Shanhong Tang

Ectopic expression of MiR-125a inhibits the proliferation and metastasis of hepatocellular carcinoma by targeting MMP11 and VEGF.

Background Studies have been shown that miR-125a plays an important role in carcinogenesis, however, the role of miR-125a in hepatocellular carcinoma (HCC) remains elusive. Methodology/Principal Real time-PCR (qRT-PCR) was performed to test the significance of miR-125a in HCC. Ectopic expression of miR-125a was used to test the influences of miR-125a on proliferation and metastasis of HCC cells in vitro and in vivo. Predicted target genes of miR-125a were determined by dual-luciferase reporting, qRT-PCR, and western blot (WB) analyses. Then immunohistochemical staining (IHC) was used to detect the expression of target genes, and the correlations and prognostic values of miR-125a and its target genes were also investigated. Conclusions/Significance Decreased miR-125a was observed in both HCC tissues and cell lines, and associated with patients’ aggressive pathologic features. Up-regulating miR-125a significantly inhibited the malignant phenotypes by repressing the expression of matrix metalloproteinase 11 (MMP11) and vascular endothelial growth factor A (VEGF-A) both in vitro and in vivo. Furthermore, miR-125a expression was inversely correlated with both MMP11 and VEGF-A expression in HCC tissues. Inhibiting miR-125a could increase both MMP11 and VEGF-A expression, and RNA interference targeting MMP11 or VEGF-A mRNA could rescue the loss of miR-125a functions. MiR-125a inhibits the proliferation and metastasis of HCC by targeting MMP11 and VEGF-A. Up-regulation of miR-125a might be a promising approach and a prognostic marker for HCC.

MicroRNA-499-5p promotes cellular invasion and tumor metastasis in colorectal cancer by targeting FOXO4 and PDCD4

MicroRNAs (miRNAs) regulate tumor progression and invasion via direct interaction with target messenger RNAs (mRNAs). We defined miRNAs involved in cancer metastasis (metastamirs) using an established in vitro colorectal cancer (CRC) model of minimally metastatic cells (SW480 line) from a colon adenocarcinoma primary lesion and highly metastatic cells (SW620 line) from a metastatic lymph node from the same patient 1 year later. We used microarray analysis to identify miRNAs differentially expressed in SW480 and SW620 cells, focusing on miR-499-5p as a novel candidate prometastatic miRNA whose functions in cancer had not been studied. We confirmed increased miR-499-5p levels in highly invasive CRC cell lines and lymph node-positive CRC specimens. Furthermore, enhancing the expression of miR-499-5p promoted CRC cell migration and invasion in vitro and lung and liver metastasis in vivo, while silencing its expression resulted in reduced migration and invasion. Additionally, we identified FOXO4 and PDCD4 as direct and functional targets of miR-499-5p. Collectively, these findings suggested that miR-499-5p promoted metastasis of CRC cells and may be useful as a new potential therapeutic target for CRC.

Gankyrin promotes the proliferation of human pancreatic cancer

Previous studies in our laboratory have suggested that gankyrin expression is correlated with a malignant phenotype in colorectal cancer. Here, we investigated the possible role of gankyrin in pancreatic carcinogenesis. Gankyrin expression was significantly increased in pancreatic cancer compared to non-cancerous tissues. This expression significantly enhanced cancer cell proliferation and growth in vitro and in vivo. Suppression of gankyrin downregulated cyclin A, cyclin D1, cyclin E, CDK2, CDK4, PCNA and p-Rb but upregulated p27, Rb and p53. However, gankyrin overexpression led to opposite results. Thus, gankyrin could enhance pancreatic cancer cell proliferation by promoting cell cycle progression and p53 degradation.

Overexpression of a novel gene gankyrin correlates with the malignant phenotype of colorectal cancer.

