Kejun Nan

MicroRNA profiling of human gastric cancer

MicroRNAs are a group of small non-coding RNAs that modulate gene expression. The de-regulation of microRNA expression has been found in several types of cancer. To study the role of microRNAs in gastric cancer (GC), we analyzed the expression profile of 847 microRNAs in GC from Chinese patients. Total RNA was used for hybridization on the miRCURY LNA Array (v. 11.0), which contains probes specific for 847 human microRNAs. The results from the miRNA microarray analysis were validated by real-time RT-PCR. A total of 24 miRNAs with a more than 2-fold change were differentially expressed between normal gastric tissue and GC. Of these, 22 miRNAs (miR-223, miR-106b, miR-147, miR-34a, miR-130b*, miR-106a, miR-18a, miR-17, miR-98, miR-616*, miR-181a-2*, miR-185, miR-1259, miR-601, miR-196a*, miR-221*, miR-302f, miR-340*, miR-337-3p, miR-520c-3p, miR-575 and miR-138) were significantly up-regulated in GC (P<0.05), whereas only miR-638 and miR-378 were significantly down-regulated in GC (P<0.05) compared to normal gastric tissue. The expression of miR-185 and miR-638, as measured by miRNA microarray analysis, was in agreement with the expression level of these microRNAs found by real-time RT-PCR in the same samples. Our results show that microRNAs are de-regulated in GC, suggesting the involvement of these genes in the development and progression of gastric cancer.

Activation of PI3 kinase/Akt/HIF-1α pathway contributes to hypoxia-induced epithelial-mesenchymal transition and chemoresistance in hepatocellular carcinoma.

Hypoxia is known to promote malignant progression and to induce chemoresistance in cancer. However, the exact mechanisms driving hypoxia induced malignance remain elusive. We found that with exposure to hypoxic condition, hepatocellular carcinoma (HCC) cells experienced epithelial-mesenchymal transition (EMT), with increased cell migration and invasion, and exhibited high resistance to chemotherapy. We demonstrated that hypoxia-induced EMT and chemoresistance were accompanied by increased HIF-1α expression and activation of Akt. HIF-1α could be blocked by PI3K inhibitor LY294002, indicating HIF-1α activation was regulated by PI3K/Akt pathway. Furthermore, we showed that inhibition of PI3K/Akt and HIF-1α enhanced the therapeutic efficacy of hypoxic chemotherapy in the HCC xenograft model. Our findings indicate that the activation of PI3K/Akt/HIF-1α pathway plays a critical role in mediating hypoxia-induced EMT and drug resistance leading to unfavorable treatment outcome. Our study provides novel insights into the malignant progression triggered by hypoxic microenvironment in HCC cells.

SDF-1/CXCR4 promotes epithelial-mesenchymal transition and progression of colorectal cancer by activation of the Wnt/β-catenin signaling pathway.

Stromal cell-derived factor 1 (SDF-1) and its receptor, CXCR4, play an important role in angiogenesis and are associated with tumor progression. This study aimed to investigate the role of SDF-1/CXCR4-mediated epithelial-mesenchymal transition (EMT) and the progression of colorectal cancer (CRC) as well as the underlying mechanisms. The data showed that expression of CXCR4 and β-catenin mRNA and protein was significantly higher in CRC tissues than in distant normal tissues. CXCR4 expression was associated with β-catenin expression in CRC tissues, whereas high CXCR4 expression was strongly associated with low E-cadherin, high N-cadherin, and high vimentin expression, suggesting a cross talk between the SDF-1/CXCR4 axis and Wnt/β-catenin signaling pathway in CRC. In vitro, SDF-1 induced CXCR4-positive colorectal cancer cell invasion and EMT by activation of the Wnt/β-catenin signaling pathway. In contrast, SDF-1/CXCR4 axis activation-induced colorectal cancer invasion and EMT was effectively inhibited by the Wnt signaling pathway inhibitor Dickkopf-1. In conclusion, CXCR4-promoted CRC progression and EMT were regulated by the Wnt/β-catenin signaling pathway. Thus, targeting of the SDF-1/CXCR4 axis could have clinical applications in suppressing CRC progression.

PTEN regulates angiogenesis and VEGF expression through phosphatase-dependent and -independent mechanisms in HepG2 cells

