miR-650 Promotes the Metastasis and Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma by Directly Inhibiting LATS2 Expression.

BACKGROUND/AIMS: Previous studies have confirmed that microRNAs are involved in the metastasis and epithelial-mesenchymal transition (EMT) of malignancies. In this study, we examined whether miR-650 promotes the migration, invasion, and EMT of hepatocellular carcinoma (HCC) cells by targeting the large tumor suppressor kinase 2 gene (LATS2). METHODS: qRT-PCR was used to detect expression of miR-650 in HCC tissues and paired normal tissues. MTT and Transwell assay were used to observe the effect of miR-650 on proliferation, migration and invasion of HCC cells. Western blot assay and Immunohistochemistry were performed to demonstrate association between miR-650 expression level and epithelial-mesenchymal transition (EMT) related protein. Mechanistically, Reporter luciferase assay was performed to reveal whether large tumor suppressor kinase 2 (LATS2) was a direct target of miR-650 in HCC cells. RESULTS: We observed that miR-650 levels were largely up-regulated in HCC tissues, and that the increased expression was closely associated with the adverse clinical features of HCC patients. Additionally, the expression of LATS2, which was identified as a direct target of miR-650, can counteract the effects of miR-650 in HCC. Furthermore, we demonstrated that high miR-650 expression levels and low LATS2 expression levels in tumors may indicate a poor prognosis for HCC patients. CONCLUSION: In conclusion, the miR-650/LATS2 pathway may serve as a novel prognostic biomarker and an attractive therapeutic target for HCC patients.

miR-103 promotes the metastasis and EMT of hepatocellular carcinoma by directly inhibiting LATS2.

Improving the long­term survival of patients with hepatocellular carcinoma (HCC) remains a challenge due to metastasis and recurrence. In this study, we demonstrate that the overexpression of miR­103 in HCC cells promotes epithelial­mesenchymal transition (EMT), and is associated with an enhanced metastasis and poor outcomes, as shown by western blot analysis and immunohistochemistry. Mechanistically, using reporter luciferase assay we reveal that the serine/threonine­protein kinase, large tumor suppressor kinase 2 (LATS2), a key component of the Hippo signaling pathway, is a direct target of miR­103 in HCC cells. Transwell assay, MTT assay and western blot analysis were performed to reveal that LATS2 can counteract the functional effects of miR­103 on HCC metastasis, growth and EMT. The analyses of clinical data indicated that a high expression of miR­103 correlated with a high expression of vimentin, but with a low expression of LATS2 and E­cadherin in HCC tissues. miR­103 also reduced yes­associated protein (YAP) phosphorylation. On the whole, the findings of this study suggest that miR­103 promotes HCC metastasis and EMT by directly inhibiting LATS2. Thus, targeting miR­103/LATS2 may prove to be a promising therapeutic strategy for HCC.

MiR-195 suppresses the metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma by inhibiting YAP.

MiR-195, a novel cancer-related microRNA, was previously reported to play an important role in many malignancies. This study aimed to investigate the role of miR-195 mediated epithelial-mesenchymal transition (EMT) and the progression of hepatocellular carcinoma (HCC) as well as the underlying mechanisms. Our result demonstrated that miR-195 were significantly down regulated in HCC and its decreased expression is associated with poor clinical features of HCC patients. Oppositely, expression level of YAP was significantly higher in HCC tissues, and the level of YAP in metastatic tissues was significantly higher. We also found that a strong inversely association between low level expression of miR-195 and high level of YAP in HCC tissues. Notably, this study confirmed that miR-195, YAP and their combination were valuable predictors for the prognosis of HCC patients. We also explored that miR-195 inhibits HCC growth and metastatic capacity. Mechanistically, we confirm that miR-195 inhibits the migration, invasion and EMT of HCC cells by suppressing YAP. Lastly, we revealed YAP was not only the downstream of miR-195 in HCC, but also mediated the promoting effects of miR-195 on the metastasis and EMT of HCC cells. Taken together, miR-195 inhibits the metastasis and EMT in HCC by targeting YAP. MiR-195/YAP pathway may potentially act as novel biomarker and attractive therapeutic target in HCC.

Potential rat model of anxiety-like gastric hypersensitivity induced by sequential stress.