Gankyrin, a small and highly conserved protein which is identical to the p28 gene product, was found to be related with the malignant phenotypes in liver and esophageal carcinoma. However, the roles of gankyrin in colorectal carcinoma (CRC) are still unknown. In the present study, the gankyrin mRNA and protein expression in human CRC cell lines and clinical tissue samples were evaluated and correlated with clinicopathological features. Possible mechanisms by which gankyrin regulates the malignant phenotype of CRC cells were also investigated. The results demonstrated that gankyrin was obviously overexpressed in CRC tissues and cell lines compared to controls, and gankyrin expression was correlated with TNM stages and metastasis of CRC. Overexpression of gankyrin by PhkitNeo-hGankyrin plasmid transfected into Lovo cells could promote the cell proliferation and tumorigenicity. This finding was further strengthened by experiments that suppressing gankyrin expression by siRNA exerted the opposite effects on CRC cells SW620. In addition, our present study showed that the co-expression of cyclinD1 and β-catenin were positive correlation with the alteration of gankyrin expression. This data suggested that gankyrin played significant roles in the pathogenesis of human CRC, and might be an important therapeutic target for CRC.

MicroRNA-501 promotes HBV replication by targeting HBXIP

MicroRNAs (miRNAs) can negatively regulate gene expression and also induce or inhibit viral replication. In the present study, we found 10 miRNAs were differentially expressed in a stable HBV-producing cell line (HepG2.2.15) compared with its control cell line (HepG2) by miRNA array analysis. miR-501 was significantly up-regulated in HepG2 cells and tissues with high-HBV replication. miR-501 expression was significantly up-regulated in hepatocellular carcinoma tissues, where HBV replication kept high. Down-regulating miR-501 could significantly inhibit HBV replication, but not influence the growth of HepG2.2.15 cells. Luciferase reporter and western blot assays revealed that HBXIP, an inhibitor of HBV replication, was a potential target of miR-501. Moreover, knockdown of HBXIP rescued the inhibition of HBV that occurred after the loss of miR-501 in HepG2.2.15 cells, suggesting that miR-501 induced HBV replication partially by targeting HBXIP. Thus, knockdown of miR-501 might provide a new mechanism and therapeutic target for inhibiting HBV replication.

MiR-199a Regulates Cell Proliferation and Survival by Targeting FZD7

A growing amount of evidence indicates that miRNAs are important regulators of multiple cellular processes and, when expressed aberrantly in different types of cancer such as hepatocellular carcinoma (HCC), play significant roles in tumorigenesis and progression. Aberrant expression of miR-199a-5p (also called miR-199a) was found to contribute to carcinogenesis in different types of cancer, including HCC. However, the precise molecular mechanism is not yet fully understood. The present study showed that miR-199a is frequently down-regulated in HCC tissues and cells. Importantly, lower expression of miR-199a was significantly correlated with the malignant potential and poor prognosis of HCC, and restoration of miR-199a in HCC cells led to inhibition of the cell proliferation and cell cycle in vitro and in vivo. Furthermore, Frizzled type 7 receptor (FZD7), the most important Wnt receptor involved in cancer development and progression, was identified as a functional target of miR-199a. In addition, these findings were further strengthened by results showing that expression of FZD7 was inversely correlated with miR-199a in both HCC tissues and cells and that over-expression of miR-199a could significantly down-regulate the expression of genes downstream of FZD7, including β-catenin, Jun, Cyclin D1 and Myc. In conclusion, these findings not only help us to better elucidate the molecular mechanisms of hepatocarcinogenesis from a fresh perspective but also provide a new theoretical basis to further investigate miR-199a as a potential biomarker and a promising approach for HCC treatment.

Distinguishing pancreatic cancer from chronic pancreatitis and healthy individuals by 1H nuclear magnetic resonance-based metabonomic profiles

OBJECTIVES To develop a noninvasive and accessible diagnostic method for pancreatic cancer (PC). DESIGN AND METHODS We presented a metabolomic method, pattern recognition techniques applied to (1)H nuclear magnetic resonance ((1)H NMR) spectra, to investigate the plasma metabolites obtained from 19 patients with PC, 20 patients with chronic pancreatitis (CP) and 20 healthy individuals. RESULTS Metabolic changes associated with PC included abnormal amino acid and lipid metabolism, and possible multiple metabolic syndrome. PC elevated plasma levels of N-acetyl glycoprotein (NAG), dimethylamine (DMA), very low density lipoprotein (VLDL), and acetone, and reduced levels of 3-hydroxybutyrate, lactate, high density lipoprotein (HDL), low density lipoprotein (LDL), citrate, alanine, glutamate, glutamine, histidine, isoleucine, lysine, and valine. These metabolites could be a biomarker group for PC that distinguishes between PC and CP patients and healthy individuals. CONCLUSIONS NMR-based metabonomic strategy appears as a promising approach for distinguishing pancreatic cancer and identifying new strategies for prevention or therapy in the clinical practice.

