Expression of TSTA3 in esophageal squamous-cell carcinoma and its significance

AIM To investigate the expression of TSTA3 in esophageal squamous cell carcinoma (ESCC) and the effects of TSTA3 on the proliferation, invasion and migration abilities of human ESCC cell lines KYSE150 and KYSE450. METHODS The expression of TSTA3 was detected by immunohistochemistry in 45 cases of ESCC and matched adjacent tissues. The mRNA expression levels of TSTA3 in ESCC cells were detected by qPCR. Over-expression of TSTA3 in KYSE150 cells and KYSE450 cells was carried out by lentivirus infection. The effects of TSTA3 on the viability and colony formation ability of ESCC cells were examined by MTT assay and colony formation assay. Transwell assays were performed to detect the effects of TSTA3 on migration and invasion abilities of ESCC cells. The effect of TSTA3 on core fucosylation modification of ESCC cells was analyzed by flow cytometry. RESULTS The expression of TSTA3 in the ESCC tissues was significantly higher than that in matched adjacent tissues (P<0.01). Over-expression of TSTA3 had no effect on the proliferation of ESCC cells (P>0.05), but promoted the migration and invasion of ESCC cells (P<0.01). Moreover, over-expression of TSTA3 increased the core fucosylation modification level of ESCC cells (P<0.01). CONCLUSION TSTA3 may promote the development and progression of ESCC via regualting fucosylation modification level, and serve as a biomarker for early diagnosis and treatment of ESCC.     

Down-regulation of lncRNA PCAT1 suppresses oral squamous cell carcinoma cell proliferation,growth, invasion,migration and epithelial-mesenchymal transition

AIM: To investigate the effects of the long non-coding RNA (lncRNA) PCAT1 on the oral squamous cell carcinoma (OSCC) cell proliferation, growth, invasion and migration, and to explore the underlying mechanisms. METHODS: The PCAT1 siRNA was transfected by Lipofectmine 2000, and RT-qPCR and Western blot were performed to determine the mRNA and protein expression of relevant genes, respectively. CCK-8 assay and colony formation assay were used to measure OSCC cell proliferation and growth, respectively. The cell invasion and migration assays were used to measure the invasive and migratory abilities of the OSCC cells, respectively. RESULTS: PCAT1 was significantly up-regulated in OSCC tissues and cells compared with normal adjacent tissues and normal human oral keratinocyte cells, respectively (P<0.05). PCAT1 siRNA transfection suppressed the expression of PCAT1 in Tca8113 and TSCCa cells (P<0.05). Knockdown of PCAT1 in Tca8133 cells and TSCCa cells significantly suppressed the cell proliferation, invasion and migration abilities (P<0.05). In addition, knockdown of PCAT1 in Tca8133 cells and TSCCa cells also suppressed the mRNA and protein levels of ZEB-1, N-cadherin and vimentin, and increased the mRNA and protein expression of E-cadherin (P<0.05). CONCLUSION: Knockdown of PCAT1 suppresses cell proliferation and migration abilities, and the effect of PCAT1 on OSCC cells may be associated with epithelial-mesenchymal transition.     

Effect of BMP9 over-expression on migration and invasion of human lung squamous-cell carcinoma NCI-H520 cells

AIM: To investigate the effect of bone morphogenetic proteins 9(BMP9) on the migration and invasion abilities of human lung squamous-cell carcinoma NCI-H520 cells and its mechanism. METHODS: The expression of BMP9 at mRNA and protein levels in the NCI-H520 cells and human bronchial epithelial (HBE) cells was detected by RT-PCR and Western blot. The NCI-H520 cells were transfected with the recombinant adenovirus AdBMP9 and the expression of BMP9 at mRNA and protein levels was validated by RT-PCR and Western blot. The migration and invasion abilities of the NCI-H520 cells were determined by wound-healing and Transwell assays. The mRNA and protein levels of the migration-related factor matrix metalloproteinase 2(MMP2) were detected by RT-PCR and Western blot. The level of phosphorylated Smad1/5(p-Smad1/5) was detected by Western blot. Meanwhile, NCI-H520 cells were treated with BMP specific antagonist AdNoggin and AdBMP9. The level of p-Smad1/5 and the cell migration ability were measured by Western blot, wound-healing and Transwell assays. RESULTS: The expression of BMP9 at mRNA and protein levels was lower in NCI-H520 cells than that in HBE cells. After AdBMP9 was stably transfected into the NCI-H520 cells, the expression of BMP9 at mRNA and protein levels was significantly up-regulated, cell migration and invasion abilities were significantly decreased, and the mRNA and protein levels of MMP2 were decreased. Meanwhile, the level of p-Smad1/5 was increased. Noggin reversed BMP9-caused the increase in p-Smad1/5 and the decrease in cell migration ability. CONCLUSION: Over-expression of BMP9 inhibits the migration and invasion abilities of lung squamous-cell carcinoma NCI-H520 cells. The activation of BMP-Smad signaling pathway may be involved in this inhibitory process.     

