Zujiang Yu

Role of MicroRNA-1 in Human Cancer and Its Therapeutic Potentials

While the mechanisms of human cancer development are not fully understood, evidence of microRNA (miRNA, miR) dysregulation has been reported in many human diseases, including cancer. miRs are small noncoding RNA molecules that regulate posttranscriptional gene expression by binding to complementary sequences in the specific region of gene mRNAs, resulting in downregulation of gene expression. Not only are certain miRs consistently dysregulated across many cancers, but they also play critical roles in many aspects of cell growth, proliferation, metastasis, apoptosis, and drug resistance. Recent studies from our group and others revealed that miR-1 is frequently downregulated in various types of cancer. Through targeting multiple oncogenes and oncogenic pathways, miR-1 has been demonstrated to be a tumor suppressor gene that represses cancer cell proliferation and metastasis and promotes apoptosis by ectopic expression. In this review, we highlight recent findings on the aberrant expression and functional significance of miR-1 in human cancers and emphasize its significant values for therapeutic potentials.

Induction of microRNA-9 mediates cytotoxicity of curcumin against SKOV3 ovarian cancer cells.

BACKGROUND Curcumin, a phenolic compound extracted from the rhizomes of Curcuma longa, has shown cytotoxic effects against a variety of cancers. The aim of this study was to identify potential microRNA (miRNA) mediators of the anticancer effects of curcumin in ovarian cancer cells. MATERIALS AND METHODS SKOV3 ovarian cancer cells were treated with curcumin (10-60 μM) and miR-9 expression, cell proliferation, and apoptosis were assessed. The effects of miR-9 depletion on curcumin-mediated growth suppression were also examined. Phosphorylation of Akt and forkhead box protein O1 (FOXO1) was measured in cells with miR-9 overexpression or curcumin treatment. RESULTS Curcumin caused a significant and dose-dependent increase of miR-9 expression in SKOV3 cells, while significantly impeding cell proliferation and stimulating apoptosis. Depletion of miR-9 significantly (p<0.05) attenuated the growth-suppressive effects of curcumin on SKOV3 cells, coupled with reduced percentages of apoptotic cells. In contrast, overexpression of miR-9 significantly enhanced the cleavage of caspase-3 and poly(ADP-ribose) polymerase and promoted apoptotic death in SKOV3 cells. Western blot analysis showed that both miR-9 overexpression and curcumin similarly caused a significant (p<0.05) decline in the phosphorylation of Akt and FOXO1, compared to untreated cells. CONCLUSIONS The present study provided evidence that curcumin exerts its cytotoxic effects against SKOV3 ovarian cancer cells largely through upregulation of miR-9 and subsequent modulation of Akt/FOXO1 axis. Further studies are needed to identify direct targets of miR-9 that mediate the anticancer effects of curcumin in ovarian cancer cells.

Overexpression of EZH2 is associated with the poor prognosis in osteosarcoma and function analysis indicates a therapeutic potential

Osteosarcoma is a primary malignant bone tumor that has a poor prognosis due to local recurrence, metastasis, and chemotherapy resistance. Therefore, there is an urgent need to develop novel potential therapeutic targets for osteosarcoma. Enhancer of zeste homologue 2 (EZH2) is a member of the polycomb group of proteins, which has important functions in epigenetic silencing and cell cycle regulation. Overexpression of EZH2 has been found in several malignancies, however, its expression and the role of EZH2 in osteosarcoma is largely unknown. In this study, we examined EZH2 expression by immunohistochemistry in a large series of osteosarcoma tissues in association with tumor characteristics and patient outcomes. EZH2 expression was also analyzed in a microarray dataset of osteosarcoma. Results showed that higher expression of EZH2 was significantly associated with more aggressive tumor behavior and poor patient outcomes of osteosarcoma. We subsequently investigated the functional and therapeutic relevance of EZH2 as a target in osteosarcoma. Immunohistochemical analysis indicated that EZH2 expression was significantly associated with more aggressive tumor behavior and poorer patient outcomes of osteosarcoma. EZH2 silencing by siRNA inhibited osteosarcoma cell growth, proliferation, migration, and invasion. Moreover, suppression of EZH2 attenuated cancer stem cell functions. Similar results were observed in osteosarcoma cells treated with EZH2 specific inhibitor 3-deazaneplanocin A (DZNep), which exhausted cellular levels of EZH2. These results suggest that EZH2 is critical for the growth and metastasis of osteosarcoma, and an epigenetic therapy that pharmacologically targets EZH2 via specific inhibitors may constitute a novel approach to the treatment of osteosarcoma.

