Long non-coding RNA PVT1 regulates effect of miR-551 on migration and invasion abilities of ovarian cancer through Wnt signaling pathway

AIM: To investigate the expression of long non-coding RNA PVT1 in ovarian cancer and the role of PVT1 in migration and invasion abilities of ovarian cancer cells.METHODS: The expression of PVT1 in ovarian cancer tissue, normal ovarian tissue and different ovarian cancer cell lines was detected by qPCR. Transwell assay was used to detect the invasion ability of ovarian cancer cells after PVT1 silencing. The migration ability of the ovarian cancer cells after PVT1 silencing was detected by scratch test. The interaction between PVT1 and microRNA (miR)-551 was analyzed by dual-luciferase reporter assay. The effect of miR-551-inhibitor on the invasion and migration abilities of ovarian cancer cells after PVT1 silencing was detected by Transwell assay and scratch test. The expression of Wnt signaling pathway-related proteins was determined by Western blot after PVT1 silencing. The effects of PVT1 silencing on tumor weight and volume of ovarian cancer were examined by subcutaneous tumor transplantation in nude mice.RESULTS: The expression of PVT1 in ovarian cancer tissue was significantly higher than that in normal ovarian tissue (P<0.05). The expression level of PVT1 in ovarian cancer cell line ES-2 was the highest. PVT1 silencing inhibited the invasion and migration abilities of the ovarian cancer cells. After PVT1 silencing, miR-551-inhibitor promoted the invasion and migration abilities of the ovarian cancer cells. The expression of Wnt signaling pathway-related proteins was decreased after PVT1 silencing (P<0.05). Compared with negative control group, the tumor volume and weight in PVT1-siRNA group were significantly decreased (P<0.05).CONCLUSION: PVT1 plays an important role in the development of ovarian cancer. PVT1 regulates the invasion and migration abilities of ovarian cancer cells through Wnt signaling pathway.     

EMMPRIN mediates matrix metalloproteinase 9 expression and monocyte migration: evidence from EMMPRIN knockdown by RNA interference

AIM: Although the evidence indicates that extracellular matrix metalloproteinase inducer (EMMPRIN) is closely associated with matrix metalloproteinase (MMP) expression in tumor cells, tumor invasion and metastasis, no direct proof that EMMPRIN regulates MMPs in monocytes, especially in the atherogenic milieu is observed. Here we tested this hypothesis by examining MMP-9 expression in macrophages/foam cells and monocyte migration through EMMPRIN knockdown by siRNA. METHODS: The methods of qPCR and Western blotting were used to detect the suppressions of EMMPRIN mRNA and protein expression in macrophages and foam cells transfected with EMMPRIN-specific siRNA. The protein expression of MMP-9 in macrophages and foam cells was also determined. Monocyte migration after EMMPRIN knockdown was observed by a Transwell assay. RESULTS: EMMPRIN knockdown by siRNA markedly abolished the MMP-9 expression by 50% and 40% in macrophages and foam cells, respectively. Migration induced by chemotactic factor MCP-1 and VEGF was significantly attenuated (P<0.05) in monocytes treated with EMMPRIN-siRNA. CONCLUSION: The protein expression and secretion of MMP-9 are down-regulated by EMMPRIN knockdown during monocyte differentiation into macrophages and foam cells. Moreover, EMMPRIN siRNA treatment also prevents monocyte migration. Thus, EMMPRIN plays a key regulatory role for MMP activity and monocyte migration, making it a potential target for pharmacological intervention of atherosclerosis.     

