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.     

Effects of TRPC1 on TGF-β1-induced migration of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 (TGF-β1). METHODS: Silencing of TRPC1 gene expression was performed by siRNA. The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The migration and invasion abilities of the 16HBE cells were detected by wound- healing assay and Transwell assay. The protein expression of E-cadherin and vimentin was determined by Western blot. RESULTS: TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups (P < 0.01). The results of CCK-8 assay and flow cytometry indicated that there were no significant difference in proliferation and apoptosis among TRPC1 siRNA group, TGF-β1 group and control group (P > 0.05). The results of wound-healing and Transwell assays showed that migration and invasion abilities in TRPC1 siRNA + TGF-β1 group were markedly suppressed compared with TGF-β1 group (P < 0.01). The protein expression of E-cadherin and vimentin induced by TGF-β1 was inhibited by TRPC1 silencing compared with TGF-β1 group (P < 0.05). CONCLUSION: TRPC1 is involved in the migration of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vimentin.     

Effects of GOLPH3 gene silencing mediated by lentivirus on proliferation,invasion and metastasis of human gastric cancer cell line SGC-7901

AIM：To investigate the effect of Golgi phosphoprotein 3 (GOLPH3) gene silencing on the proliferation, invasion and metastasis of human gastric cancer SGC-7901 cell in vitro. METHODS：SGC-7901 cells were transfected with lentiviral vector carrying GOLPH3 shRNA to construct a stable GOLPH3-silencing cell line LV-GOLPH3-RNAi. The expression of GOLPH3 at mRNA and protein levelss were detected by real-time PCR and Western blotting, respectively. The cell proliferation was analyzed by MTT assay. Transwell migration and invasion experiments were performed to measure the migration and invasion abilities, respectively. RESULTS：The stable GOLPH3-silencing cell line was successfully established. The expression of GOLPH3 at mRNA and protein levels was reduced significantly (P<0.05), leading to the inhibition of cell proliferation in LV-GOLPH3-RNAi group compared with scrambled group and blank control group, as well as the capacities of migration (56.7±1.5 vs 186.0±3.4 and 183.3±4.2, P<0.05) and invasion (33.5±3.0 vs 85.0±3.9 and 83.1±4.4, P<0.05). CONCLUSION：GOLPH3 silencing suppresses the capacities of proliferation, migration and invasion of human gastric cancer SGC-7901 cells, suggesting that GOLPH3 may be a potential tumor marker and independent prognostic factor.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

Effect of PAK4-LIMK1-cofilin signaling on non-small-cell lung cancer migration and invasion

AIM: To explore the mechanism of p21-activated kinase 4 (PAK4) on non-small-cell lung cancer (NSCLC) migration and invasion.METHODS: After A549 and NCI-H520 cell lines were transfected with PAK4-siRNA or negative control, the expression of PAK4 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The invasion and migration of A549 cells and NCI-H520 cells were measured by Matrigel invasion assay and Transwell migration assay. LIMK1, cofilin, and their respective phosphorylation were examined by Western blot. The interaction of PAK4 and LIMK1 was investigated by co-immunoprecipitation assay. The relationship between PAK4 and LIMK1 phosphorylation was examined by a protein kinase assay in the A549 cells and NCI-H520 cells. The expression of PAK4 and p-LIMK1 in 10 human NSCLC tissues was examined by Western blot. A549 cells and NCI-H520 cells were individually or commonly transfected with PAK4-siRNA or LIMK1 plasmid in order to observe the cell migration and invasion. RESULTS: After A549 cells and NCI-H520 cells were transfected with PAK4-siRNA for 48 h, the expression of PAK4 at mRNA and protein levels, and the numbers of invasion and migration cells in PAK4-siRNA group were lower than those in control group. Compared with control group, the phosphorylation of LIMK1 and cofilin was lower in PAK4-siRNA group, whereas the total expression levels of LIMK1 and cofilin did not change. The results of co-immunoprecipitation assays showed that PAK4 specifically interacted with LIMK1 in A549 and NCI-H520 cells. LIMK1 phosphorylation in the presence of PAK4 (K350M) was significantly lower than that in the presence of PAK4 (WT) or PAK4 (S445N) in the protein kinase assay. The PAK4 upregulation was positively correlated with the level of p-LIMK1 (P<0.05). After A549 cells and NCI-H520 cells were co-transfected with PAK4-siRNA and LIMK1 plasmid, the migration and invasion cell numbers in co-transfection group were higher than those in PAK4-siRNA transfection group. CONCLUSION: PAK4 promotes the invasive and migratory abilities of NSCLC, which is mediated by LIMK1 phosphorylation.     

