High expression of G-protein signaling modulator 2 in hepatocellular carcinoma facilitates tumor growth and metastasis by activating the PI3K/AKT signaling pathway.

The aim of this study was to investigate the role of G-protein signaling modulator 2 in the carcinogenesis and progression of hepatocellular carcinoma. We previously showed that G-protein signaling modulator 2 was upregulated in hepatitis B virus-related hepatocellular carcinoma tissues through a hierarchical clustering analysis. With this study, we first assessed the expression pattern of G-protein signaling modulator 2 in hepatocellular carcinoma specimens and adjacent noncancerous tissues; clinical data were analyzed, along survival times, utilizing the Kaplan-Meier method. Moreover, the functions of G-protein signaling modulator 2 were examined using small-interfering RNAs in vitro. The results showed that G-protein signaling modulator 2 was clearly overexpressed in hepatocellular carcinoma tissues and cell lines and that the G-protein signaling modulator 2 expression level was related to tumor size and hepatitis B virus infection. Furthermore, G-protein signaling modulator 2 knockdown studies suggested that G-protein signaling modulator 2 accelerates cell growth, cell cycle, migration, and invasion and inhibits apoptosis, acting as an oncogene in hepatocellular carcinoma. Western blotting indicated that silencing of G-protein signaling modulator 2 in HepG2 and SMMC-7721 cells increased the expression levels of Bax, caspase-3, and E-cadherin, while notably suppressing the cyclin-dependent kinase 4, cyclin-dependent kinase 6, CyclinD1, Snail1, Vimentin, and matrix metallopeptidase 9 expression levels, compared with that in the control groups. In addition, we found that G-protein signaling modulator 2 can affect the expression of key proteins involved in protein kinase B activation. In conclusion, high expression of G-protein signaling modulator 2 was involved in the pathological processes of hepatocellular carcinoma through activation of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, which may provide an attractive potential diagnostic biomarker and therapeutic target for treatment of hepatocellular carcinoma.

Effect of silencing NEK2 on biological behaviors of HepG2 in human hepatoma cells and MAPK signal pathway.

To investigate the expression level of NEK2 in 40 tissue specimens of primary liver cancer and to search for clues whether the effect of NEK2 depletion plays a role on biological behaviors of HepG2 cells and the relevant molecular mechanism are the objectives of this study. Real-time PCR and immunohistochemistry assessed expression level of NEK2 in specimens of cancerous tissues and carcinoma-adjacent tissues. The NEK2 expression level in HepG2, Huh7, SMMC, and 7402 cells was detected by real-time PCR and western blot to screen experimental cell line. To assess the expression levels of NEK2 mRNA and protein, an effective siRNA transfected into the HepG2 cells was designed. CCK8 and colony-forming assays were performed to verify short-term and long-term proliferative activities, respectively. Capacity of apoptosis and cell cycle changes were assessed by flow cytometry. Ability of transference and invasion was measured by Transwell Chambers. Western blot approach was used to determine the protein expression levels. There was significantly high expression level of NEK2 in cancerous tissues compared to adjacent tissues. The expression of NEK2 was higher in HepG2 cells than other cell lines. Real-time PCR and western blot shown there were obviously down-regulated NEK2 expression in the NEK2-siRNA group compared to control groups. The capacity of amplification and invasion was inhibited distinctly, and FCM revealed the apoptosis rate was increased and G1 phase was arrested in NEK2-siRNA group. Western blot indicated that low expression of NEK2 in HepG2 cells could increase the expression levels of Bax, caspase-3, P21, and TIMP-1, but significantly suppressed the c-myc, c-jun, Bcl-2, cyclinD1, CDK4, MMP2, and MMP9 expression levels and the phosphorylation levels of ERK, JNK, and P38 compared with the control groups. Our findings demonstrated that NEK2 could be a valuable carcinogenic factor and a promising therapeutic target for primary liver cancer; NEK2 may regulate proliferation, apoptosis, and other biological behaviors of HepG2 cells via MAPK signal pathway.