AIM: To establish a rat model of anxiety-like gastric hypersensitivity (GHS) of functional dyspepsia (FD) induced by novel sequential stress. METHODS: Animal pups were divided into two groups from postnatal day 2: controls and the sequential-stress-treated. The sequential-stress-treated group received maternal separation and acute gastric irritation early in life and restraint stress in adulthood; controls were reared undisturbed with their mothers. Rats in both groups were followed to adulthood (8 wk) at which point the anxiety-like behaviors and visceromotor responses to gastric distention (20-100 mmHg) and gastric emptying were tested. Meanwhile, alterations in several anxiety-related brain-stomach modulators including 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), brain-derived neurotrophic factor (BDNF) and nesfatin-1 in the rat hippocampus, plasma and gastric fundus and the 5-HT1A receptor (5-HT1AR) in the hippocampal CA1 subfield and the mucosa of the gastric fundus were examined. RESULTS: Sequential-stress-treated rats simultaneously demonstrated anxiety-like behaviors and GHS in dose-dependent manner compared with the control group. Although rats in both groups consumed similar amount of solid food, the rate of gastric emptying was lower in the sequential-stress-treated rats than in the control group. Sequential stress significantly decreased the levels of 5-HT (51.91 ± 1.88 vs 104.21 ± 2.88, P < 0.01), GABA (2.38 ± 0.16 vs 5.01 ± 0.13, P < 0.01) and BDNF (304.40 ± 10.16 vs 698.17 ± 27.91, P < 0.01) in the hippocampus but increased the content of nesfatin-1 (1961.38 ± 56.89 vs 1007.50 ± 33.05, P < 0.01) in the same site; significantly decreased the levels of 5-HT (47.82 ± 2.29 vs 89.45 ± 2.61, P < 0.01) and BDNF (257.05 ± 12.89 vs 536.71 ± 20.73, P < 0.01) in the plasma but increased the content of nesfatin-1 in it (1391.75 ± 42.77 vs 737.88 ± 33.15, P < 0.01); significantly decreased the levels of 5-HT (41.15 ± 1.81 vs 89.17 ± 2.31, P < 0.01) and BDNF (226.49 ± 12.10 vs 551.36 ± 16.47, P < 0.01) in the gastric fundus but increased the content of nesfatin-1 in the same site (1534.75 ± 38.52 vs 819.63 ± 38.04, P < 0.01). The expressions of 5-HT1AR in the hippocampal CA1 subfield and the mucosa of the gastric fundus were down-regulated measured by IHC (Optical Density value: Hippocampus 15253.50 ± 760.35 vs 21149.75 ± 834.13; gastric fundus 15865.25 ± 521.24 vs 23865.75 ± 1868.60; P < 0.05, respectively) and WB (0.38 ± 0.01 vs 0.57 ± 0.03, P < 0.01) (n = 8 in each group). CONCLUSION: Sequential stress could induce a potential rat model of anxiety-like GHS of FD, which could be used to research the mechanisms of this intractable disease.

Apoptotic pathway induced by diallyl trisulfide in pancreatic cancer cells.

AIM: To investigate the effects of diallyl trisulfide (DATS), a garlic-derived organosulfur compound, in pancreatic cancer cells. METHODS: Human pancreatic cancer cells with wild-type p53 gene (Capan-2) and normal pancreatic epithelial cells (H6C7) were cultured in RPMI1640. DATS was prepared at a concentration of 100 µmol/L. Cell viability was determined via the methyl thiazolyl tetrazolium assay. Apoptotic cells were detected by TUNEL assay. Cell cycle analysis was performed using flow cytometry. Protein expression was determined by Western blot. Bax and Bcl-2 expression was detected by immunofluorescence. Apoptosis genes and cell cycle were assessed by quantitative real-time polymerase chain reaction. RESULTS: DATS suppressed the viability of cultured human pancreatic cancer cells (Capan-2) by increasing the proportion of cells in the G2/M phase and induced apoptotic cell death. Western blot analysis indicated that DATS enhanced the expression of Fas, p21, p53 and cyclin B1, but downregulated the expression of Akt, cyclin D1, MDM2 and Bcl-2. DATS induced cell cycle inhibition which was correlated with elevated levels of cyclin B1 and p21, and reduced levels of cyclin D1 in Capan-2 cells and H6C7 cells. DATS-induced apoptosis was markedly elevated in Capan-2 cells compared with H6C7 cells, and this was correlated with elevated levels of cyclin B1 and p53, and reduced levels of Bcl-2. DATS-induced apoptosis was correlated with down-regulation of Bcl-2, Akt and cyclin D1 protein levels, and up-regulation of Bax, Fas, p53 and cyclin B protein levels in Capan-2 cells. CONCLUSION: DATS induces apoptosis of pancreatic cancer cells (Capan-2) and non-tumorigenic pancreatic ductal epithelial cells (H6C7).