Toll-like receptor 4 promotes migration and invasion abilities of human non-small-cell lung cancer cells

AIM:To evaluate the expression and biological role of Toll-like receptor 4 (TLR4) in human non-small-cell lung cancer (NSCLC) cells. METHODS:The mRNA and protein levels of TLR4 in NSCLC tissue were exa-mined by RT-qPCR, Western blot, and immunohistochemistry. After treating the A549 cells and SPC-A-1 cells with TLR4 stimulator lipopolysaccharide (LPS) and inhibitor TAK-242, RT-qPCR, Western blot and flow cytometry were performed to detect the expression of TLR4. The migration and invasion abilities were detected by Transwell assay, and the mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) was also detected. RESULTS:The mRNA and protein levels of TLR4 were higher in the NSCLC tissue than those in the noncancerous tissue (P<0.01). LPS stimulation significantly increased the mRNA and protein expression levels of TLR4 in the NSCLC cell lines A549 and SPC-A-1 (P<0.01). The LPS-induced TLR4 activation enhanced the migration and invasion abilities of A549 cells and SPC-A-1 cells (P<0.01). LPS increased the expression levels of MMP-2, MMP-9 and VEGF in the A549 cells and SPC-A-1 cells (P<0.01). Moreover, the expression levels of TLR4, MMP-2, MMP-9 and VEGF, as well as the migration and invasion abilities of the cells were blocked by TAK-242 (P<0.01). CONCLUSION:TLR4 might be involved in the migration and invasion of NSCLC cells, and TLR4 inhibition might be considered as a therapeutic target for treatment of NSCLC.     

miR-509 regulates growth,invasion and migration of human hepatocellular carcinoma LM3 cells and survival of nude mice

AIM: To investigate the effect of microRNA-509 (miR-509) on the growth, invasion and migration of human hepatocellular carcinoma (HCC) LM3 cells and survival of tumor-bearing nude mice. METHODS: LM3 cells were transferred with miR-509 mimic and pcDNA Ras-related C3 botulinum toxin substrate 1 (pcRac1), and the expression of Rac1 was measured by Western blot. The relationship between miR-509 and Rac1 was determined by luciferase reporter assay. The invasion ability was determined by Transwell assay, and the migration ability was measured by wound healing assay. Xenograft model of HCC was established by subcutaneous injection with LM3 cells into nude mice. The survival rate of the mice were recorded and the protein level of Rac1 was determined by Western blot. RESULTS: miR-509 mimic inhibited the expression of Rac1 in the LM3 cells (P<0.05). pcRac1 attenuated the effect of miR-509 on Rac1. miR-509 also alleviated luciferase activity of wild Rac1 (P<0.05). Meanwhile, miR-509 mimic decreased the number of invasive LM3 cells and inhibited the migration of LM3 cells (P<0.05). In addition, over-expression of miR-509 up-regulated survival rate of model mice and decreased the protein level of Rac1 in the tumor tissue (P<0.01). CONCLUSION: miR-509 inhibits the invasion and migration of HCC cells and promotes the survival of tumor-bearing nude mice through inhibiting the expression of Rac1.     

MCP-1/CCR2 signaling pathway mediates alcohol-induced angiogenesis in breast cancer

AIM To investigate the role of monocyte chemoattractant protein-1 (MCP-1) and its receptor CC chemokine receptor 2 (CCR2) in ethanol-promoted breast cancer angiogenesis and the underlying mechanism. METH?ODS: A mouse model of transplanted breast tumor with moderate alcohol consumption was established. The correlations between the expression of MCP-1/CCR2 and the expression of angiogenesis markers [platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor (VEGF)] in tumor tissues were examined by immunohistochemistry. In vitro, a 3D tumor-endothelial co-culture system was established to observe tumor angiogenesis and the role of MCP-1/CCR2 signaling pathway in alcohol-mediated angiogenesis. The cell migration ability was detected to clarify whether MCP-1/CCR2 enhanced cell mobility to form new vessels. RESULTS MCP-1 and CCR2 were both highly expressed in the breast tumor tissues of tumor-bearing mice consuming alcohol, and their expression levels were consistent with the angiogenic markers PECAM-1 and VEGF (P<0.05). The interaction between mouse breast cancer E0771 cells and endothelial cells was observed to promote angiogenesis in the 3D tumor-endothelial co-culture system with or without alcohol stimulation. MCP-1 promoted this kind of tumor angiogenesis, while CCR2 antagonist effectively inhibited the tumor angiogenesis and especially blocked alcohol-induced angiogenesis. Activation of MCP-1/CCR2 signaling pathway enhanced the migration ability of endothelial cells. CONCLUSION The MCP-1/CCR2 signaling pathway plays an important role in promoting the angiogenesis of breast cancer stimulated by alcohol. The mechanism might be that MCP-1 improves the migration of endothelial cells and then promotes angiogenesis.     