Inhibition of 12-lipoxygenase reduces proliferation and induces apoptosis of hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND: 12-lipoxygenase (12-LOX) has been reported to be an important gene in cancer cell proliferation and survival, and tumor metastasis. However, its role in hepatocellular carcinoma (HCC) cells remains unknown. METHODS: Expression of 12-LOX was assessed in a diethyl-nitrosamine-induced rat HCC model, and in SMMC-7721, HepG2 and L-02 cells using immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). GST-π and Ki-67 were determined in vivo by immunohistochemical staining. Apoptosis was evaluated by TUNEL assay. Cell viability and apoptosis were determined by MTT assay and flow cytometry, respectively. Apoptosis-related proteins in SMMC-7721 and HepG2 cells were detected by Western blotting. RESULTS: Immunohistochemical staining and RT-PCR showed that 12-LOX was over-expressed in rat HCC and two HCC cell lines, while the expression was inhibited by baicalein, a specific inhibitor of 12-LOX. Baicalein inhibited cell proliferation and induced apoptosis in rat HCC and both cell lines in a dose- and time-dependent manner. Our in vivo study demonstrated that baicalein also reduced neoplastic nodules. Mechanistically, baicalein reduced Bcl-2 protein expression coupled with a slight increase of the expression of Bax and activation of caspase-3. Furthermore, baicalein inhibited the activation of ERK-1/2 (phosphorylated). Interestingly, the effects of baicalein were reversed by 12(S)-HETE, a metabolite of 12-LOX. CONCLUSIONS: Inhibition of 12-LOX leads to reduced numbers of HCC cells, partially caused by increased apoptosis. 12-LOX may be a potential molecular target for HCC prevention and treatment.

MicroRNA-223-3p Protect Against Radiation-Induced Cardiac Toxicity by Alleviating Myocardial Oxidative Stress and Programmed Cell Death via Targeting the AMPK Pathway.

Objectives: Radiotherapy improves the survival rate of cancer patients, yet it also involves some inevitable complications. Radiation-induced heart disease (RIHD) is one of the most serious complications, especially the radiotherapy of thoracic tumors, which is characterized by cardiac oxidative stress disorder and programmed cell death. At present, there is no effective treatment strategy for RIHD; in addition, it cannot be reversed when it progresses. This study aims to explore the role and potential mechanism of microRNA-223-3p (miR-223-3p) in RIHD. Methods: Mice were injected with miR-223-3p mimic, inhibitor, or their respective controls in the tail vein and received a single dose of 20 Gy whole-heart irradiation (WHI) for 16 weeks after 3 days to construct a RIHD mouse model. To inhibit adenosine monophosphate activated protein kinase (AMPK) or phosphodiesterase 4D (PDE4D), compound C (CompC) and AAV9-shPDE4D were used. Results: WHI treatment significantly inhibited the expression of miR-223-3p in the hearts; furthermore, the levels of miR-223-3p decreased in a radiation time-dependent manner. miR-223-3p mimic significantly relieved, while miR-223-3p inhibitor aggravated apoptosis, oxidative damage, and cardiac dysfunction in RIHD mice. In addition, we found that miR-223-3p mimic improves WHI-induced myocardial injury by activating AMPK and that the inhibition of AMPK by CompC completely blocks these protective effects of miR-223-3p mimic. Further studies found that miR-223-3p lowers the protein levels of PDE4D and inhibiting PDE4D by AAV9-shPDE4D blocks the WHI-induced myocardial injury mediated by miR-223-3p inhibitor. Conclusion: miR-223-3p ameliorates WHI-induced RIHD through anti-oxidant and anti-programmed cell death mechanisms via activating AMPK by PDE4D regulation. miR-223-3p mimic exhibits potential value in the treatment of RIHD.

LncRNA LINC00460 promotes tumor growth of human lung adenocarcinoma by targeting miR-302c-5p/FOXA1 axis.