Cyclin-dependent kinase 11p110 (CDK11p110) is crucial for human breast cancer cell proliferation and growth

Cyclin-dependent kinases (CDKs) play important roles in the development of many types of cancers by binding with their paired cyclins. However, the function of CDK11 larger protein isomer, CDK11p110, in the tumorigenesis of human breast cancer remains unclear. In the present study, we explored the effects and molecular mechanisms of CDK11p110 in the proliferation and growth of breast cancer cells by determining the expression of CDK11p110 in breast tumor tissues and examining the phenotypic changes of breast cancer cells after CDK11p110 knockdown. We found that CDK11p110 was highly expressed in breast tumor tissues and cell lines. Tissue microarray analysis showed that elevated CDK11p110 expression in breast cancer tissues significantly correlated with poor differentiation, and was also associated with advanced TNM stage and poor clinical prognosis for breast cancer patients. In vitro knockdown of CDK11p110 by siRNA significantly inhibited cell growth and migration, and dramatically induced apoptosis in breast cancer cells. Flow cytometry demonstrated that cells were markedly arrested in G1 phase of the cell cycle after CDK11p110 downregulation. These findings suggest that CDK11p110 is critical for the proliferation and growth of breast cancer cells, which highlights CDK11p110 may be a promising therapeutic target for the treatment of breast cancer.

Hyperammonaemia induces hepatic injury with alteration of gene expression profiles

Hyperammonaemia is a serious metabolic disorder commonly observed in patients with hepatic failure. However, it is unknown whether hyperammonaemia has a direct adverse effect on the hepatocytes and thereby serves as both a cause and effect of hepatic failure.

Ammonium chloride inhibits autophagy of hepatocellular carcinoma cells through SMAD2 signaling.

Autophagy is a cellular degradation process for the clearance of damaged or superfluous proteins and organelles, the recycling of which serves as an alternative energy source during periods of metabolic stress to maintain cell homeostasis and viability. The anti-necrotic function of autophagy is critical for tumorigenesis of many tumor cells, including hepatocellular carcinoma (HCC). However, the underlying mechanism is not clarified yet. Ammonium chloride (NH4Cl) is a well-known autophagy inhibitor, whereas its interaction with SMAD2 signaling pathway has not been reported previously. Here, we show that NH4Cl significantly inhibited rapamycin-induced autophagy in HCC cells through decreasing the levels of Beclin-1, autophagy-related protein 7 (ATG7), p62, and autophagosome marker LC3 and significantly decreased the level of phosphorylated SMAD2 in rapamycin-treated HCC cells. In order to find out whether NH4Cl may inhibit the autophagy in rapamycin-treated HCC cells through inhibition of SMAD2 signaling, we used transforming growth factor β1 (TGFβ1) to induce phosphorylation of SMAD2 in HCC cells. We found that induction of SMAD2 in HCC cells completely abolished the inhibitory effect of NH4Cl on rapamycin-induced autophagy in HCC cells, suggesting that NH4Cl inhibits autophagy of HCC cells through inhibiting SMAD2 signaling.

Ammonia-induced energy disorders interfere with bilirubin metabolism in hepatocytes.

Hyperammonemia and jaundice are the most common clinical symptoms of hepatic failure. Decreasing the level of ammonia in the blood is often accompanied by a reduction in bilirubin in patients with hepatic failure. Previous studies have shown that hyperammonemia can cause bilirubin metabolism disorders, however it is unclear exactly how hyperammonemia interferes with bilirubin metabolism in hepatocytes. The purpose of the current study was to determine the mechanism or mechanisms by which hyperammonemia interferes with bilirubin metabolism in hepatocytes. Cell viability and apoptosis were analyzed in primary hepatocytes that had been exposed to ammonium chloride. Mitochondrial morphology and permeability were observed and analyzed, intermediates of the tricarboxylic acid (TCA) cycle were determined and changes in the expression of enzymes related to bilirubin metabolism were analyzed after ammonia exposure. Hyperammonemia inhibited cell growth, induced apoptosis, damaged the mitochondria and hindered the TCA cycle in hepatocytes. This led to a reduction in energy synthesis, eventually affecting the expression of enzymes related to bilirubin metabolism, which then caused further problems with bilirubin metabolism. These effects were significant, but could be reversed with the addition of adenosine triphosphate (ATP). This study demonstrates that ammonia can cause problems with bilirubin metabolism by interfering with energy synthesis.

