High mobility group box 1 protein promotes proliferation,migration and invasion of colon cancer cells

AIM: To investigate the expression of high mobility group box 1 protein (HMGB1) in colon can-cer cells, and to determine its regulatory roles in colon cancer cell proliferation, migration and invasion. METHODS: qPCR and Western blot were used to quantify the mRNA and protein expression levels of HMGB1 in human colon cancer SW620 cells and normal colonic epithelial FHC cells. HMGB1 shRNA was transfected into the SW620 cells to establish the stable HMGB1-downregulating colon cancer cells (shHMGB1 group), and negative control (shNC) group and blank control (blank) group were also set up. The proliferation, migration and invasion of the cells were determined by CCK-8 assay, colony formation experiment and Transwell chamber assays. Western blot was used to determine the protein levels of p-ERK, ERK, c-Myc, matrix metalloproteinase (MMP)-2/9, E-cadherin, N-cadherin, Bcl-2 and Bax. RESULTS: Both of the mRNA and protein levels of HMGB1 in colon cancer cells were higher than those in the normal colonic epithelial cells (P<0.05). HMGB1 gene was successfully knocked down in SW620 cells. Compared with blank group and shNC group, the proliferation, migration and invasion abilities of the cells in shHMGB1 group were significantly inhibited (P<0.05). The protein levels of MMP-2, MMP-9, N-cadherin, c-Myc, Bcl-2 and p-ERK were reduced notably, while the expression of Bax protein was increased (P<0.05) in shHMGB1 group compared with shNC group and blank group.CONCLUSION: HMGB1 effectively promotes the proliferation, migration and invasion of colon cancer cells through ERK/c-Myc signaling pathway.     

Sinomenine inhibits viability,migration and invasion of human ovarian cancer SKOV3 cells

AIM:To investigate the effect of sinomenine on the viability, migration and invasion of human ovarian cancer SKOV3 cells and its possible mechanism. METHODS:The SKOV3 cells were treated with sinomenine at different concentrations for 12 h, 24 h and 48 h. CCK-8 assay was employed to detect the effects of sinomenine on the viability of the SKOV3 cells. Flow cytometry was used to analyze the cell cycle distribution. The cell migration and invasion abilities were measured by Transwell assay. Western blot was used to determine the protein levels of cyclin A, cyclin D1, E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS:Sinomenine remarkably inhibited the viability of SKOV3 cells and IOSE80 cells in a time-dependent and dose-dependent manner (P<0.05), and the IC₅₀ values of 48 h were 2.12 mmol/L and 17.35 mmol/L, respectively. In a dose-dependent manner, sinomenine induced G₀/G₁ and S phase arrest in SKOV3 cells (P<0.05), suppressed the migration and invasion abilities of SKOV3 cells (P<0.05), down-regulated the protein levels of cyclin A, cyclin D1 and MMP-9 (P<0.05), and up-regulated the protein level of E-cadherin (P<0.05). CONCLUSION:Sinomenine inhibits the viability, migration and invasion of human ovarian cancer SKOV3 cells most likely via down-regulation of the protein levels of cyclin A, cyclin D1 and MMP-9, and up-regulation of the protein level of E-cadherin.     

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.     

Effect of DEC1 gene over-expression on proliferation and invasion abilities of human esophageal cancer ECA109 cells

AIM: To investigate the effect of DEC1 gene over-expression on the proliferation and invasion abilities of human esophageal cancer ECA109 cells.METHODS: ECA109 cells were transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group). The mRNA and protein levels of DEC1, cyclin D1 and MMP-9 were evaluated by real-time PCR and Western blot, respectively. The effects of DEC1 over-expression on the proliferation and invasion abilities of the ECA109 cells were evaluated by CCK-8 assay, colony formation assay and Transwell test respectively.RESULTS: The DEC1 expression level in ECA109 cells in DEC1 group was significantly higher than that in vector group (P<0.01), but the levels of MMP9 and cyclin D1 expression were opposite (P<0.01). However, both the proliferation and invasion abilities of ECA109 cells in DEC1 groups decreased significantly as compared with those in vector group (P<0.05).CONCLUSION: The over-expression of DEC1 significantly inhibits the proliferation and invasion of ECA109 cells, which may be involved in the expression of cyclin D1 and MMP9.     