[Induction of active antitumor immune response by myeloma idiotype protein-pulsed dendritic cells].

BACKGROUND & OBJECTIVE: Most multiple myeloma (MM) patients could not be cured by high-dose chemotherapy and bone marrow transplantation. This study was designed to investigate in vitro killing effect of tumor-specific cytotoxic T lymphocytes (CTLs) stimulated by idiotype protein (Id)-pulsed dendritic cells (DCs) on autologous MM cells. METHODS: DCs were generated from peripheral blood monocytes of 6 MM patients using interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). After cultured for 5 days, immature DCs were pulsed with Idû tumor necrosis factor-alpha (TNF-alpha) was added at the 7th day. Id-pulsed DCs were cocultured with autologous T cells for 3 days to induce tumor-specific CTLs. MTT assay was used to detect proliferation of autologous T cells, and evaluate killing effect of CTLs on autologous MM cells. RESULTS: Mature DCs were successfully induced. Id-pulsed DCs markedly increased proliferation of autologous T cells in a dose-dependent manner; stimulation index (SI) of Id-pulsed DCs was the highest [(39.1+/-6.0)%] when the radio of DCs to T cells was 10:1, which was significantly higher than those of unpulsed mature DCs [(19.3+/-7.7)%], Id-pulsed immature DCs [(15.9+/-6.1)%], and unpulsed immature DCs [(11.4+/-4.9)%] (P < 0.01). Id-pulsed DCs induced anti-MM activity of CTLs in a dose-dependent manner. Unpulsed mature DCs also induced cytotoxicity of CTLs against autologous MM cellsû however, when DC:T was 30:1, killing rate of MM cells was significantly higher in Id-pulsed mature DCs group than in unpulsed mature DCs group [(70.1+/-7.9)% vs. (40.8+/-7.8)%,P < 0.05]. KRN7000-pulsed mature DCs stimulated proliferation of allogeneic T cells in a dose-dependent manner; when DC:T was 1:10, SI was significantly higher in KRN7000-pulsed mature DCs group than in unpulsed mature DCs group [(38.5+/-5.7)% vs. (20.2+/-5.7)%, P < 0.05]. CONCLUSIONS: Mature DCs could be induced and Id with biological activity could be extracted from peripheral blood of MM patients. Id-pulsed DCs could induce antitumor immune response. KRN7000 could improve the immune function of in vitro cultured DCs.

[In vitro anti-myeloma effects induced by myeloma idiotype-protein pulsed dendritic cell vaccine].

OBJECTIVE: To investigate the specific antitumor immune response induced by idiotype protein (Id)-pulsed dendritic cells (DC) in vitro. METHODS: DC was generated from peripheral blood monocytes of the multiple myeloma (MM) patients using GM-CSF, IL-4, and TNF-alpha. The DCs were pulsed with idiotypic fragment, the F(ab')2 fragment of M protein from MM patient at the immature stage. The morphologic characteristics of the cells were observed with light and electron microscopes. The phenotypic features were analyzed with FACS, MTT assay was employed to evaluate the proliferation of autologous T cells and the inhibition rate of MM cells. RESULTS: DC precursors in peripheral blood could be induced to typical mature DC in medium containing GM-CSF, IL-4 and TNF-alpha. Mature DC with Id could increase the proliferation of the autologous T cells and activate naive T cells to become tumor specialized cytotoxic T lymphocytes (CTL). The CTL at different doses showed significant inhibition on or killing ability to autologous MM cells in vitro. CONCLUSIONS: In a suitable cytokine environment, the DC precursors from peripheral blood of MM patients could be induced to functional DC, and vaccination of Id-pulsed DC could induce active antitumor immune response.