Reverse effect of FOXC2 silencing on epithelial-mesenchymal transition induced by TGF-β1 in MCF-7 cells

AIM: To study the reverse effect of FOXC2 silencing on epithelial-mesenchymal transition (EMT) induced by transforming growth factor β1(TGF-β1) in MCF-7 cells. METHODS: Cultured MCF-7 cells were treated with TGF-β1 at concentration of 5 μg/L for 6 d. The cell morphological changes were observed under phase-contrast microscope. The changes of EMT-related marker proteins were assessed by immunofluorescence staining assay. TGF-β1-induced MCF-7 cells were transfected with FOXC2-siRNA mediated by recombinant lentivirus. In addition, the expression levels of FOXC2 and EMT-related marker proteins E-cadherin, claudin-1 and fibronectin-1 were also measured by RT-PCR and Western blotting. The invasion of MCF-7 cells was detected by Transwell assay. RESULTS: TGF-β1 induced the morphological alteration in MCF-7 cells from epithelial phenotype to mesenchymal phenotype,up-regulated the expression of mesenchymal marker fibronectin-1, and down-regulated the expression of epithelial markers E-cadherin and claudin-1. FOXC2 silencing reversed and restored the mesenchymal MCF-7 cells to epithelial phenotype and reduced the tumor invasion. CONCLUSION: EMT model induced by TGF-β1 in breast cancer MCF-7 cells is successfully established, which increases the invasion of MCF-7 cells. The effect of TGF-β1 is reversed by FOXC2-siRNA and the invasion of the cells is reduced.     

Effects of BCL6B on proliferation and migration of human colorectal carcinoma LoVo cells and its potential mechanism

AIM: To detect the endogenous expression of B-cell leukemia/lymphoma 6 member B (BCL6B) in FHC and LoVo cells, and to investigate the effects of BCL6B on proliferation and migration of LoVo cells for further exploring the underlying mechanism. METHODS: The endogenous expression of BCL6B in the FHC and LoVo cells was detected by RT-PCR and Western blot. The methods of MTT assay, colony formation assay, wound healing assay and Transwell chamber experiment were employed to examine the biological functions of BCL6B in the LoVo cells. The mRNA and protein levels of BCL6B, cyclin D1 and matrix metalloproteinase-9 (MMP-9) were determined by RT-PCR and Western blot, respectively. The level of phosphorylated protein kinase B (p-AKT) was detected by Western blot. RESULTS: BCL6B expression was notably repressed in the LoVo cells as compared with the FHC cells, which were significantly increased by transfection with pcDNA3.1-BCL6B. The abilities of proliferation and migration of the LoVo cells at 72 h were inhibited by 28.33%(P<0.01) and 36.11%(P<0.05) in BCL6B group. The mRNA levels of cyclin D1 and MMP-9 in the cells of BCL6B group were decreased by 39.90%(P<0.01) and 77.36% (P<0.05), and the protein levels of cyclin D1, MMP-9 and p-AKT were reduced by 44.00%(P<0.05), 47.06%(P<0.01) and 32.88% (P<0.05), respectively. CONCLUSION: BCL6B inhibits proliferation and migration of the LoVo cells, and the PI3K/AKT signaling pathway is involved in this process.     