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.     

Effect of FGFR1 expression knock-down on biological characteristics of infantile hemangioma endothelial cells

AIM: To study the effect of fibroblast growth factor receptor 1 (FGFR1) expression knock-down on the viability, apoptosis, invasion and migration of infantile hemangioma endothelial cells (HemECs). METHODS: FGFR1 was down-regulated by FGFR1 small interfering RNA (si-FGFR1) transfection. The viability of the cells was measured by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry and the invasion and migration abilities were determined by Transwell assay. The protein levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and phosphorylated AKT (p-AKT) were examined by Western blot. RESULTS: Transfection of si-FGFR1 into HemECs had significant effects on inhibiting cell viability (P<0.05), promoting apoptosis (P<0.05), and decreasing cell invasion and migration abilities (P<0.05). The results of Western blot showed that knockdown of FGFR1 gene expression in the cells reduced the protein levels of PI3K and p-AKT (P<0.05), and had no significant effect on AKT protein level. CONCLUSION: Knock-down of FGFR1 expression changes the biological characteristics of endothelial cells in infantile hemangiomas by regulating PI3K/AKT signaling pathway.     

Construction of SETD2 gene knockout nasopharyngeal carcinoma cell strains using CRISPR/Cas9 technique and analysis of their proliferation property

AIM:To establish SETD2 gene knockout nasopharyngeal carcinoma (NPC) cell strains based on CRIPSR/Cas9 technique and to analyze their proliferation characteristics. METHODS:Sub-quantitative RT-PCR and Western blot were used to detect the expression of SETD2 in immortalized nasopharyngeal epithelial cell line NP-69, well differentiated NPC cell line CNE1, poorly-differentiated NPC cell line CNE2Z and undifferentiated NPC cell line C666-1, and the SETD2 high expression cell line CNE1 was screened. The proliferation ability of CNE1 cells before and after the SETD2 gene knockout was analyzed by CCK-8 and colony formation assay. The cell cycle distribution was detected by flow cytometry, and the expression of cell cycle-related proteins was detected by Western blot. RESULTS:Compared with NP-69 cells, the expression of SETD2 was decreased gradually in CNE1, CNE2Z and C666-1 cells (P<0.01). Based on the CRISPR/Cas9 technique, 2 monoclonal cell strains with SETD2 gene stable knockout, named CNE1-SETD2-KO-#5 and #9, were successfully screened from total 15 monoclones. The results of CCK-8 and plate colony formation assay confirmed that the proliferation ability of CNE1-SETD2-KO-#5 and #9 cells was significantly enhanced compared with CNE1-WT cells (P<0.05). The results of flow cytometry analysis showed that the G₁ phase of CNE1-SETD2-KO-#5 and #9 cells was decreased, while the G₂/M and S phases were increased significantly (P<0.05). The results of Western blot confirmed the increases in the protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin B1, cyclin A2, cyclin E1, cyclin-dependent kinases 2 (CDK2) and CDK4, and the decrease in the protein level of p21 after SETD2 gene knockout (P<0.05). CONCLUSION:The NPC cell strains with SETD2 gene knockout were successfully constructed based on CRISPR/Cas9 technique. SETD2 expression correlates with cell differentiation status in the NPC cells. SETD2 gene knockout promotes NPC cell proliferation by up-regulating cyclin D1, cyclin B1, cyclin A2, cyclin E1, CDK2 and CDK4, and down-regulating p21 expression.     

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.     