Hepatocellular carcinoma (HCC) is a typical hypervascular tumor, and increased levels of vascular endothelial growth factor (VEGF) are associated with progression of HCC. Tumor suppression gene PTEN (phosphatase and tensin homolog deleted on chromosome 10), an important antagonist of the phosphoinositide-3-kinase (PI3K)/adenosine triphosphate-dependent tyrosine kinase (Akt) pathway, is also commonly lost or mutated in HCC. However, the effect of PTEN on VEGF-mediated angiogenesis in HCC remains unknown. To explore this relationship, we expressed a panel of PTEN mutants in human HCC cells with low expression of PTEN (HepG2 cells). Overexpression of PTEN in HepG2 cells resulted in the downregulation of proliferation and migration of cocultured endothelial cells and decreased expression of hypoxia-inducible factor 1 (HIF-1) and VEGF. Similarly, using a nude mouse model, we demonstrated that PTEN decreased expression of HIF-1 and VEGF and suppressed HepG2-induced angiogenesis. This inhibitory effect was not observed in cells expressing a phosphatase-deficient PTEN mutant, suggesting that PTEN inhibits angiogenesis and VEGF through a phosphatase-dependent pathway. Strikingly, reintroducing the C2 domain of PTEN also resulted in a significant decrease in angiogenesis and VEGF expression, although it did not affect Akt phosphorylation or HIF-1 expression. In summary, this study suggests the novel viewpoint that PTEN suppresses angiogenesis and VEGF expression in HCC through both phosphatase-dependent and -independent mechanisms.

Over-expression of CircRNA_100876 in non-small cell lung cancer and its prognostic value

Circular RNAs (circRNAs) are a novel type of endogenous RNAs featuring stable structure and high tissue-specific expression. Recently, accumulating evidence has revealed that aberrant circRNAs expression plays important roles in carcinogenesis and tumor progression. However, the expression pattern and biological function of circRNAs in non small cell lung cancer (NSCLC) remain largely unknown. Therefore, the purpose of this study was to clarify the possible role of one of typical circRNAs, circRNA_100876 in NSCLC and to define its prognostic value in NSCLC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circRNA_100876 in tumor tissues and their adjacent nontumorous tissues in 101 patients with NSCLC. We found that the expression level of circRNA_100876 was significantly elevated in NSCLC tissues when compared with their adjacent nontumorous tissues (P=0.000). Moreover, there was a close correlation between the circRNA_100876 up-regulation expression and lymph node metastasis (P=0.001) and tumor staging (P=0.001) in NSCLC. In addition, Kaplan-Meier survival analysis demonstrated that the overall survival time of NSCLC patients with high circRNA_100876 expression was significantly shorter than those patients with low circRNA_100876 expression (P=0.000). In conclusion, our findings indicate that circRNA_100876 is closely related to the carcinogenesis of NSCLC and it might be served as a potential prognostic biomarker and therapeutic target for NSCLC.

Oxidative stress in depressive patients with gastric adenocarcinoma

Recent data from several studies suggest that oxidative stress is involved in the biochemical mechanisms that underlie neuropsychiatric disorders. The present study was designed to investigate oxidative stress status in depressive patients with gastric adenocarcinoma (GA) at TNM stage III. Oxidative stress, depression and expression of specific genes were monitored during a pretreatment period. Serum total antioxidant capacity, catalase, superoxide dismutase concentrations, and antisuperoxide anion capacity (A-ASC) were significantly decreased in depressive patients compared to control subjects, whereas serum malondialdehyde (MDA) levels were significantly increased. Importantly, the formation of 8-hydroxy-deoxyguanosine (8-OHdG) accumulated. Furthermore, SYBR Green real-time PCR revealed that the expression levels of human oxoguanine glycosylase 1 and APEX nuclease 1 (APEX1) were increased in depressive patients. Pearson correlation analysis revealed that depression was positively correlated with SAS, SCL-90, MDA, 8-OHdG and APEX1, but negatively correlated with A-ASC. Thus, this study confirms oxidative imbalance in depressive patients with GA, and oxidative stress may play a role in the onset and exacerbation of depression.

Oxidative stress upregulates PDCD4 expression in patients with gastric cancer via miR-21

Reactive oxygen species (ROS) plays a key role in carcinogenesis by aberrantly inducing signaling networks that initiatiate tumorigenesis and stimulate tumor progression. MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate approximately 30% of the genes in a cell via degradation or translational inhibition of their target mRNAs. However, the effects of ROS on miRNAs expression and the role of miRNAs in ROS-mediated injury on carcinogenesis are uncertain. Using UV spectrophotometry and enzyme-linked immunosorbent assay (ELISA), we examined tissues from human gastric cancers and tissues adjascent to gastric cancer and normal gastric tissues and found that total anti-oxidation competence (T-AOC), superoxide dismutase (SOD) and catalase (CAT) concentrations were lower in gastric cancer patients compared to the control subjects, while the concentrations of DNA oxidative damage product 8-oxo-deoxyguanosine (8-OHdG) was higher. To determine the potential role of miRNA in gastric carcinogenesis, real-time quantitative polymerase chain reaction (QPCR) analysis was performed. We found that human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) mRNA and miR-21 expression were significantly upregulated in gastric cancer tissues than in the adjacent normal gastric tissues. Furthermore, the expression of programmed cell death 4 protein (PDCD4) in gastric cancer tissues was significantly lower than in adjacent normal gastric tissues. The expression of miR-21 and PDCD4 was highly correlated with the degree of differentiation, tumor staging, local lymphatic node metastasis and remote metastasis. Expression of miR-21 was negatively correlated with T-AOC, SOD and CAT, but positively correlated with 8-OHdG and hOGG1mRNA. In addition, the relative expression of PDCD4 was negatively correlated with miR-21. These results suggest that the defensive balance of oxidation and antioxidant system in patients with GC was impaired, resulting in enhanced oxidative tissue injury, which may directly contribute to gastric carcinogenesis. Thus we conclude that ROS promotes gastric carcinogenesis via upregulating miR-21 expression which in turn down-regulates the expression of PDCD4 in gastric cancer cells.

