siRNA targeting TRIM29 gene induces apoptosis of nasopharyngeal carcinoma cells by inhibiting PI3K/AKT signaling pathway

AIM:To investigate the effect of TRIM29 gene expression silencing on the apoptosis and PI3K/AKT signaling pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were divided into blank group, negative control (NC) group (transfected negative control siRNA) and si-TRIM29 group (transfected TRIM29 specific siRNA). The viability of the 5-8F cells transfected with si-TRIM29 for 0~96 h was measured by CCK-8 assay. The apoptotic rate and the protein levels of TRIM29, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, t-AKT and p-AKT in the 5-8F cells transfected with si-TRIM29 for 48 h were determined by flow cytometry and Western blot, respectively. PI3K/AKT signal specific inhibitor LY294002 at 10 μmol/L and si-TRIM29 alone or in combination were treated with the 5-8F cells, and the cells were divided into blank group, LY294002 group and LY294002+si-TRIM29 group. The apoptotic rates in the 3 groups were detected by flow cytometry. RESULTS:The protein expression of TRIM29 in the 5-8F cells transfected with TRIM29 siRNA was significantly lower than that in blank group (P<0.05). Compared with blank group, the cell viability was significantly decreased, the apoptotic rate was significantly increased, the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax were significantly increased, and the protein levels of Bcl-2 and p-AKT were significantly decreased in si-TRIM29 group (P<0.05). The apoptotic rate in LY294002 group was higher than that in blank group, while that in LY294002+si-TRIM29 group was even higher than that in LY294002 group (P<0.05). CONCLUSION:Silencing of TRIM29 gene expression induces apoptosis of nasopharyngeal carcinoma 5-8F cells by inhibiting PI3K/AKT signaling pathway.     

Over-expression of SATB1 mediates invasion and metastasis of nasopharyngeal carcinoma cells through epithelial-mesenchymal transition

AIM:To analyze the high expression of special AT-rich sequence-binding protein 1 (SATB1) in nasopharyngeal carcinoma (NPC) and its role in tumor invasion and metastasis. METHODS:The method of immunohistochemistry was used to detect the expression of SATB1 and epithelial-mesenchymal transition (EMT)-related molecules E-cadherin and vimentin in 76 cases of NPC and 61 cases of nasopharyngeal chronic inflammation (NPI), and the correlations of over-expression of SATB1 with NPC patients' clinical parameters as well as the expression of E-cadherin and vi-mentin were analyzed. Variously differentiated NPC cell lines CNE1, CNE2Z and C666-1 were cultured in vitro, and then SATB1-overexpressing cell line was screened. After interfering with SATB1 expression by siRNA, the expression of EMT-related molecules and the change of cell invasiveness were analyzed. RESULTS:The expression of SATB1 in the nasopharyngeal tissue was dominantly localized in the nuclei. The positive rate of SATB1 in NPC group was significantly higher than that in NPI group (P<0.01). E-cadherin was membrane-positive in NPI epithelial cells, while membrane E-cadherin in NPC was decreased but cytoplasmic expression was increased. The positive expression rate of membrane E-cadherin in NPI was significantly higher than that of NPC (P<0.01). Vimentin was localized in cytoplasm and negative in NPI epithelial cells, but the positive rate in NPC parenchymal cells was significant higher than that in NPI (P<0.01). The high expression of SATB1 in NPC was not related to the patents' sex, age, clinical classification and N classification, but positively correlated with T and M classification (P<0.05). Besides, high expression of SATB1 was positively correlated with vi-mentin in NPC tissues (r=0.358, P=0.009). SATB1 expression in NPC cell lines was negatively correlated with the levels of cell differentiation. Knockdown of SATB1 expression in C666-1 cells with siRNA was accompanied by an increase in E-cadherin and a decrease in vimentin levels, as well as a decrease in cell invasiveness. CONCLUSION:High expression of SATB1 promotes the clinical progress of NPC through EMT mechanism.     

miRNA-7 inhibits proliferation of human nasopharyngeal 5-8F carcinoma cells via EGFR/PI3K/Akt pathway