Effect of hirsutine on hypoxia-induced migration and invasion abilities in human breast cancer MCF-7 cells

AIM: To investigate the effect of hirsutine on hypoxia-induced migration and invasion abilities of human breast cancer MCF-7 cells and its possible mechanism. METHODS: CCK-8 assay was employed to detect the cytotoxic effect of hirsutine on the MCF-7 cells. Cell migration was observed by wound healing assay, and cell invasion ability was measured by Transwell invasion assay. Western blot was used to analyze the protein levels of hypoxia-inducible factor-1α (HIF-1α), Snail, E-cadherin and matrix metalloproteinase-9 (MMP-9). The mRNA levels of HIF-1α was detected by RT-PCR. RESULTS: Hirsutine remarkably reduced the cell viability from 32 μmol/L (P<0.05), and the IC₅₀ value was 62.82 μmol/L. In hypoxia state, MCF-7 cells showed more powerful capabilities of migration and invasion (P<0.05), higher protein levels of HIF-1α, Snail and MMP-9 (P<0.05), lower protein level of E-cadherin (P<0.05), and higher mRNA level of HIF-1α (P<0.05). These hypoxia-induced effects were all inhibited by hirsutine at 16 μmol/L (P<0.05), apart from the mRNA level of HIF-1α. CONCLUSION: Hirsutine inhibits hypoxia-induced migration and invasion in human breast cancer MCF-7 cells most likely via down-regulation of the protein levels of HIF-1α, Snail and MMP-9, and up-regulation of the protein level of E-cadherin.     

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-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.     

miR-556-3p regulates viability,migration and invasion of endometrial cancer cells by targeting SASH1

AIM To investigate whether microRNA-556-3p （miR-556-3p） regulates the viability, migration and invasion of endometrial cancer cells by targeting SASH1 gene. METHODS The expression of miR-556-3p, and the mRNA and protein levels of SASH1 in endometrial cancer tissues were detected by RT-qPCR and Western blot. Anti-miR-556-3p or pcDNA-SASH1 was transfected into endometrial cancer Ishikawa cells. The cell viability was detected by MTT assay, the migration and invasion abilities of the cells were detected by Transwell chamber method, and the protein expression levels of cyclin D1, p21, matrix metalloproteinase-2 （MMP-2） and MMP-9 were detected by Western blot. StarBase prediction and dual-luciferase reporter experiments were used to analyze the targeting relationship between miR-556-3p and SASH1. Anti-miR-556-3p and si-SASH1 were co-transfected into the Ishikawa cells, and their effects on cell viability, migration and invasion were examined by the methods described above. RESULTS Compared with adjacent tissues, the expression of miR-556-3p in endometrial cancer tissues was increased significantly, and the expression of SASH1 at mRNA and protein levels was decreased significantly （P<0.05）. Inhibition of miR-556-3p expression or induction of SASH1 over-expression obviously reduced the viability of Ishikawa cells, the number of migratory cells, the number of invasive cells and the protein levels of cyclin D1, MMP-2 and MMP-9, and dramatically increased the protein level of p21 （P<0.05）. miR-556-3p targeted SASH1 and negatively regulated its expression. Knock-down of SASH1 expression reversed the inhibitory effect of miR-556-3p expression inhibition on the viability, migration and invasion of Ishikawa cells. CONCLUSION Inhibition of miR-556-3p expression suppresses the viability, migration and invasion of endometrial cancer cells. The mechanism is related to the regulation of its target gene SASH1.     

Over-expression of SCUBE2 suppresses epithelial-mesenchymal transition through Wnt/β-catenin signaling pathway in colorectal cancer cells

AIM: To study the effect of SCUBE2 on epithelial-mesenchymal transition (EMT) in colorectal cancer cells and its mechanism. METHODS: The expression of SCUBE2 in human colorectal cancer cell line HCT116 and normal colonic cell line FHC was detected by real-time PCR and Western blot. HCT116 cells were transfected with GV144-SCUBE2 to over-express SCUBE2, and then the cell viability, migration, and apoptosis were determined by MTT assay, Transwell assay and flow cytometry, respectively. The expression of EMT markers (E-cadherin, vimentin, and Snail), β-catenin, c-Myc and cyclin D1 in the HCT116 cells was analyzed by real-time PCR or Western blot after transfection with GV144-SCUBE2 for 6 h, followed by the stimulation of 10 μg/L recombinant TGF-β1 protein for 48 h. Additionally, the EMT process of HCT116 cells, which were stimulated by TGF-β1, over-expressed SCUBE2, and treated with Wnt/β-catenin pathway activator lithium chloride (LiCl) or inhibitor XAV93920, was analyzed by Western blot. RESULTS: Compared with FHC cells, the expression of SCUBE2 in the HCT116 cells was significantly decreased. The viability and migration ability of the HCT116 cells were suppressed by SCUBE2 over-expression, but the apoptosis was not markedly changed. Elevated expression of SCUBE2 increased E-cadherin expression, and decreased the expression of vimentin, Snail, β-catenin, c-Myc and cyclin D1 induced by TGF-β1. Treatment with LiCl blocked but treatment with XAV93920 enhanced the effects of SCUBE2 on EMT. CONCLUSION: Over-expression of SCUBE2 may inhibit the cell growth and migration, and suppress EMT through Wnt/β-catenin signaling pathway.     