Effects of oridonin on invasion and migration of human hepatocellular carcinoma MHCC-97H cells

AIM: To investigate the effects of oridonin on the invasion and migration of hepatocelluar carcinoma cells. METHODS: Human hepatocelluar carcinoma MHCC-97H cells were cultured and treated with 5, 10 or 20 μmol/L oridonin. The migration ability was measured by wound healing assay. The invasion ability was examined by Transwell invasion assay. The adhesion capabilities were evaluated by adhesion assay. The protein levels of LIM kinase-1 (LIMK-1), cofilin and phosphorylated cofilin (p-cofilin) were determined by Western blot. RESULTS: Oridonin inhibited the migration, invasion and adhesion abilities of MHCC-97H cells in a dose-dependent manner (P<0.05). After oridonin treatment, the expression of cofilin had no obvious change, but the protein levels of LIMK-1 and p-cofilin decreased significantly. CONCLUSION: Oridonin inhibits the invasion and migration of MHCC-97H cells. The mechanism may be related with the regulatory effect of oridonin on LIMK-1/cofilin signal transduction pathway.     

Cordycepin inhibits proliferation and migration of gallbladder cancer cell SNU-308 by ERK1/2, Ezrin and Akt signaling pathways

AIM: To investigate the effects of cordycepin on the proliferation and migration abilities of gallbladder cancer cell line SNU-308 and its molecular mechanism. METHODS: The viability of SNU-308 cells treated with cordycepin at different concentrations was measured by MTT assay and the colony formation ability was also detected. The effect of cordycepin on apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The protein levels of apoptosis and autophagy markers, and the phosphorylation level of Akt, ERK1/2 and Ezrin were evaluated by Western blot. Immunofluorescence staining was also used to analyze the expression level of LC3 after cordycepin treatment. Wound healing assay and Transwell assay were performed to evaluate the migration ability of the SNU-308 cells after cordycepin treatment. Wound healing assay was also used to evaluate the effects of Akt inhibitor, ERK1/2 inhibitor and Ezrin knockdown on the changes of migration ability. RESULTS: Cordycepin significantly inhibited the viability and the ability of colony formation of gallbladder cancer cells (P<0.05). Induction of apoptosis by cordycepin were revealed by flow cytometry (P<0.05). The protein expression of Bcl-2 was down-regulated, while the protein levels of Bax, cytochrome C (Cyto C), Fas, FasL and cleaved caspase-3 were increased and the autophagy marker beclin 1 and the ratio of LC3-Ⅱ/I were upregulated by Western blot analysis (P<0.05). LC3 accumulation in the cytoplasm after cordycepin treatment was demonstrated by immunofluorescence staining. Cordycepin treatment resulted in the inhibition of cell migration were detected by Transwell assay and wound healing assay (P<0.05). The protein levels of p-Akt, p-ERK1/2 and p-Ezrin were down-regulated after cordycepin treatment (P<0.05). Besides, Ezrin knockdown, Akti-1/2 and GDC-0994 all resulted in the inhibition of migration ability (P<0.05). CONCLUSION: Cordycepin induces apoptosis and autophagy to inhibit gallbladder can-cer cell proliferation and migration by regulating ERK1/2, Ezrin and Akt signaling pathways.     

PGRN gene silencing on proliferation and migration abilities of human non-small-cell lung cancer A549 cells

AIM: To investigate the effect of small interfering RNA (siRNA)-mediated progranulin (PGRN) gene silencing on the proliferation and migration abilities of human non-small-cell lung cancer A549 cells and its mechanism. METHODS: The mRNA and protein expression levels of PGRN in the A549 cells and human bronchial epithelial (HBE) cells were detected by qPCR and Western blot. A549 cells were transfected with PGRN-siRNA by liposome method. The expression of PGRN at mRNA and protein levels in the A549 cells transfected with PGRN-siRNA was detected by qPCR and Western blot, respectively. The cell viability was measured by MTT assay. The cell proliferation ability was measured by living cells counting and crystal violet staining assays. The cell migration ability was measured by wound-healing and Transwell assays. The protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1, Bcl-2 and Bax were determined by Western blot. The protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated protein kinase B (p-Akt) were also determined by Western blot. RESULTS: The expression of PGRN at mRNA and protein levels was higher in the A549 cells than that in the HBE cells (P<0.05). The expression of PGRN at mRNA and protein levels in the A549 cells transfected with PGRN-siRNA was significantly decreased, and the cell proliferation and migration abilities were significantly decreased. The protein expression levels of PCNA, cyclin D1 and Bcl-2 were significantly reduced and the protein expression level of Bax was significantly increased (P<0.05). Meanwhile, the protein levels of p-ERK1/2 and p-Akt were down-regulated (P<0.05). CONCLUSION: PGRN gene silencing obviously inhibits the proliferation and migration abilities of human non-small-cell lung cancer A549 cells. The PI3K/Akt and MAPK/ERK signaling pathways may play an important role in these processes.     