Expression of miRNA-22 in ovarian cancer and effect of miRNA-22 over-expression on SKOV-3 ovarian cancer cell proliferation,migration and invasion

AIM: To examine the expression of miRNA-22 in the ovarian tissues and the effect of miRNA-22 over-expression on the proliferation, migration and invasion in SKOV-3 cells. METHODS: The expression levels of miRNA-22 in different ovarian tissues and SKOV-3 cells were determined by qPCR. miRNA-22 was over-expressed by transfection of miRNA-22 mimic. The cell viability was examined by CCK-8 assay. The cell migration was measured by wound healing test. The cell invasion was analyzed by Transwell assay. The protein expression levels of VEGF and P53 were determined by Western blot. RESULTS: Compared with the normal ovarian tissue, the expression level of miRNA-22 was remarkably decreased in the ovarian tumor tissues. After transfection with miRNA-22 mimic, the expression level of miRNA-22 in the SKOV-3 cells was significantly increased, while the cell viability, migration and invasion were obviously decreased. Moreover, the protein expression of VEGF and P53 was dramatically inhibited after over-expression of miRNA-22. CONCLUSION: The decreased miRNA-22 expression may be correlated with the development of ovarian can-cer. Over-expression of miRNA-22 decreases the cell viability, migration and invasion by reducing the protein expression of VEGF and P53.     

Galectin-9 regulates SHH signaling pathway to influence apoptosis of colorectal cancer HT29 cells

AIM: To investigate the effect of galectin-9 on the apoptosis of colorectal cancer HT29 cells. METHODS: Galectin-9 over-expression vector (pcDNA3.1-Galectin-9) or control vector (pcDNA3.1) was transfected into the HT29 cells. The galectin-9 over-expression was detected by real-time PCR and Western blot. Annexin V-FITC/PI double staining was used to detect the apoptosis. The protein level of activated caspase-3 and the expression of SHH signaling pathway-related proteins Smo, Gli1 and SHH in the HT29 cells were determined by Western blot. SHH signaling pathway specific inhibitor cyclopamine was used to treat the HT29 cells with up-regulated galectin-9 expression, and the apoptosis, the protein level of cleaved caspase-3 and the expression of SHH signaling pathway-related proteins Smo, Gli1 and SHH in the HT29 cells were detected by the above methods. RESULTS: Transfection with pcDNA3.1-Galectin-9 up-regulated galectin-9 expression at mRNA and protein levels in the colorectal cancer HT29 cells (P<0.05). The apoptotic rate of the HT29 cells was increased after galectin-9 up-regulation. The protein level of cleaved caspase-3 in the cells was increased, while the expression levels of SHH signaling pathway-related proteins Smo, Gli1 and SHH were decreased. Cyclopamine treatment further induced the apoptosis of the HT29 cells with up-regulation of galectin-9, increased the protein le-vels of cleaved caspase-3, and decreased the activation level of SHH signaling pathway in the HT29 cells (P<0.05). CONCLUSION: Galectin-9 induces the apoptosis of colorectal cancer HT29 cells by inhibiting SHH signaling pathway.     

Gene silencing of indoleamine 2,3-dioxygenase 2 influences proliferation,migration and invasion of B16-BL6 melanoma cells

AIM: To investigate the effects of indoleamine 2, 3-dioxygenase 2 (IDO2) silencing on proliferation, migration and invasion of B16-BL6 melanoma cells.METHODS: IDO2-siRNA was transfected into the B16-BL6 melanoma cells in vitro. The expression of IDO2 or IDO1 at mRNA and protein levels was detected by real-time PCR and Western blot. Colony formation assay was performed to analyze the proliferation of IDO2-silencing tumor cells. The migration ability of B16-BL6 cells after silencing of IDO2 was measured by wound healing assay and Transwell cell migration assay. The invasion ability of the tumor cells was detected by Transwell cell invasion assay.RESULTS: IDO2-siRNA signi-ficantly down-regulated IDO2 expression in B16-BL6 melanoma cells, and did not affect IDO1 expression. Compared with control group, the colony formation ability, the migratory distance measured by wound healing assay, and the migration and the invasion cell numbers detected by Transwell assay all remarkably decreased in the IDO2-silencing cells.CONCLUSION: IDO2 silencing affects the proliferation, migration and invasion abilities of the B16-BL6 melanoma cells.     