The influence of TLR4 agonist lipopolysaccharides on hepatocellular carcinoma cells and the feasibility of its application in treating liver cancer

Objective This study was designed to explore the influence of Toll-like receptor 4 (TLR4) agonist lipopolysaccharides (LPS) on liver cancer cell and the feasibility to perform liver cancer adjuvant therapy. Methods Human liver cancer cell lines HepG2, H7402, and PLC/PRF/5 were taken as models, and the expression of TLRs mRNA was detected by real time-polymerase chain reaction method semiquantitatively. WST-1 method was used to detect the influence of LPS on the proliferation ability of liver cancer cells; propidium iodide (PI) single staining and Annexin V/PI double staining were used to test the influence of LPS on the cell cycle and apoptosis, respectively, on human liver cancer cell line H7402. Fluorescent quantitative polymerase chain reaction and Western blot method were used to determine the change of expression of Cyclin D1. Results The results demonstrated that most TLRs were expressed in liver cancer cells; stimulating TLR4 by LPS could upregulate TLR4 mRNA and the protein level, activate NF-κB signaling pathway downstream of TLR4, and mediate the generation of inflammatory factors IL-6, IL-8, and TNF-α; LPS was found to be able to strengthen the proliferation ability of liver cancer cells, especially H7402 cells; the expression of Cyclin D1 rose and H7402 cells were promoted to transit from G1 stage to S stage under the stimulation of LPS, but cell apoptosis was not affected. It was also found that LPS was able to activate signal transducer and activator of transcription -3 (STAT3) signaling pathway in H7402 cells and meanwhile significantly increase the initiation activity of STAT3; proliferation promoting effect of LPS to liver cancer cells remarkably lowered once STAT3 was blocked or inhibited. Conclusion Thus, TLR4 agonist LPS is proved to be able to induce liver cancer cells to express inflammation factors and mediate liver cancer cell proliferation and generation of multidrug resistance by activating the cyclooxygenase-2/prostaglandin signal axis as well as the STAT3 pathway.

miR-935 Promotes Liver Cancer Cell Proliferation and Migration by Targeting SOX7.

Hepatocellular carcinoma (HCC) is the most common cancer in the world. MicroRNAs (miRNAs) are a type of small noncoding RNA that can regulate the expression of target genes under physiological and pathophysiological conditions. Aberrant expression of microRNA-935 (miR-935) has been reported in cancer studies. However, its expression and mechanism in HCC remain unclear. In our study, we found that miR-935 was upregulated in liver cancer tissues and cells. Overexpression of miR-935 in liver cells promoted cell proliferation, tumorigenesis, and cell cycle progression, whereas inhibition of miR-935 reduced cell proliferation, tumorigenicity, and cell cycle progression. These changes in the properties of HCC cells were associated with upregulation of two well-known cellular G1/S transitional regulators: cyclin D1 and c-Myc. Additionally, we identified SOX7 as a direct target of miR-935. Overexpression of miR-935 inhibited SOX7 expression but promoted the levels of c-Myc and cyclin D1, which promotes cell proliferation and tumorigenesis; knockdown of miR-935 increased SOX7 level and inhibited c-Myc and cyclin D1 expression, whereas SOX7 silencing could promote cell proliferation, cell motility, and invasiveness in vitro. Our findings suggest that miR-935 represents a biomarker and a potential new target in HCC progression by suppressing SOX7 expression.

Hepatic injury is associated with cell cycle arrest and apoptosis with alteration of cyclin A and D1 in ammonium chloride-induced hyperammonemic rats.

Hyperammonemia is considered to be central to the pathophysiology of hepatic encephalopathy in patients exhibiting hepatic failure (HF). It has previously been determined that hyperammonemia is a serious metabolic disorder commonly observed in patients with HF. However, it is unclear whether hyperammonemia has a direct adverse effect on hepatic cells or serves as a cause and effect of HF. The present study investigated whether hepatic injury is caused by hyperammonemia, and aimed to provide an insight into the causes and mechanisms of HF. Hyperammonemic rats were established via intragastric administration of ammonium chloride solution. Hepatic tissues were assessed using biochemistry, histology, immunohistochemistry, flow cytometry (FCM), semi-quantitative reverse transcription-polymerase chain reaction and western blot analysis. Hyperammonemic rats exhibited significantly increased levels of liver function markers, including alanine transaminase (P<0.01), aspartate aminotransferase (P<0.01), blood ammonia (P<0.01) and direct bilirubin (P<0.05), which indicated hepatic injury. A pathological assessment revealed mild hydropic degeneration, but no necrosis or inflammatory cell infiltration. However, terminal deoxynucleotidyl transferase dUTP nick end-labeling assays confirmed a significant increase in the rate of cellular apoptosis in hyperammonemic rat livers (P<0.01). FCM analysis revealed that there were significantly more cells in the S phase and fewer in the G2/M phase (P<0.01), and the expression levels of cyclin A and D1 mRNA and proteins were significantly increased (P<0.01). In summary, cell cycle arrest, apoptosis and an alteration of cyclin A and D1 levels were all markers of hyperammonemia-induced hepatic injury. These findings provide an insight into the potential mechanisms underlying hyperammonemia-induced hepatic injury, and may be used as potential targets for treating or preventing hepatic damage caused by hyperammonemia, including hepatic encephalopathy.