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.     

Effect of ABCE 1 gene silencing on proliferation and migration of human bladder cancer cell line T24

AIM: To investigate the effect of small interfering RNA (siRNA)-mediated ABCE1 knockdown on the survival, cell cycle and invasion of human bladder cancer cell line T24. METHODS: The siRNA against ABCE1 was constructed and transfected into the T24 cells with Lipofectamine^TM 2000. The expression of ABCE1 was detected by RT-PCR and Western blot. Flow cytometry was used to detect the cell cycle. The effects of ABCE1 gene silencing on proliferation, migration and invasion of T24 cells were evaluated by CCK-8 assay, wound-healing assay and Transwell invasion assay, respectively. RESULTS: Compared with control group and blank group, the mRNA and protein levels of ABCE1 were significantly decreased in experimental group after transfected with ABCE1 siRNA. The cell cycle was arrested at G₀/G₁ phase and the cell number in S phase was decreased in the T24 cells. Compared with control group and blank group, the proliferation of T24 cells in experimental group was inhibited significantly, and the migration and invasion abilities of T24 cells in experimental group decreased significantly. CONCLUSION: Knockdown of ABCE1 gene may decrease migration, invasion and proliferation abilities in T24 cells.     

miRNA-126 inhibits proliferation,migration and invasion of human lung cancer A549 cells via EGFR/AKT/mTOR pathway

AIM: To investigate the effects of microRNA(miRNA)-126 on the proliferation, migration and invasion of human lung cancer cell lines, and to explore its mechanism. METHODS: The A549 cells were transfected with miRNA-126 agomir by Lipofectamine 2000. The expression of miRNA-126 was detected by real-time PCR. The cell activity was detected by MTT assay. The number of viable A549 cells was counted by the method of Trypan blue exclusion. The cell colony-forming capability was determined by cell colony formation test. The cell migration and invasion abilities were assayed by wound healing and Transwell methods, respectively. The protein levels of p-EGFR, EGFR, p-AKT, AKT, p-mTOR and mTOR were determined by Western blot. RESULTS: The expression level of miRNA-126 was significantly increased in the A549 cells compared with negative control(NC) group and control group(P<0.01). The proliferation of A549 cells was decreased extremely after transfected with the miRNA-126 agomir(P<0.01), so did the result of the cell colony-formation test. The migration and invasion abilities of the lung cancer cells were also significantly inhibited. The protein levels of p-EGFR, p-AKT and p-mTOR were significantly down-regulated compared with NC group and control group(P<0.01). CONCLUSION: Over-expression of miRNA-126 significantly inhibits the proliferation, migration and invasion ability of human lung cancer A549 cells by down-regulation of EGFR/AKT/mTOR pathway.     