Effects of propofol on biological characteristics of colorectal cancer cells

AIM: To investigate the effect of propofol on the viability, invasion ability and apoptosis of colorectal cancer cells.METHODS: Propofol at 10, 25, 50 and 100 μmol/L was used to treat LoVo cells for 72 h, and propofol at 100 μmol/L was used to treat the LoVo cells for 12, 24, 48 and 72 h. The cell viability was measured by CCK-8 assay. The invasion ability of the LoVo cells treated with propofol at 100 μmol/L for 72 h was detected by Transwell assay. The cell cycle distribution and cell apoptotic rate were analyzed by flow cytometry. The protein levels of matrix metalloproteinase (MMP)-2, MMP-9, cleaved caspase-3, Notch1 and hairy and enhancer of split 1 (Hes1) were determined by Western blot.RESULTS: Propofol inhibited LoVo cell viability. The cell invasion ability, S stage cells, and the protein levels of MMP-2, MMP-9, Notch1 and Hes1 in propofol group were significantly lower than those in control group, and the apoptotic rate, G₀/G₁ cells and the protein level of cleaved caspase-3 were significantly higher than those in control group (P<0.01).CONCLUSION: Propofol inhibits the viability and invasion ability of colorectal cancer LoVo cells, blocks cell cycle and induces apoptosis. The mechanism is related to down-regulation of Notch1 signaling pathway.     

Effect of di-indolyl thiozoline on proliferation of lung cancer A549 cells

AIM: To investigate the effect of di-indolyl thiozoline (DIIT) on the proliferation of human lung cancer A549 cells. METHODS: The effects of DIIT on the proliferation of human lung cancer A549 cell line were determined by CCK-8 assay and EdU assay. The effects of DIIT on the expression of cyclin-dependent kinase 4 (CDK4), cyclin D1, and the phosphorylation of Akt and mTOR were determined by Western blot. RESULTS: After the A549 cells were treated with DIIT at 12.5, 25, 50 and 100 mg/L, the cell viability detected by CCK-8 assay was decreased by 12%, 27% (P<0.01), 33% (P<0.01) and 52% (P<0.01), respectively, compared with DMSO control group. The EdU positive cell number determined by EdU assay was decreased by 10%, 21% (P<0.05), 26% (P<0.05) and 34% (P<0.01), respectively, compared with DMSO control group. Compared with DMSO control group, DIIT inhibited the phosphorylation of Akt and mTOR and the expression of cyclin CDK4 and cyclin D1 (P<0.05). CONCLUSION: Di-indolyl thiozoline inhibits the proliferation of A549 cells, which may be related to the decreases in phosphorylation levels of Akt and mTOR and the inhibition of cell cycle-related protein expression.     

Effects of kaempferol-3-O-rutinoside on proliferation,migration and TGFBR1 signaling pathway activation in vascular smooth muscle cells

AIM:To investigate the effects of kaempferol-3-O-rutinoside (KR) on the proliferation, migration of vascular smooth muscle cells (VSMC) and the activation of transforming growth factor β receptor 1 (TGFBR1) signaling pathway in the cells. METHODS:The viability of VSMC was detected by MTT assay. The proliferation of VSMC was measured by EdU staining. The migration ability of VSMC was examined by Transwell assay. The protein levels of the migration-associated proteins matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) were detected by Western blot. Molecular docking study was conducted to explore the interaction between KR and TGFBR1. The protein le-vels of the phosphorylated TGFBR1, Smad2 and Smad3 were determined by Western blot. RESULTS:KR inhibited the viability of VSMC in a dose-and time-dependent manner. KR reduced the ratio of EdU-positive cells in a dose-dependent manner. KR dose-dependently suppressed the migration ability of VSMC and decreased the protein levels of MMP2 and MMP9 (P<0.05). KR docked into TGFBR1 with the binding energy of -9.804 kcal/mol by forming hydrogen bonds with SER-280, ARG-215, ASP-290 and LYS-335 of TGBFR1. KR dose-dependently suppressed the activation of TGFBR1 and its downstream proteins Smad2 and Smad3 (P<0.05). CONCLUSION:KR inhibits the proliferation and migration of VSMC, possibly via blocking the TGFBR1 signaling pathway.     