AIM:To investigate the relationship of microRNA-7 (miRNA-7) over-expression and epidermal growth factor receptor (EGFR)/phosphatidylinositol kinase-3 (PI3K)/protein kinase B (PKB, also called Akt) pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were transfected with miRNA-7 mimics (carrying by Lipofectamine 2000). The expression of miRNA-7 was detected by real-time PCR. The cell proliferation was analyzed by CCK-8 assay. The cell colony-forming capability was determined by cell colony formation test. The expression of EGFR/PI3K/Akt at mRNA and protein levels was examined by real-time PCR and Western blotting. RESULTS:The expression level of miRNA-7 was significantly increased in 5-8F cells compared with negative control (NC) group and control group (P<0.01). The proliferation of NPC 5-8F cells was decreased extremely after tansfected with the miRNA-7 mimics (P<0.01), so did the result of the cell colony-formation test. The expression of EGFR/PI3K/Akt at mRNA and protein levels was significantly down-regulated compared with NC group and control group (P<0.01). CONCLUSION: Over-expression of miRNA-7 significantly inhibits the proliferation and colony-forming ability of nasopharyngeal carcinoma 5-8F cells by down-regulation of EGFR/PI3K/Akt pathway.     

Muscarinic receptor 3 agonist promotes epithelial-meschymal transition in human lung cancer A549 cells by activating PI3K/AKT signaling pathway

AIM: To investigate the possible signaling pathway that promotes epithelial-mesenchymal transition (EMT) of the lung cancer A549 cells stimulated with muscarinic receptor 3 (M3R) agonist carbachol. METHODS: The lung cancer cells A549 were treated with 400 μmol/L carbachol. The morphological changes of the cells were observed under inverted phase contrast microscope. The migration and invasion abilites were measured by Wound healing and Transwell assays. qPCR was used to detect the mRNA level of vimentin and E-cadherin. The protein levels of p-AKT, vimentin and E-cadherin were determined by Western blot. RESULTS: After treatment with carbachol, the A549 cells showed loss of the close connection and the cell morphology was transformed from irregular polygon to spindle-like cells. The results of Wound healing and Transwell assays showed that the migration and invasion abilites of the A549 cells were enhanced. Carbachol increased the vimentin expression and decreased the E-cadherin expression at mRNA and protein level (P<0.05). The phosphorylation of AKT in the A549 cells was up-regulated (P<0.05). These changes was inhibited by M3R antagonist 4-DAMP. CONCLUSION: Carbachol promotes EMT in the human lung cancer A549 cells by activating PI3K/AKT signaling pathway.     

Rip2 induces autophagy and its mechanism in human pancreatic cancer cells

AIM:To explore whether receptor-interacting protein 2 (Rip2) induces autophagy and its under-lying mechanisms in human pancreatic cancer cell line Panc-1. METHODS:The empty plasmid pEGFP-C2 or recombinant plasmid pEGFP-Rip2 was transfected into the Panc-1 cells by jetPRIME reagent. The untreated cells served as control group. The protein levels of Rip2, autophagy-related molecules (beclin-1 and LC3-Ⅱ), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway-related proteins were determined by Western blot 48 h after transfection. The morphological changes of the autophagosomes were observed by transmission electron microscopy. RESULTS:The protein level of Rip2 was significantly increased in the Panc-1 cells transfected with pEGFP-Rip2 plasmid. The protein expression of beclin-1 and LC3-Ⅱ in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group (all P<0.01). An increased number of autophagosomes was observed under transmission electron microscope in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group. Furthermore, the protein levels of p-mTOR and p-AKT in pEGFP-Rip2 group were lower than those in control group and pEGFP-C2 group (all P<0.01), while no significant difference of the total mTOR and AKT protein levels was found among the 3 groups. CONCLUSION:Rip2 induces autophagy in the Panc-1 cells and its mechanism may be related to inhibiting the activation of PI3K/Akt/mTOR pathway.     

A 3D cell culture system to study epithelial-mesenchymal transition and drug resistance of lung cancer