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.     

Effects of zinc on biological behavior of hepatocellular carcinoma cell line BEL-7404

AIM: To observe the effects of exogenous zinc on the biological behavior of hepatocellular carcinoma (HCC) cell line BEL-7404. METHODS: BEL-7404 cells were cultured with zinc sulfate at various concentrations. The intracellular concentration of zinc, cell viability, cell cycle, cell apoptosis and migration and invasion abilities were measured by TSQ fluorescent probe, MTT assay, DNA ploid analysis, acridine orange/ethidium bromide fluorescence staining and Transwell assay, respectively. The mRNA and protein expression levels of albumin in the BEL-7404 cells were determined by real-time PCR and Western blot, respectively. RESULTS: With the elevated concentration of zinc in culture condition, the concentration of zinc in the BEL-7404 cells was increased (P<0.05). The cell viability and migration and invasion abilities were decreased, while the apoptotic rate was increased (P<0.05). The cells in G₀/G₁ phase were decreased, while the cells in G₂/M phase were increased. Additionally, the mRNA and protein expression of albumin also increased (P<0.05). CONCLUSION: The zinc ion inhibits the cell viability as well as migration and invasion abilities, blocks the cells in G₂/M phase, and may reduce cell malignant phenotype.     

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.     

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.     

Effect of toosendanin on invasion and migration of human ovarian cancer cells

AIM: To investigate the effect of toosendanin (TSN) on invasion and migration abilities of human ovarian cancer cells and the related mechanism. METHODS: The human ovarian cancer cell lines CAVO-3 and SKVO-3 were treated with TSN at different concentrations. The cell viabilty at 12, 24, 48, 72 and 96 h after TSN treatment was measured by CCK-8 assay. Scratch wound healing assay and Transwell assay were employed to measure the invasion and migration abilities of CAVO-3 cells. The protein expression of nuclear factor-κB (NF-κB) p65, E-cadherin, N-cadherin, vimentin and Snail was determined by Western blot. RESULTS: TSN significantly inhibited the viability of CAVO-3 and SKVO-3 cells (P<0.05). Compared with control group, the migration and invasion abilities of CAVO-3 cells in TSN group decreased significantly (P<0.05). In addition, the expression of NF-κB p65 and E-cadherin protein increased notably, followed with N-cadherin, vimentin and Snail protein decreased significantly (P<0.05). However, the inhibitor of NF-κB BAY11-7082 reversed the impact above. Compared with TSN group, the migration and invasion abilities in TSN+BAY11-7082 group increased significantly (P<0.05). The protein expression of E-cadherin also decreased notably, followed with the protein expression of N-cadherin, vimentin and Snail increased significantly (P<0.05). CONCLUSION: TSN inhibits the invasion and migration abilities of human ovarian cancer cells, which is related to the inhibition of epithelial-mesenchymal transition process mediated by NF-κB/Snail signaling pathway.     

Mechanism of miR-30c over-expression inhibiting malignant phenotypes of cervical cancer cells

AIM:To investigate the molecule mechanism of microRNA (miR)-30c over-expression inhibiting malignant phenotypes of cervical cancer cells. METHODS:Cervical cancer cell lines C33A, HeLa, SiHa and CaSki were transfected with pGenesil-1-miR-30c plasmid using Lipofectamine 2000 kit, and the expression of miR-30c was determined by TaqMan real-time PCR. The cell viability inhibition rate, colony formation ability, migration rate and apoptotic rate were measured by MTT assay, colony formation assay, Transwell experiment, and flow cytometry with Annexin V-FITC staining. The protein expression of Bax, Bcl-2, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloprotei-nase-1 (TIMP-1) was detected by Western blot. RESULTS:The expression levels of miRNA-30c in the cervical cancer cell lines transfected with pGenesil-1-miR-30c plasmid were significantly higher than those in negative control groups (cell lines transfected with pGenesil-1 plasmid) (P<0.01). Significantly increased cell viability inhibition rate, and decreased colony formation ability and migration rate were found in the cervical cancer cell lines over-expressing miR-30c as compared with negative control groups (P<0.05). The apoptotic rate in the cervical cancer cell lines over-expressing miR-30c was dramatically higher than that in control groups (P<0.05). Over-expression of miR-30c in cervical cancer cells promoted the protein expression of Bax and TIMP-1, and decreased the protein expression of Bcl-2 and MMP-13 (P<0.05 or P<0.01). CONCLUSION:Over-expression of miR-30c significantly inhibits the viability and migration, and induces apoptosis of cervical cancer cells. The mechanism may be related to activating apoptosis pathway and inhibiting MMP-13 protein expression.     