Accumulating evidence has shown that long non-coding RNAs (lncRNAs) had malfunctioning roles in the development of human cancers, especially lung adenocarcinoma (LC). In the present study, we aimed to investigate the role and potential mechanism of lncRNA long intergenic non-protein coding RNA 460 (LINC00460) in LC progression using human tissues and cell lines. We observed that LINC00460 was increased in lung adenocarcinoma tissues and cells in comparison to their corresponding controls. Moreover, overexpression of LINC00460 indicated the poor prognosis of lung adenocarcinoma patients. In addition, silencing LINC00460 was able to suppress lung adenocarcinoma cell growth in vitro and in vivo. Rescue assay confirmed that LINC00460 contributed to lung adenocarcinoma progression by regulating miR-302c-5p/FOXA1 signal pathway. In conclusion, LINC00460 promotes LC progression by competitively binding miR-302c-5p and regulating FOXA1 signal pathway. Our findings reveal that LINC00460 may be a potential prognostic biomarker and a candidate target for LC therapy.

Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells.

AIM: To investigate whether autophagic cell death is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells, and to explore the underlying mechanism. METHODS: Human hepatocellular carcinoma cells were treated with hyperthermia and ionizing radiation. MTT and clonogenic assays were performed to determine cell survival. Cell autophagy was detected using acridine orange staining and flow cytometric analysis, and the expression of autophagy-associated proteins, LC3 and p62, was determined by Western blot analysis. Intracellular reactive oxygen species (ROS) were quantified using the fluorescent probe DCFH-DA. RESULTS: Treatment with hyperthermia and ionizing radiation significantly decreased cell viability and surviving fraction as compared with hyperthermia or ionizing radiation alone. Cell autophagy was significantly increased after ionizing radiation combined with hyperthermia treatment, as evidenced by increased formation of acidic vesicular organelles, increased expression of LC3II and decreased expression of p62. Intracellular ROS were also increased after combined treatment with hyperthermia and ionizing radiation. Pretreatment with N-acetylcysteine, an ROS scavenger, markedly inhibited the cytotoxicity and cell autophagy induced by hyperthermia and ionizing radiation. CONCLUSION: Autophagic cell death is involved in hyperthermic sensitization of cancer cells to ionizing radiation, and its induction may be due to the increased intracellular ROS.

Twist mediates an aggressive phenotype in human colorectal cancer cells.

Epithelial-mesenchymal transition (EMT) is a crucial process providing cancer cells with the ability to migrate and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype and chemoresistance. Twist is a transcription factor that regulates EMT in a various cancer cells, including colorectal cancer (CRC). Our study was done to determine the role of Twist in mediating aggressive phenotype in CRC. Human CRC cell lines were transduced with a retroviral Twist construct or vector control. Migration and invasion abilities were determined in vitro using modified Boyden chamber assays. Mammosphere formation assay was performed to detect CSC characteristics. EMT and CSC markers were detected using western blotting and RT-PCR. Chemosensitivity to oxaliplatin of the transfected cells were determined by the MTT assay. Human CRC specimens were stained for Twist and P-gp expression. Twist overexpression triggered EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration, invasion and mammosphere formation abilities. In addition, Twist-overexpressing CRC cells were more chemo-resistant to oxaliplatin than control cells. Furthermore, Twist over-expression increased P-gp expression in CRC cells, which is a transmembrane glycoprotein conferred multidrug-resistance phenotype to various cancer cells. Importantly, Twist and P-gp were expressed correlatively in human CRC specimens. Thus, Twist is a potential therapeutic target in metastatic CRC.

Long noncoding RNAs in hepatitis B virus-related hepatocellular carcinoma.