AIM To explore the molecular mechanism of transforming growth factor-β1 （TGF-β1）-induced epithelial-mesenchymal transition （EMT） and cisplatin resistance in three-dimensionally cultured lung cancer cells. METHODS Under three-dimensional culture condition, the morphological changes and protein expression changes of human non-small-cell lung cancer 95D cells were observed by inversed fluorescence microscopy, scanning electron microscopy, laser scanning confocal microscopy and Western blot before or after TGF-β1 stimulation. The cisplatin sensitivity was determined by MTT assay. RESULTS Under the three-dimensional culture condition, the structure of 95D cell spheroids after TGF-β1 stimulation collapsed, the cells were dispersed and migrating, and the spheroids merged with each other. The results of laser confocal microscopy showed that E-cadherin protein expression in the 95D cells did not changed after TGF-β1 stimulation, and the protein expression of N-cadherin and vimentin was significantly up-regulated. The results of Western blot showed that the expression of E-cadherin was down-regulated after TGF-β1 stimulation, and the protein levels of N-cadherin, vimentin, phosphorylated AKT and phosphorylated mTOR were up-regulated. LY294002 and rapamycin reversed TGF-β1-induced expression of the above proteins. The results of MTT assay showed that TGF-β1 reduced the sensitivity of three-dimensionally cultured 95D cells to cisplatin, while LY294002 and rapamycin reversed the cisplatin resistance of the 95D cells stimulated by TGF-β1. CONCLUSION TGF-β1 induces the EMT and cisplatin resistance of three-dimensionally cultured lung cancer cells through the activation of PI3K/AKT/mTOR signaling pathway.     

Radiosensitizing effect of baicalin on human cervical carcinoma HeLa cells

AIM: To investigate the effect of baicalin on the radiosensitization of HeLa cells. METHODS: The cell activity was determined by MTT assay. The radiosensitivity of HeLa cells was detected by colony formation assay. The cell cycle was analyzed by flow cytometry. The protein levels of Akt, p-Akt, Bad and p-Bad were examined by Western blot. RESULTS: The cell growth of the HeLa cells was inhibited by baicalin dose-dependently and IC₅₀ was 43.65 mg/L. The results of colony formation assay showed that combination of 8 mg/L baicalin and radiotherapy further improved survival curve and decreased the value of D0 and Dq, as compared with radiotherapy alone (P<0.05). Furthermore, baicalin enhanced the effect of radiotherapy on cell cycle, as evidenced by the increase in cell percentage in G₂/M phase (P<0.05). Additionally, after incubation with baicalin, radiotherapy-induced phosphorylation of Akt and Bad were further augmented (P<0.05). CONCLUSION: Baicalin augments radiosensitivity of HeLa cells through the inhibition of cell cycle transition and activation of PI3K/Akt signaling pathway.     

Role of HMGA2 in epithelial-mesenchymal transition of gastric cancer cells

AIM:To investigate the role of HMGA2 in the epithelial-mesenchymal transition (EMT) in gastric cancer cells. METHODS:The expression of HMGA2 in human gastric cancer cell lines with different degrees of differen-tiation (MKN45, MKN28 and SGC7901) and immortalized human gastric epithelial cell line GES-1 was determined by Western blot and RT-qPCR. pcDNA3.0-HMGA2 plasmid was transfected into the MKN28 cells by liposome method. Transfection of si-HMGA2 interference fragments into MKN45 cells was also performed. The transfection efficiency was evaluated by Western blot and RT-qPCR. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the cell viability were measured by CCK-8 assay. The effects of HMGA2 over-expression in the MKN28 cells on the cell migration and invasion abilities were detected by wound healing and Transwell invasion assays. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the expression of EMT-related markers E-cadherin, N-cadherin, vimentin at mRNA and protein levels were determined by RT-qPCR and Western blot. The changes of Wnt/β-catenin signaling pathway-related molecules in the MKN28 cells with HMGA2 over-expression were also determined by RT-qPCR. RESULTS:The expression levels of HMGA2 were quite different in different differentiation levels of gastric cancer cells (P<0.05). The increased expression level of HMGA2 in MKN28 cells inhibited the cell viability (P<0.05), while the decreased expression level of HMGA2 in MKN45 cells promoted the cell viability (P<0.05). The increased expression level of HMGA2 in MKN28 cells promoted cell migration and invasion (P<0.05), changed the expression of EMT-related markers (P<0.05), while the decreased expression level of HMGA2 in the MKN45 cells changed the expression of EMT-related markers (P<0.05). The increased expression level of HMGA2 in the MKN28 cells significantly increased the mRNA levels of β-catenin in the Wnt/β-catenin pathway and the downstream molecules c-Myc and cyclin D1 (P<0.05). CONCLUSION:HMGA2 is closely related to the migration and invasion abilities of gastric cancer cells. Moreover, it promotes the EMT process of gastric cancer cells by activating Wnt/β-catenin pathway.     