Calreticulin promotes migration and invasion of nasopharyngeal carcinoma cells by inducing cell EMT

AIM:To observe the expression of calreticulin (CRT) in nasopharyngeal carcinoma tissues, analyze the significance of clinical pathology and the influence on epithelial-mesencymal transition (EMT) of CNE2 cells. METHODS:The expression of calreticulin was detected by immunohistochemistry in 52 nasopharyngeal carcinoma and 57 nasopharyngeal benign tissues, and the significance of clinical pathology was evaluated. The calreticulin gene-specific small interfering RNA was constructed, and then was transfected into the NPC cell line CNE2 using the cationic liposome method. The effect of CRT on the morphological changes of the CNE2 cells was observed under light microscope. The effect of CRT on the cell migration and invasion abilities of the CNE2 cells was detected by Transwell migration and invasion assays. The expression of EMT-related proteins E-cadherin, vimentin, transforming growth factor (TGF)-β and matrix metalloproteinase (MMP)-9 in the CNE2 cells was determined by Western blot. RESULTS:The positive expression rate of CRT in the benign lesion tissues was 19.29% (11/57), which was significantly increased in the nasopharyngeal carcinoma tissues as 82.69% (43/52). The expression rate of CRT was positively correlated with the stage of nasopharyngeal carcinoma and lymph node metastasis (P<0.05). Knockdown of CRT expression made the CNE2 cells showing a spindle shape to a flat, cobblestone-like epithelial state change, arranged more compact, and the migration and invasion abilities were significantly decreased (P<0.05). Knockdown of CRT expression resulted in significant increase in the protein expression of E-cadhe-rin, and the decreases in the protein expression of vimentin, TGF-β and MMP-9 in the CNE2 cells (P<0.05). CONCLUSION:Calreticulin expression in nasopharyngeal carcinoma is significantly higher and positively correlated with nasopharyngeal carcinoma stage and lymph node metastasis. Calreticulin promotes cell migration and invasion of nasopharyngeal carcinoma CNE2 cells by inducing EMT.     

Reduced expression of SIRT2 in serous ovarian carcinoma promotes cell proliferation,migration and invasion

AIM: To compare the expression of SIRT2 in ovarian surface epithelial (OSE) cell line and serous ovarian carcinoma (SOC) cell lines, and to investigate the effects of SIRT2 on the cell proliferation, migration and invasion. METHODS: The expression levels of SIRT2 in the OSE cell line and the SOC cell lines were determined by Western blot. The SIRT2 siRNAs and overexpression construct were designed and verified. Transient transfection of SIRT2 siRNAs or overexpression construct was performed, and the effect of SIRT2 on the cell proliferation, migration and invasion was evaluated. RESULTS: SIRT2 levels in the 5 strains of SOC cell lines were significantly lower than that in the OSE cell line. SIRT2 knockdown in HOSEpiC cells significantly enhanced the ability of cell colony formation and accelerated the cell growth rate. On the contrary, overexpression of SIRT2 in HO8910 cells dramatically repressed the number of cell colonies and cell activity. SIRT2 significantly changed the ability of ovarian cell migration. Knockdown of SIRT2 facilitated the cell invasion. CONCLUSION: The expression of SIRT2 in the SOC cells is significantly down-regulated. In the OSE cells, SIRT2 acts as a tumor suppressor and mediates the inhibition of cell proliferation, migration and invasion.     

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.     