Antitumor effect of chlorophyllin in vitro

AIM: To study the effect and mechanism of chlorophyllin (CHL) inhibiting HT29 cells. METHODS: IC₅₀ value and growth curve of HT29 cells were detected with MTT method. Apoptosis was detected with Wright-Giemsa staining, FCM and DNA electrophoresis. Telomerase was detected by PCR-ELISA, and protein and mRNA expression of COX-2 gene were detected through RT-PCR and Western blot. RESULTS: CHL inhibited the growth of HT29 in a dose-dependent manner. CHL blocked HT29 cells in G₁ phase but did not induce apoptosis. Different concentration of CHL inhibits the expression of telomerase and COX-2 in HT29 cells. CONCLUSION: CHL inhibited the growth of HT29 cells by inhibiting the expression of telomerase and COX-2 and blocking cells in G₁ phase.     

Knockdown of RRM2 gene by siRNA inhibits human breast cancer cell proliferation,migration and tumor growth in nude mice

AIM: To explore the effect of ribonucleotide reductase M2 (RRM2) gene knockdown by siRNA on the proliferation and migration of human breast cancer MCF-7 cells and the tumor growth in BALB/c nude mice. METHODS: The mRNA and protein expression leves of RRM2 in human breast cancer cell line MCF-7 and human normal breast cell line MCF-10A were determined by real-time PCR and Western blotting. siRNA-RRM2 was constructed and transfected into MCF-7 cells at different time points and different concentrations. The silencing efficiency of RRM2 gene was detected by real-time PCR. The cell proliferation was measured by CCK-8 assay. The migration was observed using Transwell cell migration system. The effect of siRNA-RRM2 on the tumor growth was determined in nude mice. RESULTS: The mRNA and protein levels of RRM2 were higher in MCF-7 cells than those in MCF-10A cells. siRNA-RRM2 down-regulated the expression of RRM2 in MCF-7 cells in a time-and concentration-dependent manner. The results of CCK-8 assay showed that siRNA-RRM2 inhibited the proliferation ability of MCF-7 cells, but not that of MCF-10A cells. The results of Transwell assay indicated that siRNA-RRM2 inhibited the migration ability of MCF-7 cells. siRNA-RRM2 also inhibited the tumor growth in nude mice. CONCLUSION: RRM2 overexpression is associated with the breast cancer proliferation and migration. Suppression of RRM2 function is a potential therapeutic strategy for treating breast cancer.     

Microwave ablation provides effective antigen for induction of anti-tumor immune response

AIM: To investigate the efficacy of microwave ablation (MWA)-coagulated mouse hepatoma tissue in inducing antitumor immune response.METHODS: A model of MWA for s.c. hepatoma in C57L/J mice was established, the anti-tumor effect of MWA-treated mice against Hepa1-6 cells rechallenge and the cytotoxicity of spleen lymphocytes by use of crystal-violet analysis were observed. Mice were immunized with MWA vaccine consisting of MWA-coagulated hepatoma tissue mince, granulocyte-macrophage-colony stimulating factor (GM-CSF) and interleukin-2 (IL-2) slow-release microparticles, the protective anti-tumor immunity induced by MWA vaccine was evaluated.RESULTS: After s.c. challenge of Hepa1-6 cells, hepatoma were observed in 100% mice of control group and 17％ of MWA-treated mice. The tumor volumes of tumor-bearing MWA-treated mice were significantly smaller than those in control group, while MWA-treated mice survived longer than control mice. Spleen lymphocytes deprived from tumor-free MWA-treated mice were specific cytotoxic to Hepa1-6 cells. MWA vaccine protected 50% more mice than that using MWA-coagulated tumor tissue alone, and showed similar anti-tumor effect against hepatoma to formalin-fixed tumor vaccine.CONCLUSION: In situ MWA provides effective tumor antigen for induction of tumor-specific antitumor immune response, and the response is amplified by additional use of cytokines.     