New insights into metronomic chemotherapy-induced immunoregulation

Chemotherapy is the mainstay of treatment in mid-advanced tumors. Considering their toxicity on hematopoietic cells, chemotherapeutics are often considered as immunosuppressive inducers. However, recently accumulating data indicate that some chemotherapeutic agents with specific administration schedules also display some positive immunological effect to contribute to tumor elimination. For instance, metronomic chemotherapy could promote tumor eradication not only by inhibiting tumor angiogenesis but also by selectively eliminating immunosuppressive cells and improving anti-tumor immune responses. In this review, we summarize what is currently known regarding metronomic chemotherapy-induced immunoregulation. Understanding of the molecular mechanisms of metronomic chemotherapy-induced immunoregulation may yield invaluable information for the optimal design of immunochemotherapies.

Autophagy facilitates lung adenocarcinoma resistance to cisplatin treatment by activation of AMPK/mTOR signaling pathway

Resistance to cisplatin-based therapy is a major challenge in the control of lung cancer progression. However, the underlying mechanisms remain largely unclear. Autophagy is closely associated with resistance to lung cancer therapy, but the function of autophagy in cisplatin treatment is still controversial. Here, we investigated whether autophagy was involved in lung adenocarcinoma resistance to cisplatin and further elucidated the underlying molecular mechanisms. Cisplatin-refractory lung adenocarcinoma cells increased autophagic vacuole formation detected by monodansylcadaverine staining. When exposed to cisplatin, lung adeno-carcinoma cells demonstrated increased levels of autophagy detected by MAP1A/1B LC3B and mammalian homologue of yeast Atg6 (Beclin-1) expression using Western blot analysis. Activation of cisplatin-induced autophagic flux was increased by using chloroquine (CQ), which can accumulate LC3B-II protein and increase punctate distribution of LC3B localization. The combination of cisplatin with CQ was more potent than cisplatin alone in inhibiting lung adenocarcinoma cell growth, which also increased cisplatin-induced apoptosis. Compared to cisplatin treatment alone, the combination of cisplatin and CQ decreased p-AMPK and increased p-mTOR protein expressions, in addition, the AMPK inhibitor Compound C plus cisplatin downregulated p-AMPK and upregulated p-mTOR as well as depressed LC3B cleavage. These findings demonstrate that activation of autophagy is a hallmark of cisplatin exposure in human lung adenocarcinoma cells, and that there is a cisplatin-induced autophagic response via activation of the AMPK/mTOR signaling pathway. We speculate that autophagy can be used as a novel therapeutic target to overcome cisplatin-resistant lung adenocarcinoma.

miRNA-302b Suppresses Human Hepatocellular Carcinoma by Targeting AKT2

miRNAs (miR) play a critical role in human cancers, including hepatocellular carcinoma. Although miR-302b has been suggested to function as a tumor repressor in other cancers, its role in hepatocellular carcinoma is unknown. This study investigated the expression and functional role of miR-302b in human hepatocellular carcinoma. The expression level of miR-302b is dramatically decreased in clinical hepatocellular carcinoma specimens, as compared with their respective nonneoplastic counterparts, and in hepatocellular carcinoma cell lines. Overexpression of miR-302b suppressed hepatocellular carcinoma cell proliferation and G1–S transition in vitro, whereas inhibition of miR-302b promoted hepatocellular carcinoma cell proliferation and G1–S transition. Using a luciferase reporter assay, AKT2 was determined to be a direct target of miR-302b. Subsequent investigation revealed that miR-302b expression was inversely correlated with AKT2 expression in hepatocellular carcinoma tissue samples. Importantly, silencing AKT2 recapitulated the cellular and molecular effects seen upon miR-302b overexpression, which included inhibiting hepatocellular carcinoma cell proliferation, suppressing G1 regulators (Cyclin A, Cyclin D1, CDK2) and increasing p27Kip1 phosphorylation at Ser10. Restoration of AKT2 counteracted the effects of miR-302b expression. Moreover, miR-302b was able to repress tumor growth of hepatocellular carcinoma cells in vivo. Implications: Taken together, miR-302b inhibits HCC cell proliferation and growth in vitro and in vivo by targeting AKT2. Mol Cancer Res; 12(2); 190–202. ©2013 AACR.