Effects of miRNA-25 on proliferation of human esophageal squamous-cell carcinoma cell line TE1

AIM: To investigate the effects and mechanisms of microRNA-25(miRNA-25) on the proliferation of human esophageal squamous-cell carcinoma cell line TE1. METHODS: The abundance of miRNA-25 in different tissues was measured by RT-PCR. After silencing or over-expression of miRNA-25 with mimics or inhibitor in TE1 cells, the cell proliferation, cell cycle distribution and the expression of cyclin E1 and cyclin-dependent kinase 2(CDK2) at mRNA and protein levels were measured by CCK-8 assay, BrdU detection, flow cytometry, RT-PCR and Western blot, respectively. RESULTS: miRNA-25 was prominent in esophageal mucosal tissue and highly expressed in TE1 cells (P<0.05). Over-expression of miRNA-25 increased TE1 cell proliferation, promoted the cell cycle progression and enhanced the entrance of the cells into S phase (P<0.05). Inverse results were obtained after down-regulation of miRNA-25(P<0.05). Furthermore, the expression of cyclin E1 and CDK2 at mRNA and protein levels was significantly increased after over-expression of miRNA-25, but decreased after down-regulation of miRNA-25(P<0.05). CONCLUSION: miRNA-25 enhances cell cycle transition by increasing the expression of cyclin E1 and CDK2, thus accelerating TE1 cell proliferation. This study provides a novel mechanism by which miRNA-25 increases the proliferation of human esophageal squamous-cell carcinoma cell line TE1, suggesting that down-regulation of miRNA-25 may be a potential new therapeutic strategy for treating esophageal squamous-cell carcinoma.     

Baicalein inhibits proliferation and migration of gastric cancer MGC-803 cells

AIM: To investigate the effects of baicalein (BAI) on the proliferation and migration of gastric cancer MGC-803 cells and the mechanisms. METHODS: After MGC-803 cells were treated with BAI at different concentrations, the viability of the MGC-803 cells was tested by MTT assay. The cell colony formation ability were detected by plate colony formation assay. Wound-healing and Transwell cell migration assays were used to test the migration ability of the MGC-803 cells. The concentration of 12-hydroxyeicosatetraenoic acid (12-HETE) was measured by ELISA. The protein levels of platelet type 12-lipoxygenase (p12-LOX), vascular endothelial growth factor (VEGF), p-ezrin and epithelial-mesenchymal transition (EMT) markers in MGC-803 cells were determined by Western blot. RESULTS: BAI significantly inhibited the proliferation, plate colony formation and migration abilities of the MGC-803 cells (P<0.05 or P<0.01), down-regulated the concentration of p12-LOX metabolite 12-HETE significantly (P<0.05 or P<0.01), decreased the protein levels of p12-LOX, VEGF, p-ezrin, vimentin and Snail (P<0.05 or P<0.01), and increased the protein expression of E-cadherin (P<0.01). CONCLUSION: BAI suppresses the proliferation and migration abilities of gastric cancer MGC-803 cells effectively. These effects of BAI may be related to regulating the protein levels of p12-LOX, VEGF, p-ezrin and EMT-related proteins.     

Effect of digoxin on hypoxia-induced epithelial-mesenchymal transition and invasion in human breast carcinoma MCF-7 cells

AIM: To investigate the effect of digoxin on hypoxia-induced epithelial-mesenchymal transition (EMT), migration and invasion in human breast carcinoma MCF-7 cells. METHODS: MCF-7 cells were treated in vitro with a chemical hypoxia inducer cobalt chloride (CoCl₂) to imitate hypoxia. Cell migration was observed by wound healing assay, and cell invasion was measured by Transwell invasion assay. The protein levels of hypoxia-inducible factor-1α (HIF-1α), Snail, E-cadherin and vimentin in MCF-7 cells were detected by Western blot. RESULTS: Digoxin inhibited CoCl₂-induced EMT and reversed the mesenchymal phenotype. CoCl₂ enhanced the abilities of migration and invasion (P<0.01), significantly decreased the expression of E-cadherin and increased the expression of HIF-1α, Snail and vimentin (P<0.01), but these effects were blocked by digoxin. CONCLUSION: Digoxin inhibits CoCl₂-induced EMT and invasion most likely via HIF1-α-Snail signaling pathway.     