CUDC-907 induces DNA damage and autophagy in glioma cells

AIM:To investigate the effect of CUDC-907, a dual histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K) inhibitor, on the DNA damage, cell cycle distribution and autophagy in human glioma U251 cells. METHODS:U251 cells were treated with CUDC-907 of different concentrations, and the cell viability was detected by MTT assay. The quantitative γ-H2AX foci were determined by laser scanning confocal microscopy. The cell cycle distribution of U251 cells was examined by flow cytometry. The protein expression was determined by Western blot analysis. RESULTS:CUDC-907 inhibited the cell viability and the phosphorylation of Akt and p70 ribosomal protein S6 kinase (p70s6K) in the U251 cells (P<0.05). In CUDC-907-treated cells, the number of γ-H2AX foci and protein expression of γ-H2AX were increased significantly (P<0.05). CUDC-907 also induced cell arrest in the G₂/M phase by up-regulating the expression of p21, and inhibiting the protein level of cyclin B1 and the phosphorylation of cell division cycle protein 2 (Cdc2). In addition, CUDC-907 triggered cell autophagy, and inhibition of autophagy increased CUDC-907-induced DNA damage of U251 cells. CONCLUSION:CUDC-907 significantly inhibits PI3K/Akt signaling pathway, induces DNA damage and arrests cell cycle in G₂/M phase. Blockage of autophagy promotes CUDC-907-induced DNA damage of U251 cells.     

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.     

Shikonin induces apoptosis and autophagy of human cervical cancer HeLa cells by PI3K/Akt/mTOR signaling pathway

AIM:To observe the effects of shikonin on the apoptosis and autophagy of human cervical cancer HeLa cells, and to explore the possible role of PI3K/Akt/mTOR signaling pathway in these processes. METHODS:The HeLa cells were treated with shikonin, and the cell viability was detected by CCK-8 assay. The apoptosis was detected by Annexin V/PI double staining. The autophagosome was observed by transfection with GFP-LC3 into the HeLa cells. After the treatment with shikonin combined with autophagy inhibitor 3-methyladenine (3-MA) or apoptosis inhibitor Z-DEVD-FMK, the protein levels of autophagy-and apoptosis-related molecules microtuble-associated protein 1 light chain 3 (LC3) and cleaved caspase-3 in the HeLa cells were determined by Western blot. The protein levels of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR) were also determined by Western blot. RESULTS:Shikonin significantly inhibited the viability of HeLa cells (P<0.05). Compared with control group, shikonin significantly induced apoptosis of HeLa cells (P<0.05). The results of GFP-LC3 plasmid transfection analysis showed that green dot-like congregate autophagosomes appeared in the cytoplasm of the HeLa cells after shikonin treatment, while the autophagosomes were rarely observed in control group. Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly decreased and cleaved caspase-3 was significantly increased in shikonin+3-MA group (P<0.05). Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly increased and cleaved caspase-3 was significantly decreased in shikonin+Z-DEVD-FMK group (P<0.05). Compared with control group, shikonin significantly decreased the protein levels of p-PI3K, p-Akt and p-mTOR (P<0.05). CONCLUSION:The apoptosis and autophagy of the HeLa cells are induced by shikonin, these two processes are complementary. The mechanism may be related to inhibition of PI3K/Akt/mTOR signaling pathway.     

Juglone inhibits epithelial-mesenchymal transition of prostate cancer cells by regulating Wnt/β-catenin/Snail signaling pathway

AIM: To investigate the mechanism of juglone on epithelial-mesenchymal transition in prostate cancer cells. METHODS: Human prostate cancer LNCaP cells were divided into control group (without juglone), 12.5 μmol/L juglone group and 25 μmol/L juglone group. LNCaP cells in the latter 2 groups were treated with juglone for 24 h. The invasion ability of the LNCaP cells was detected by Transwell assay. The protein expression of E-cadherin, vimentin, Snail and β-catenin was determined by Western blot. The LNCaP cells were treated with LiCl and juglone in combination for 24 h, and the protein expression of Snail and E-cadherin was detected by Western blot.RESULTS: The results of Trans-well invasion assay showed that the invasion ability in juglone groups was significantly decreased (P<0.01). The protein expression of E-cadherin in the LNCaP cells treated with juglone was increased, and the expression levels of vimentin and β-catenin were reduced (P<0.01). Treatment with LiCl significantly attenuated the inhibitory effect of juglone on Snail expression and subsequent down-regulation of E-cadherin expression. CONCLUSION: Juglone inhibits the epithelial-mesenchymal transition by inhibiting the Wnt/β-catenin/Snail signaling pathway in the LNCaP cells.     