EGCG depresses migration and invasion of human glioma cell line by downregulating COX-2 and MMP-2 expression

AIM: To investigate the mechanism that epigallocatechin-3-gallate (EGCG) depresses the migration and invasion in human glioma cell line SWO-38 by downregulation of cyclocxygenase-2(COX-2) and matrix metalloproteinase-2(MMP-2). METHODS: The effect of EGCG on the apoptosis of SWO-38 cell line was examined by the method of MTT. The migration and invasion of the SWO-38 cells were determined by Transwell assay. The expression of COX-2 and MMP-2 was measured by Western blotting. Meanwhile, TNF-α was used to stimulate the expression of COX-2 for determining if the mechanism of COX-2 pathway is involved in the inhibitory effect of EGCG on the migration and invasion of the tumor cells. RESULTS: After treated with EGCG for 24 h, the migration and invasion abilities of SWO-38 cells were lower than that of the cells before treatment. The results of Western blotting revealed that the 24 h treatment of EGCG on SWO-38 cell line inhibited the expression levels of COX-2 and MMP-2, indicating that the degradation of the extracellular matrix in SWO-38 cells was related to the COX-2 signaling pathway. CONCLUSION: EGCG inhibits the migration and invasion of SWO-38 cell line. The correlation between COX-2 expression and enzymatic degradation in the extracellular matrix determines the abilities of migration and invasion of tumor cells.     

Combined targeting blockage of miR-29b/92b/106b inhibits gastric can-cer cell growth and migration

AIM: To investigate the metastasis-associated microRNAs (miRNAs, miR) in gastric cancer, and to determine their biological and therapeutic roles in this malignancy. METHODS: The tumor tissue samples from gastric cancer patients with or without lymph node metastasis were collected (n=3 each). miRNA microarray was used to determine the metastasis-associated miRNAs. Gastic cancer cell line BGC-823 was transfected with locked nucleic acidmodified antisense oligonucleotides of candidate miRNAs and subsequently used for functional assays including CCK-8 assay, flow cytometry, wound healing assay and Transwell migration assay. Furthermore, the in vivo xenograft mice were used to evaluate the tumor suppressive effect of collaborative inhibition of the candidate miRNAs. RESULTS: miR-29b, miR-92b and miR-106b were up-regulated in the tumor tissues from the gastric cancer patients with lymph node metastasis. The functional assays showed that blockage of miR-29b, miR-92b and miR-106b by antisense oligonucleotides in the BGC-823 cells significantly inhibited cell growth and migration, and induced apoptosis. Furthermore, the collaborative inhibition of these triple miRNAs remarkably suppressed tumor growth in vivo. CONCLUSION: miR-29b, miR-92b and miR-106b are metastasis-associated miRNAs. These miRNAs may provide promising therapeutic targets in gastric cancer.     