AIM: To study the expression of long noncoding RNAs (lncRNAs) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: The lncRNA profiles between HBV-related HCC tissues and corresponding normal liver tissues were generated using microarray analysis. Datasets were analyzed using multiple algorithms to depict alterations in gene expression on the basis of gene ontology (GO), pathway analysis, and lncRNA levels. RESULTS: The microarray revealed that 1772 lncRNAs and 2508 mRNAs were differently expressed. The pathway analysis demonstrated that the cell cycle, cytokine-cytokine receptor interaction, chemokine signaling pathway, and phosphoinositide 3-kinase-protein kinase B signaling pathway may play important roles in HCC. Several GO terms, such as cell cycle, DNA replication, immune response, and signal transduction, were enriched in gene lists, suggesting a potential correlation with HBV-related HCC. The upregulated large intergenic noncoding RNA ULK4P2 was physically combined with enhancer of zeste homolog 2. Therefore, the lncRNAs may participate in regulating HBV-related HCC. CONCLUSION: lncRNAs play important roles in HCC, future studies should verify whether large intergenic noncoding ULK4P2 functions by combining with enhancer of zeste homolog 2 in HCC.

5-Lipoxygenase contributes to the progression of hepatocellular carcinoma.

5-Lipoxygenase (5-LOX) has been implicated in the development and progression of lung, pancreatic and esophageal cancers. However, its role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to explore the role of 5-LOX in the pathogenesis of HCC. The expression of 5-LOX was detected in human HCC, HepG2 cells and diethylnitrosamine (DEN)-induced rat HCC using immunohistochemistry (IHC) staining or reverse transcriptase-polymerase chain reaction. Apoptosis in rat HCC was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. Cell viability and apoptosis were determined in HepG2 cells by MTT assay and flow cytometry, respectively. IHC staining showed that the 5-LOX protein was highly expressed in human HCC, HepG2 cells and rat HCC, but not in the normal liver tissues. 5-LOX mRNA expression in human and rat HCC was also significantly increased compared to normal liver tissues. Zileuton, a 5-LOX inhibitor, reduced the nodule incidence and the mean number of nodules per nodule-bearing liver in DEN-induced rats. Further study using TUNEL assay showed that zileuton treatment induced apoptosis in the liver as the result of inhibition on 5-LOX levels. This result is consistent with our observation of significantly higher apoptotic indices in rats treated with DEN/zileuton, which were significantly higher compared to those from the control groups. In addition, zileuton reduced cell viability and induced apoptosis in a concentration- and time-dependent manner as detected using HepG2 cells in our in vitro analysis. In conclusion, 5-LOX is expressed in HCC, and the inhibition of 5-LOX blocks the development of HCC via the induction of apoptosis in tumor cells.

Hepatic oval cells activated by hepatocyte apoptosis in diethylnitrosamine-induced rat liver cirrhosis.

OBJECTIVE: To investigate whether hepatic oval cells are activated in diethylnitrosamine (DEN)-induced rat liver cirrhosis, and to explore its mechanism. METHODS: Liver cirrhosis was induced in rats (n=8) by weekly intraperitoneal injections of DEN at a dose of 50mg/kg body weight for 12 weeks followed by a 2-week wash out period. Rats (n=5) that received isovolumic vehicle served as the control group. Liver pathology was examined. Apoptotic hepatocytes were identified and quantified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. Oval cells were detected using immunohistochemical staining for pyruvate kinase type M2 (M2PK) and cytokeratin 19 (CK19). The work was carried out at Renmin Hospital of Wuhan University, Wuhan, Hubei, China from February to December 2009. RESULTS: Liver cirrhosis developed in rats subjected to DEN administration. The TUNEL and morphology assay showed that a substantial number of hepatocytes underwent apoptosis. The apoptotic index in rats subjected to DEN administration (0.75 0.15) was much higher than normal control rats (0.10 0.05). Both CK19 and M2PK were moderately expressed in the rat liver cirrhosis, and the expression was dispersed or forming small cords in the liver; but the expression was hardly detected in the liver tissue of normal control rats. CONCLUSION: In the DEN-induced rat liver cirrhosis, oval cells are activated and stimulated to proliferation, the mechanism of which may be related to substantial hepatocyte apoptosis in the model.