Rab1A knockdown inhibits malignant biological behaviors of breast carcinoma cells by inhibiting phosphorylation of AKT

AIM: To investigate the role of Rab1A gene in the malignant biological behaviors of breast carcinoma cells. METHODS: The expression levels of Rab1A in breast carcinoma tissues and normal adjacent tissues, and the basic expression level of Rab1A in different breast carcinoma cell lines were measured by Western blot. Small interfering RNA (siRNA) targeting Rab1A was designed, synthetized and transfected into the breast carcinoma MDA-MB-231 cells. After validation of efficiency of Rab1A gene expression knock-down, the malignant biological behaviors of the MDA-MB-231 cells were measured by CCK-8 assay, wound healing assay, Transwell assay and flow cytometry. The protein levels were determined by Western blot. RESULTS: Rab1A was expressed in normal breast tissue and cells at low level, and at high level in the cancer tissues and cancer cells (P<0.05). Compare with control group, after knock-down of Rab1A expression, the viability of MDA-MB-231 cells was significantly inhibited (P<005), the abilities of migration and invasion were reduced (P<0.05), the apoptosis was decreased (P<0.05), the percentage of G²/M phase was increased, the protein levels of p53, Bax, cleaved caspase-3 and PTEN were significantly increased (P<0.05), and the protein levels of Bcl-2, cyclin D1, cyclin B1, matrix metalloproteinase 2 (MMP2), p-AKT and mTOR were significantly decreased (P<0.05). CONCLUSION: Rab1A modulates the breast carcinoma cell viability, inhibits the migration and invasion abilities, induces G² arrest and effectively regulates the cell growth-, cell cycle-and apoptosis-related proteins. Knock-down of Rab1A expression inhibits the evolution and development of breast cancer by inhibiting the phosphorylation of AKT pathway, and Rab1A may function as a potential target in breast carcinoma treatment.     

Linarin inhibits migration and invasion abilities of human breast cancer MDA-MB-231 cells through IKK/NF-κB signaling pathway

AIM: To investigate the effect of linarin (LIN) on the migration and invasion abilities of human breast cancer MDA-MB-231 cells and its underlying mechanism. METHODS: MCF-7, MDA-MB-231 and MCF-10A cells were cultured in vitro and treated with LIN at 5, 10, 20, 40, 80 and 160 μmol/L for 24 h, and the cell proliferation was measured by CCK-8 assay and colony formation assay. The protein levels of Snail, E-cadherin, matrix metalloproteinase-9 (MMP-9), IκBα, p-IKKα/β and p-p65 were determined by Western blot. RESULTS: LIN remarkably reduced the viability of MDA-MB-231 cells in a dose-dependent manner (P<0.05), and the IC₅₀ was 55.89 μmol/L for 24 h. LIN decreased the colony formation rate of MDA-MB-231 cells at the concentration of 20 μmol/L (P<0.05). After exposed to LIN at 5 μmol/L and 10 μmol/L for 24 h, the migration and invasion abilities of the MDA-MB-231 cells were significantly reduced (P<0.05), the protein expression levels of E-cadherin and IκBα were up-regulated (P<0.05), the protein expression levels of Snail and MMP-9 were down-regulated (P<0.05), and the phosphorylation levels of IKKα/β and p65 were decreased (P<0.05) in comparison with the control group. Meanwhile, IKK-16 (IKKα/β inhibitor) and PDTC (NF-κB inhibitor) also down-regulated the protein expression levels of Snail and MMP-9 (P<0.05), and up-regulated the protein expression level of E-cadherin (P<0.05). CONCLUSION: LIN down-regulates the protein expression levels of Snail and MMP-9, and up-regulates the protein expression level of E-cadherin most likely through inhibiting IKK/NF-κB signaling pathway, and ultimately lead to decreases in the migration and invasion abilities of MDA-MB-231 cells.     

PTBP1 promotes migration and invasion of liver cancer cells through EMT pathway

AIM: To investigate the molecular mechanism of polypyrimidine tract-binding protein 1 (PTBP1) promoting the migration and invasion of hepatoma cells. METHODS: The differentially expressed splicing proteins in different cell lines were screened by qPCR and Western blot. The difference of the expression of PTBP1 between liver cancer and normal liver tissues was analyzed by bioinformatics. Wound-healing and Transwell assays were used to study the effect of PTBP1 on the migration and invasion of hepatoma cells, and Western blot was used to detect the effect of PTBP1 on epithelial-mesenchymal transition (EMT) signaling pathway. RESULTS: Compared with the HepG2 cells, the expression of splicing factor PTBP1 was significantly increased in the HCCLM3 cells with high metastatic ability (P<0.05), and the expression level of PTBP1 in hepatocellular carcinoma tissues was significantly higher than that in normal tissues (P<0.05). Over-expression of PTBP1 significantly increased the migration and invasion of HCCLM3 cells (P<0.05), increased the expression of mesenchymal marker proteins N-cadherin and vimentin (P<0.05), and promoted the EMT process of liver cancer cells. CONCLUSION: PTBP1 promotes the migration and invasion of liver cancer cells by promoting the EMT pathway of liver cancer cells.     