PPARγ mediates effects of diosgenin on proliferation and apoptosis in human glioblastoma U87MG cells

AIM:To investigate the effect of diosgenin (Dio) on the proliferation, apoptosis and expression of peroxisome proliferator-activated receptor γ (PPARγ) in human glioblastoma U87MG cells and its possible mechanism. METHODS:Human astrocytes (HA) and U87MG cells were cultured in vitro and treated with Dio (0, 10, 20, 30, 40 and 50 μmol/L) and GW9662 (5 μmol/L) for 48 h, and then the cell viability was detected by CCK-8 assay. Cell colony formation assay was used to assess the proliferation potential. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. The mRNA expression level of PPARγ was measured by RT-PCR. Western blot was used to determine the protein levels of PPARγ, cyclin D1, cyclin E1, Bcl-2 and Bax. RESULTS:Dio had no significant influence on the viabi-lity of HA (P>0.05). However, Dio remarkably reduced the viability of U87MG cells in a dose-dependent manner (P<0.05) with IC₅₀ of 24.31 μmol/L. Meanwhile, Dio remarkably diminished colony formation ability (P<0.05), induced G₀/G₁ phase arrest of the cell cycle and apoptosis (P<0.05), up-regulated the expression of PPARγ at mRNA and protein levels, increased the protein level of Bax (P<0.05), and down-regulated the protein levels of cyclin D1, cyclin E1 and Bcl-2 (P<0.05) in a dose-dependent manner. However, these effects induced by Dio were inhibited by GW9662 (P<0.05), a specific inhibitor of PPARγ. CONCLUSION:Dio may inhibit proliferation and induce apoptosis in human glioblastoma U87MG cells most likely via up-regulating the expression of PPARγ, and then down-regulating the protein levels of cyclin D1, cyclin E1 and Bcl-2, and up-regulating the protein level of Bax.     

Effects of down-regulation of AEG-1 expression on cell cycle and invasion of human cervical carcinoma cells

AIM: To investigate the effects of down-regulation of astrocyte elevated gene-1 (AEG-1) expression on cell cycle and invasion of human cervical carcinoma SiHa cells.METHODS: The protein expression of AEG-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, HeLa cells, SiHa cells and CaSki cells. Control siRNA or AEG-1 siRNA was transfected into SiHa cells, and the protein expression of AEG-1 in SiHa cells was detected by Western blotting. The changes of cell cycle distribution and cell invasion were determined by flow cytometry and Boyden chamber, respectively. The protein levels of cyclin D1, cyclin-dependent kinase 2(CDK2) and matrix metalloproteinase-9 (MMP-9) were analyzed by Western blotting.RESULTS: The protein expression of AEG-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues (P<0.05). Meanwhile, the protein expression of AEG-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which SiHa cells displayed the highest AEG-1 protein level (P<0.05). In addition, AEG-1 siRNA effectively down-regulated the protein expression of AEG-1 in SiHa cells, which led to increase the percentage at G0/G1 phase and reduced the invasion of SiHa cells. Furthermore, the protein levels of cyclin D1, CDK2 and MMP-9 in AEG-1 siRNA group were markedly lower than those in non-treatment group and control siRNA group (P<0.05).CONCLUSION: Over-expression of AEG-1 may be closely associated with the occurrence and development of cervical carcinoma, and the AEG-1 down-regulation-mediated cell cycle arrest and attenuation of invasion may be tightly related to the down-regulations of cyclin D1, CDK2 and MMP-9 at protein levels.