Effects of tangeretin on growth and invasion of human non-small-cell lung cancer cells

AIM: To study the influences of tangeretin (TGN) on the growth and invasion of non-small-cell lung cancer (NSCLC) cells, and to explore the molecular mechanisms. METHODS: The A549 cells were treated with different concentrations of TGN in vitro. The relative cell activity was determined by MTT assay. The apoptotic rate was analyzed by flow cytometry with Annexin V-FITC/PI staining. The number of the invasive cells was measured by Transwell assay. The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR, and the protein levels of Ki67, Cyt C, caspase-3, cleaved caspase-3, MMP-2, MMP-9, Akt, p-Akt and p-PI3K were determined by Western blotting analysis. RESULTS: TGN inhibited the proliferation of A549 cells in a dose-dependent manner (P<0.05) along with the low expression level of proliferation biomarker Ki67. TGN up-regulated the protein levels of Cyt C, caspase-3 and cleaved caspase-3 (P<0.01) and promoted the apoptosis of A549 cells in a dose-dependent manner. Moreover, TGN down-regulated the invasion-related molecules MMP-2 and MMP-9 at the mRNA and protein levels, and the number of invasive cells reduced with the increase in the concentration of TGN. The protein levels of p-Akt and p-PI3K in the A549 cells was reduced (P<0.05), and no difference of the cell viability in the cells treated with different concentrations of TGN was observed after blocking PI3K/Akt signaling pathway using LY294002. CONCLUSION: TGN inhibits the growth and invasion of A549 cells and promotes the cell apoptosis by potentially inhibiting PI3K/Akt signaling pathway activation. Therefore, this study will provide a new target for the prevention and control of NSCLC.     

NVP-BEZ235 induces autophagy of polycystic kidney rat cholangiocytes

AIM: To explore the effect of dual PI3K/Akt/mTOR inhibitor NVP-BEZ235 on autophagy of polycystic kidney (PCK) rat cholangiocytes. METHODS: The protein levels of p-mTOR and p-Akt in the bile duct epithelial cells were examined by immunohistochemistry. The effect of NVP-BEZ235 on the viability of cholangiocytes was detected by WST-1 assay. The levels of PI3K/Akt/mTOR signaling pathway and autophagy-related proteins with NVP-BEZ235 treatment were determined by Western blot. The effects of LC3 and Beclin 1 silencing, and authophagy-specific inhibitor 3-methyladenine (3-MA) on the cell viability were analyzed by WST-1 assay. RESULTS: The protein levels of p-Akt and p-mTOR were highly increased in the bile duct epithelium of the PCK rats. NVP-BEZ235 significantly inhibited the viability of the cholangiocytes in a dose- and time-dependent manner (P<0.05). NVP-BEZ235 significantly reduced the levels of PI3K/Akt/mTOR signaling pathway-related proteins in the PCK rat cholangiocytes. NVP-BEZ235 upregulated the autophagy-specific proteins LC3 II and Beclin 1. The inhibitory effect of NVP-BEZ235 on the cell viability was weakened by treatment with 3-MA and knockdown of LC3 and Beclin 1 (P<0.01).CONCLUSION: The PI3K/Akt/mTOR inhibitor NVP-BEZ235 suppresses the viability of PCK rat cholangiocytes, and the mechanism is closely related with autophagy.     

Antitumor mechanism of danusertib in human AML HL-60 cells

AIM: To investigate the effects of danusertib, a pan-inhibitor of Aurora kinases, on the viability, cell cycle, apoptosis and autophagy of human acute myelocytic leukemia (AML) HL-60 cells.METHODS: The effect of danusertib on the viability of HL-60 cells was examined by MTT assay. The effect of danusertib on apoptosis of HL-60 cells was quantitated by the flow cytometry using an Annexin V/7-AAD apoptosis detection kit. The effect of danusertib on autophagy in the HL-60 cells was assessed by flow cytometry and confocal microscopic analysis. The levels of various proteins related to the cell cycle, apoptosis and autophagy were determined by Western blot.RESULTS: Danusertib decreased the viability of human AML HL-60 cells and induced the cell cycle arrest in G₂/M phase. Danusertib also induced mitochon-drium-dependent apoptosis by activation of caspase-3 and autophagy in the HL-60 cells via inhibition of PI3K/Akt/mTOR signaling pathway.CONCLUSION: Danusertib shows effective antitumor ability for promising AML treatment.     