Effect of long non-coding RNA MEG3 on invasion and migration of colorectal cancer cells

AIM: To investigate the expression of long non-coding RNA maternally expressed gene 3(MEG3) in colorectal cancer(CRC) cells, and to observe the effect of MEG3 on the invasion and migration of CRC cells. METHODS: The levels of MEG3 in human normal colon cell NCM460 and CRC cells SW48 and LoVo were detected by real- time PCR. MEG3 was over-expressed by plasmid transfection, and the effects of MEG3 on the invasion and migration of SW48 and LoVo cells were analyzed by Transwell assay and wound healing assay. The expression of matrix metalloproteinase(MMP) family proteins was determined by Western blotting. RESULTS: The level of MEG3 was down-regulated in CRC cells compared with normal colon cell NCM460. The invasion and migration of CRC cells were reduced after MEG3 over-expression. Transwell invasion and migration assays showed that the numbers of transmembrane SW48 and LoVo cells were smaller in MEG3 over-expression group than control group(CONCLUSION: The expression of MEG3 is down-regulated in CRC cells. Over-expression of MEG3 inhibits the invasion and migration of CRC cells. TIMP-2, MMP-2 and MMP-9 might play an important role in this regulation.     

miRNA-101-3p inhibits proliferation and migration of gastric cancer cells by targeting EZH2

AIM: To investigate the role of microRNA-101-3p (miRNA-101-3p) on the proliferation, apoptosis and invasion of gastric cancer cells and the possible regulatory mechanisms. METHODS: The expression of miRNA-101-3p in two kinds of gastric cancer cells and a gastric mucosal cell line was detected by real-time PCR. The miRNA-101-3p was overexpressed by Lipofectamine 2000 transfection with miRNA-101-3p mimics. The effects of miRNA-101-3p on cell cycle distribution and apoptosis were analyzed by flow cytometry. The effects of miRNA-101-3p on cell proliferation and migration abilities were detected by CCK-8 assay, trypan blue exclusion test and Transwell assay. The protein expression of enhancer of zeste homolog 2 (EZH2) was determined by Western blot. RESULTS: The expression of miRNA-101-3p in gastric cancer cells was lower than that in gastric mucosal cells (P<0.05). The gastric cancer cell MGC-803 had the lowest expression level of miRNA-101-3p. The result of flow cytometry showed that the population of S phase was reduced, and the population of G₀/G₁ phase and the early stage apoptotic rate were increased after the expression of miRNA-101-3p was overexpressed (P<0.05). The results of CCK-8 assay, trypan blue exclusion test and Transwell assay showed that overexpression of miRNA-101-3p significantly reduced the proliferation and migration abilities of gastric cancer cells (P<0.05). Overexpression of miRNA-101-3p decreased the protein level of EZH2 (P<0.05). CONCLUSION: miRNA-101-3p may suppresses the gastric cancer cell proliferation and migration, and promotes the gastric cancer cell apotosis by down-regulation of EZH2.     

Effect of GPNMB on proliferation,apoptosis and invasion of human hepatoma cells

AIM：To investigate the effect of glycoprotein nonmetastatic melanoma protein B (GPNMB) on the proliferation, apoptosis and invasion of human hepatoma HepG2 cells and its molecular mechanisms. METHODS：The GPNMB siRNA and GPNMB-overexpressing vector were constructed, and then transfected into HepG2 cells. MTT assay, flow cytometry and Transwell chamber were used to determine the effects of GPNMB down-regulation and up-regulation on the proliferation, apoptosis and invasive ability of HepG2 cells. RESULTS：The proliferation of HepG2 cells was obviously promoted by the up-regulation of GPNMB. No effect of GPNMB on the apoptosis of HepG2 cells was observed. The invasion of HepG2 cells was also significantly promoted by the up-regulation of GPNMB. When integrin β1 was silenced by siRNA, the promoting effect of GPNMB on the proliferation and invasive ability of HepG2 cells was significantly suppressed. CONCLUSION: GPNMB may promote the proliferation and invasion of HepG2 cells by the interaction with integrin β1, and may be used as a potential therapeutic target in liver cancer.     

