCC tissues compared with 3.9% in normal liver tissues. Low CMTM5 expression was significantly correlated with poor overall survival in patients with HCC (P = 0.009). Restoring CMTM5 expression in Huh7 cells significantly inhibited cell growth, promoted cell apoptosis, and reduced cell metastatic and invasion ability compared with mock transfected cells in vitro. Overexpression of CMTM5 also suppressed xenograft tumour growth in vivo in a HCC xenograft model. Reduced cell growth and metastasis ability mediated by CMTM5 overexpression was associated with downregulation of PI3K/AKT and its downstream Bcl2, cyclinD1, cyclinE, MMP2 and MMP9 expressions, and an upregulation of p21, Bax, Bad, cleaved caspase3 expressions.

Conclusions

Our data suggest that CMTM5 might function as a tumour suppressor in human HCC, and represent a valuable potential therapeutic target for HCC.

     

MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma

In our in-depth analysis carried out by the Illumina Solexa massive parallel signature sequencing, microRNA-99a (miR-99a) was found to be the sixth abundant microRNA in the miRNome of normal human liver but was markedly down-regulated in hepatocellular carcinoma (HCC). Compelling evidence has suggested the important roles of microRNAs in HCC development. However, the biological function of miR-99a deregulation in HCC remains unknown. In this study, we found that miR-99a was remarkably decreased in HCC tissues and cell lines. Importantly, lower miR-99a expression in HCC tissues significantly correlated with shorter survival of HCC patients, and miR-99a was identified to be an independent predictor for the prognosis of HCC patients. Furthermore, restoration of miR-99a dramatically suppressed HCC cell growth in vitro by inducing the G(1) phase cell cycle arrest. Intratumoral injection of cholesterol-conjugated miR-99a mimics significantly inhibited tumor growth and reduced the α-fetoprotein level in HCC-bearing nude mice. Insulin-like growth factor 1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) were further characterized as the direct targets of miR-99a. Furthermore, protein levels of IGF-1R and mTOR were found to be inversely correlated with miR-99a expression in HCC tissues. miR-99a mimics inhibited IGF-1R and mTOR pathways and subsequently suppressed expression of cell cycle-related proteins, including cyclin D1 in HCC cells. Conclusively, miR-99a expression was frequently down-regulated in HCC tissues and correlates with the prognosis of HCC patients, thus proposing miR-99a as a prospective prognosis predictor of HCC. miR-99a suppresses HCC growth by inducing cell cycle arrest, suggesting miR-99a as potential tumor suppressor for HCC therapeutics.     

Upregulation of cystatin SN promotes hepatocellular carcinoma progression and predicts a poor prognosis

Cystatin SN, a specific cysteine protease inhibitor, is thought to be involved in various malignant tumors. Therefore, we evaluated the role of cystatin SN in hepatocellular carcinoma (HCC). Notably, cystatin SN was elevated in tumorous samples and cells. Moreover, overexpression of cystatin SN was correlated with tumor diameter and TNM stage. Cox multivariate analysis displayed that cystatin SN was an independent prognosis indicator and that high cystatin SN level was associated with a dismal prognosis. Moreover, cystatin SN enhancement facilitated the proliferation, migratory, and invasive potential of Huh7 and HCCLM3 cells, whereas cystatin SN knockdown caused the opposite effect. Cystatin SN also modulated the epithelial-mesenchymal transition progression through the PI3K/AKT pathway. In vivo cystatin SN promoted HCCLM3 cell growth and metastasis in xenograft mice model. Thus, cystatin SN was involved in HCC progression and could be a latent target for HCC treatment.     

Down-regulation of SLC25A20 promotes hepatocellular carcinoma growth and metastasis through suppression of fatty-acid oxidation

Solute carrier family 25 member 20 (SLC25A20) is a mitochondrial-membrane–carrier protein involved in the transport of acylcarnitines into mitochondrial matrix for oxidation. A previous-integrated-proteogenomic study had identified SLC25A20 as one of the top-three prognostic biomarkers in HCC. However, the expression and the biological function of SLC25A20 have not yet been investigated in HCC. In the present study, we found that SLC25A20 expression is frequently down-regulated in HCC cells mainly due to the up-regulation of miR-132-3p. Down-regulation of SLC25A20 is associated with a poor prognosis in patients with HCC. SLC25A20 suppressed HCC growth and metastasis, both in vitro and in vivo, by suppression of G1–S cell transition, epithelial-to-mesenchymal transition (EMT), and induction of cell apoptosis. Mechanistically, SLC25A20 down-regulation promoted HCC growth and metastasis through suppression of fatty-acid oxidation. Altogether, SLC25A20 plays a critical tumor-suppressive role in carcinogenesis of HCC; SLC25A20 may serve as a novel prognostic factor and therapeutic target for patients with HCC.Subject terms: Liver cancer, Liver cancer     

Dependence on glucose limitation of the pCO2 influences on CHO cell growth, metabolism and IgG production

The culture levels of glucose and CO(2) have been reported to independently have important influences on mammalian cell processes. In this work the combined effects of glucose limitation and CO(2) partial pressure (pCO(2)) on monoclonal antibody (IgG) producing Chinese Hamster Ovary cells were investigated in a perfusion reactor operated with controlled cell specific medium feed rate, pH and osmolality. Under high glucose conditions (14.3 +/- 0.8 mM), the apparent growth rate decreased (from 0.021 to 0.009 h(-1)) as the pCO(2) increased to approximately 220 mmHg, while the cell specific IgG productivity was almost unchanged. The lactate yield from glucose was not affected by pCO(2) up to approximately 220 mmHg and glucose was mainly converted to lactate. A feed medium modification from high (33 mM) to low (6 mM) glucose resulted in <0.1 mM glucose in the culture. As a result of apparently shifting metabolism towards the conversion of pyruvate to CO(2), both the ratio of lactate to glucose and the alanine production rate were lowered (1.51-1.14 and 17.7-0.56 nmol/10(6) cells h, respectively). Interestingly, when the pCO(2) was increased to approximately 140 mmHg, limiting glucose resulted in 1.7-fold higher growth rates, compared to high glucose conditions. However, at approximately 220 mmHg pCO(2) this beneficial effect of glucose limitation on these CHO cells was lost as the growth rate dropped dramatically to 0.008 h(-1) and the IgG productivity was lowered by 15% (P < 0.01) relative to the high glucose condition. The IgG galactosylation increased under glucose- limited compared to high-glucose conditions.