Expression of microRNA-1284 in gastric cancer and underlying mechanism

AIM: To evaluate the correlation between microRNA-1284 (miR-1284) and gastric cancer, and to investigate the underlying mechanism. METHODS: The expression of miR-1284 was examined by real-time PCR in 63 gastric cancer (GC) tissue samples and 63 non-malignant adjacent tissue samples. The correlation between miR-1284 and the clinicopathological feature of GC was analyzed. Lentiviral vector containing miR-1284 was constructed and transfected into GC SGC-7901 cells. After transfection, the expression of miR-1284 was examined by real-time PCR. The cell activity was evaluated by CCK-8 assay. The cell cycle and apoptosis were determined by flow cytometry. The ability of cell migration was measured by wound-healing assay. The potential target gene of miR-1284 was predicted by online bioinformatic softwares. The expression of JAG1 mRNA was examined by real-time PCR. The protein levels of JAG1, Notch1 and NF-κB were analyzed by Western blotting. RESULTS: Compared with non-malignant adjacent tissue samples, the results of real-time PCR showed significant downregulation of miR-1284 in 42 GC tissue samples (P<0.05). The expression level of miR-1284 was not significantly associated with age and gender of the patients, tumor size, TNM staging and lymph node metastases (P>0.05), but significantly associated with histologic grading (P<0.05). Compared with LV-NC-GFP group and control group, after transfection of miR-1284 in LV-miR-1284 group, the expression of miR-1284 was significantly increased (P<0.05), the percentages of apoptotic cells and the cells in G₀/G₁ phase were significantly increased (P<0.05), the cells activity and ability of migration were significantly decreased (P<0.05), and the expression of JAG1, Notch1 and NF-κB was significantly decreased (P<0.05). CONCLUSION: The inhibitory effect of miR-1284 on gastric cancer may be associated with the regulation of its targeting gene JAG1.     

EGCG attenuates the inhibitory effect of high TNF-α level on the process of wound healing in dermal fibroblasts

AIM: To explore the effect of high tumor necrosis factor α (TNF-α) level and pre-treatment of epigallocathechin-3 gallate (EGCG) on the process of wound healing in dermal fibroblasts. METHODS: Primary dermal fibroblasts were cultured in vitro. The cells were treated with TNF-α at α concentration of 10 μg/L for 24 h or co-treated with EGCG (40 μmol/L). The cell counting assay was used to observe the proliferation. The cell migration was assessed by wound healing assay. Western blotting was used to observe the expression of collagen type I. RESULTS: High TNF-α level significantly inhibited the proliferation and migration of dermal fibroblasts. However, EGCG pre-treatment attenuated the inhibitory effect of TNF-α on the proliferation in a dose-dependent manner. The inhibited cell migration was also improved by EGCG. The expression of collagen type I was down-regulated by TNF-α and recovered by EGCG pre-treatment. CONCLUSION: EGCG abrogates the inhibitory effect of TNF-α on the proliferation and migration of dermal fibroblasts in wound healing. The expression of collagen type I is also improved. The results suggest that EGCG has protective effect on wound healing.     

Influence of basic fibroblast growth factor on ECV304 cell migration

AIM: To observe the influence of basic fibroblast growth factor (bFGF) at different concentrations on migration of ECV304 cells after scratch wound. METHODS: Cell migration of ECV304 was monitored by computer assisted video microscopy with an image analysis system after treatment with bFGF at concentrations of 0, 5, 10 and 15 μg/L. The morphological changes induced by bFGF were observed under light microscope and scanning electron microscope. RESULTS: bFGF increased the cell migration at low concentration (5 μg/L), and inhibited at high concentration (15 μg/L). The surface of migration cells exhibited numerous filopodia. CONCLUSION: bFGF has double direction adjustment effects on migration of ECV304 cells. The surface of migration cells exhibits affluent pseudopods, which are mainly filopodia.     

The expression and significance of Apaf-1 in papillary thyroid carcinoma

AIM: To investigate the mRNA and protein expression of apoptotic protease-activating factor 1 (Apaf-1) in papillary thyroid carcinoma (PTC) and its relationship with cell proliferation.METHODS: The methods of real-time PCR, Western blot and immunohistochemistry were used to detect the mRNA and protein expression of Apaf-1 in PTC and tumor-adjacent tissues. The relationship between Apaf-1 expression and clinicopathological characteristics was analyzed. The effect of Apaf-1 on cell proliferation was verified by down-regulating the expression of Apaf-1 in CGTHW-3 cells.RESULTS: The mRNA and protein expression levels of Apaf-1 in the PTC tissue were significantly lower than those in the tumor-adjacent tissue (P<0.05). Down-regulation of Apaf-1 expression enhanced the proliferation of CGTHW-3 cells (P<0.05).CONCLUSION: The expression of Apaf-1 is low in PTC, and inhibition of its expression enhances the proliferation of CGTHW-3 cells. Apaf-1 may play a tumor suppressor role in PTC.