miR-10b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells by repressing KLF10 expression

AIM:To explore the effect and the underlying mechanisms of microRNA-10b (miR-10b) on high glucose-stimulated epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. METHODS:The expression level of miR-10b was examined by RT-qPCR in the kidney tissues of the type 2 diabetes patients with kidney fibrosis. The EMT model of HK-2 cells was induced by high glucose stimulation and the miR-10b expression in the process was detected by RT-qPCR. The morphological changes of the HK-2 cells were observed using a microscope. EMT markers, such as fibronectin and N-cadherin, were examined by Western blot. The online database predicted that the 3'-UTR of KLF10 bound to miR-10b and their direct interaction was confirmed by dual luciferase report assay. RESULTS:Compared with the para-carcinoma normal tissues, the expression level of miR-10b was up-regulated in the tissues of type 2 diabetes patients with kidney fibrosis (P<0.01). In high glucose-stimulated HK-2 cells, the expression level of miR-10b was increased in a time-dependent manner (P<0.01). miR-10b inhibitor reversed the morphological changes and the increases expression of the EMT markers including fibronectin, SLUG, N-cadherin and SNAI1 induced by high glucose stimulation. Online database showed miR-10b was able to bind with the 3'-UTR in the promoter region of KLF10, thus negatively regulating its expression. Meanwhile, over-expression of KLF10 inhibited the EMT induced by high glucose. Inhibition of TGF-β/Smad3 activation was observed during the process of KLF10-repressed EMT. CONCLUSION:miR-10b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells may through repressing KLF10 expression.     

miRNA-7-mediated Bax/Bcl-2 expression promotes apoptosis of human nasopharyngeal carcinoma CNE-1 cells

AIM: To investigate the relationship of microRNA-7 (miRNA-7) over-expression and Bax/Bcl-2 expression in human nasopharyngeal carcinoma CNE-1 cells.METHODS: The CNE-1 cells were transfected with miRNA-7 mimics using Lipofectamine 2000. The expression of miRNA-7 was detected by real-time PCR. CCK-8 assay and Hoechst 33258 staining were used to detect the cell activity and apoptosis. The expression of Bax/Bcl-2 at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: The expression of miRNA-7 was increased significantly in the CNE-1 cells compared with negative control group and mock group (P<0.01). The activity of CNE-1 cells were extremely decreased after tansfected with miRNA-7 mimics (P<0.01). The typical apoptotic nuclear morphological changes were observed in the CNE-1 cells under the fluorescence microscope with Hoechst 33258 staining. The expression of Bax at mRNA and protein levels was significantly increased compared with the other 2 groups (P<0.01), while the Bcl-2 expression at mRNA and protein levels was significantly down-regulated (P<0.01).CONCLUSION: Over-expression of miRNA-7 significantly inhibits the growth and promotes the apoptosis of nasopharyngeal carcinoma CNE-1 cells by increasing the expression of Bax and down-regulating Bcl-2.     

Effects of andrographolide on invasion and apoptosis of ovarian cancer SKOV-3 cells

AIM:To investigate the effects of andrographolide on the invasion and apoptosis of ovarian cancer cell line SKOV-3,and to explore the possible mechanisms.METHODS:SKOV-3 cells were treated with different concentrations (0,5,10,20 or 40 μmol/L) of andrographolide for different time (12,24,36 or 48 h),and then the cell viability was determined by CCK-8 assay.The cell invasion ability was analyzed by Transwell assay and cell apoptosis was detected by TUNEL staining.The protein levels of p-PI3K,p-Akt and p-mTOR were examined by Western blot.RESULTS:The results of CCK-8 assay revealed that andrographolide inhibited the growth of SKOV-3 cells in a dose-and time-dependent manner.Treatment with andrographolide at 20 μmol/L for 36 h significantly decreased the invasion ability of SKOV-3 cells,while increased cell apoptosis.In addition,the protein levels of p-PI3K,p-Akt and p-mTOR were reduced after andrographolide treatment.CONCLUSION:Andrographolide inhibits the growth and invasion of ovarian cancer SKOV-3 cells by suppression of PI3K/Akt/mTOR signaling pathway.