Curcumin inhibits migration and invasion of lung cancer PC-9 cells via down-regulation of nectin-4 expression

AIM: To investigate the effects of curcumin on the abilities of migration and invasion in the lung cancer PC-9 cells, and to observe the relationship between curcumin and nectin-4 expression.METHODS: The viability, migration and invasion of lung cancer PC-9 cells treated with curcumin or transfected with siNectin-4 were measured by MTT assay, wound healing test and Transwell assay, respectively. The protein levels of nectin-4, p-AKT and AKT in the PC-9 cells treated with curcumin or transfected with siNectin-4 were detected by Western blot.RESULTS: Curcumin inhibited the viability of PC-9 cells. The wound healing rates and the numbers of the transmembrane cells in curcumin 10 μmol/L and 20 μmol/L groups were decreased compared with control group without curcumin treatment. The expression level of nectin-4 was reduced after curcumin treatment for 24 h. The viability of the PC-9 cells was significantly inhibited after transfected with siNectin-4 for 48 h or 72 h (P<0.01), and the wound healing rates was decreased in siNectin-4 group compared with NC group (P<0.01). The numbers of the transmembrane cells in siNectin-4 group was significantly reduced (P<0.01). Curcumin and knockdown of nectin-4 suppressed the activation of AKT pathway in PC-9 cells. In siNectin-4+curcumin group, the cell viability reduced compared with curcumin group, and wound healing rates, cell invasive ability and AKT phosphorylation levels were decreased.CONCLUSION: Curcumin inhibits migration and invasion of the lung cancer PC-9 cells via down-regulation of nectin-4 expression and inhibition of AKT pathway.     

Effect of reversing methylation of SARI gene by 5-azacytidine on proliferation and invasion of human breast cancer cells

AIM: To observe whether reversing methylation of SARI (suppressor of activator protein-1 regulated by interferon) gene by 5-azacytidine (5-Aza) inhibits the proliferation and invasion of human breast cancer MDA-MB-231 cells. METHODS: MDA-MB-231 cells were treated with 5-Aza at 5 and 10 μmol/L. The methylation of SARI gene promoter was detected by methylation-specific PCR (MSP), and the mRNA expression of SARI was detected by RT-PCR. The protein expression of SARI was determined by Western blot. The cell growth was detected by MTT assay and colony formation assay. The cell invasion ability was detected by Transwell invasion assay. RESULTS: The results of MSP detection showed that the methylation levels of SARI promoter were significantly decreased after 5-Aza treatment (P<0.01). The results of RT-PCR and Western blot showed that the expression of SARI were significantly increased after 5-Aza treatment (P<0.05). The results of MTT and colony formation assays showed that the cell proliferation was significantly decreased after 5-Aza treatment (P<0.05). The results of Transwell invasion test showed that the invasive ability of the cells was significantly decreased after 5-Aza treatment (P<0.05). CONCLUSION: 5-Aza reverses the methylation status of SARI promoter in MDA-MB-231 cells, up-regulates the expression of SARI, and restores its ability to inhibit tumor cell growth and invasion.     

Effects of CENP-W down-regulation on human glioma U87 cells

AIM: To study the effect of centromere protein W (CENP-W) down-regulation on human glioma U87 cells.METHODS: Small interfering RNA (siRNA) was used to inhibit the expression of CENP-W in the U87 cells. The interference effect of siRNA was evaluated by RT-qPCR and Western blot. The proliferation of the cells was analyzed by MTT assay, BrdU staining and colony formation experiment. Transwell chamber assay was used to detect the invasion ability of the cells. The cell migration ability was measured by a scratch test. The changes of the cell cycle distribution and apoptosis were analyzed by flow cytometry.RESULTS: The results of MTT assay, colony formation experiment and BrdU staining showed that the cell proliferation and colony formation abilities in experimental group were significantly lower than those in control group and negative control group. The results of Transwell and scratch experiments showed that the migration and invasion abilities in experimental group were weaker than those in blank control group and negative control group. The results of flow cytometry analysis showed that the cell cycle distribution in experimental group was arrested in G₀/G₁ phase. The percentage of apoptotic cells in experimental group was higher than that in control group (P<0.05).CONCLUSION: Down-regulation of CENP-W expression inhibits the proliferation, migration and invasion of human glioma cells and promotes the apoptosis of the cells, suggesting that CENP-W may be a potential target of gene therapy for human glioma.