SOX2, a predictor of survival in gastric cancer, inhibits cell proliferation and metastasis by regulating PTEN

Inconsistent results of SOX2 expression have been reported in gastric cancer (GC). Here, we demonstrated that SOX2 was progressively downregulated during GC development via immunochemistry in 755 human gastric specimens. Low SOX2 levels were associated with pathological stage and clinical outcome. Multivariate analysis indicated that SOX2 protein expression served as an independent prognostic marker for GC. Gain-and loss-of function studies showed the anti-proliferative, anti-metastatic, and pro-apoptotic effects of SOX2 in GC. PTEN was selected as SOX2 targets by cDNA microarray and ChIP-DSL, further identified by luciferase assays, EMSA and ChIP-PCR. PTEN upregulation in response to SOX2-enforced expression suppressed GC malignancy via regulating Akt dephosphorylation. PTEN inhibition reversed SOX2-induced anticancer effects. Moreover, concordant positivity of SOX2 and PTEN proteins in nontumorous tissues but lost in matched GC specimens predicted a worse patient prognosis. Thus, SOX2 proved to be a new marker for evaluating GC outcome.

A peptide derived from phage display library exhibits anti-tumor activity by targeting GRP78 in gastric cancer multidrug resistance cells

Multidrug resistance (MDR) remains a significant challenge to the clinical treatment of gastric cancer (GC). In the present study, using a phage display approach combined with MTT assays, we screened a specific peptide GMBP1 (Gastric cancer MDR cell-specific binding peptide), ETAPLSTMLSPY, which could bind to the surface of GC MDR cells specifically and reverse their MDR phenotypes. Immunocytochemical staining showed that the potential receptor of GMBP1 was located at the membrane and cytoplasm of MDR cells. In vitro and in vivo drug sensitivity assays, FACS analysis and Western blotting confirmed that GMBP1 was able to re-sensitize MDR cells to chemical drugs. Western blotting and proteomic approaches were used to screen the receptor of GMBP1, and GRP78, a MDR-related protein, was identified as a receptor of GMBP1. This result was further supported by immunofluoresence microscopy and Western blot. Additionally, Western blotting demonstrated that pre-incubation of GMBP1 in MDR cells greatly diminished MDR1, Bcl-2 and GRP78 expression but increased the expression of Bax, whereas downregulation of GRP78, function as a receptor and directly target for GMBP1, only inhibited MDR1 expression. Our findings suggest that GMBP1 could re-sensitize GC MDR cells to a variety of chemotherapeutic agents and this role might be mediated partly through down-regulating GRP78 expression and then inhibiting MDR1 expression. These findings indicate that peptide GMBP1 likely recognizes a novel GRP78 receptor and mediates cellular activities associated with the MDR phenotype, which provides new insight into research on the management of MDR in gastric cancer cells.

Nuclear expression of Twist promotes lymphatic metastasis in esophageal squamous cell carcinoma.

Twist-1 protein (also called Twist) has been suggested to be involved in tumor epithelial-mesenchymal transition (EMT) related progression, however, the mechanism by which twist promotes lymph node metastasis is not fully understood. In the present study, we found that nuclear twist expression is clearly correlated with lymph node (LN) metastasis as determined by immunohistochemistry (IHC). A highly invasive EC109 cell subline, EC109-P, was established by repeated in vitro transwell isolations for the cell model. Immunofluorescence (IF) assay demonstrated that nuclear twist expression was markedly higher in the highly invasive EC109-P cell line when compared with EC109 and EC9706 cells. Based on our cell model, the function and mechanism by which twist regulates LN metastasis in ESCC was investigated. The results showed that the overexpression of Twist could significantly increase the invasion and VEGF-C expression of EC9706 cells, whereas the knockdown of twist expression results in the opposite effects. This finding was further strengthened by the results of the analysis of co-expression of twist and VEGF-C by IHC in ESCC clinical samples. In summary, our study indicates that nuclear twist plays an important role in ESCC lymphatic metastasis by increasing the expression of VEGF-C. The combination of twist and VEGF-C detection could be a reliable prediction of LN metastasis in ESCC.