miR-105 inhibits cell proliferation,migration and invasion abilities in non-small-cell lung cancer cells by targeting and negative regulation of KIFC1

AIM:To study the effects of microRNA-105(miR-105) on the cell proliferation, migration and invasion abilities of non-small-cell lung cancer (NSCLC) H460 cells, and further to explore its mechanism. METHODS:The expression of miR-105 and kinesin family member C1 (KIFC1) mRNA in the NSCLC tissues and adjacent tissues and cells was detected by RT-qPCR. The protein expression of KIFC1 in the NSCLC tissues, adjacent normal tissues and cells was determined by Western blot. The H460 cells were divided into miR-105 group (transfection with miR-105 mimics), miR-negative control (NC) group (transfection with miR-NC), inhibitor-NC group (transfection with NC of inhibitor), inhibitor-miR-105 group (transfection with miR-105 inhibitor), si-NC group (transfection with NC siRNA), si-KIFC1 group (transfection with KIFC1 siRNA), miR-105+vector group (miR-105 mimics and pcDNA 3.1 co-transfection) and miR-105+KIFC1 group (miR-105 mimics and pcDNA 3.1-KIFC1 co-transfection). The cell proliferation was measured by MTT assay and colony formation assay. The migration and invasion abilities were detected by Transwell methods. The relative luciferase acitivity was evaluated by double luciferase reporter assay. RESULTS:Compared with the adjacent tissues, the expression of miR-105 was significantly decreased and the expression of KIFC1 was significantly increased in NSCLC tissues (P<0.05). Compared with human normal embryonic lung fibroblasts MRC-5, the expression of miR-105 in the H460 cells was significantly decreased, and the expression of KIFC1 was significantly increased (P<0.05). miR-105 inhibited the relative luciferase activity of H460 cells with wild-type KIFC1 and negatively regulated the protein expression of KIFC1. Over-expression of miR-105 and knockdown of KIFC1 expression significantly inhibited the proliferation, migration and invasion abilities of H460 cells. Over-expression of KIFC1 reversed the inhibitory effect of miR-105 on the cell proliferation, migration and invasion abilities of H460 cells. CONCLUSION:miR-105 inhibits the proliferation, migration and invasion abilities of NSCLC cells. The mechanism may be related to targeting and negatively regulating expression of KIFC1.     

Effect of sirtuin 6 on proliferation of hepatocellular carcinoma cells

AIM: To illuminate the effect of sirtuin 6 (SIRT6) on the proliferation of hepatocellular carcinoma (HCC) cells. METHODS: The mRNA expression of SIRT6 in the peripheral blood from 200 cases of HCC patients and 50 cases of healthy people was detected by real-time quantitative PCR (RT-qPCR). The mRNA expression levels of SIRT6 in the peripheral blood from 200 cases of HCC patients were combined with multiple clinicopathologic parameters for statistical analysis. The protein expression of SIRT6 in primary hepatocytes, immortalized hepatocytes and 4 hepatoma cell lines were determined by Western blotting. The silencing of SIRT6 was conducted by transfection of vector expressing short hairpin RNA targeting on SIRT6, and the protein level of SIRT6 was measured by Western blotting. The viability of HCC cells was tested by MTS assay. DNA synthesis was analyzed by Cell-Light^TM EdU Apollo® 488 In Vitro Imaging Kit. The abilities of colony formation and anchorage-dependent growth were measured by colony formation assay and soft agar assay, respectively. RESULTS: The mRNA expression of SIRT6 in the peripheral blood of HCC patients was significantly higher than that in the healthy people, and its expression was highly associated with tumor size, tumor grade and vascular invasion. SIRT6 expression in 4 hepatoma cell lines was significantly higher than that in the others. SIRT6 silencing led to a significant decrease in the cell viability as tested by MTS assay. EdU staining revealed that SIRT6 silencing reduced DNA synthesis. SIRT6 silencing reduced the ability of colony formation and anchorage-dependent growth as determined by colony formation assay and soft agar assay, respectively. CONCLUSION: Sirtuin 6 promotes the proliferation and malignant transformation of HCC cells.