Thiazol Research Articles

Effect of long non-coding RNA colorectal neoplasia differentially expressed targeting microRNA-384 on invasion and migration of lung cancer SPC-A1 cells

Objective To investigate the effect of long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) targeting microRNA (miRNA, miR)-384 on invasion and migration of lung cancer SPC-A1 cells. Methods Small interfering RNA (siRNA) CRNDE was transfected into lung cancer SPC-A1 cells, and the interference effect was determined by real-time quantitative polymerase chain reaction (Real-time PCR). Cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay, cell invasion and migration were measured by Transwell chamber, and Western blotting was used to detect the expression of matrix metalloproteinase (MMP)-9, MMP-2, E-cadherin and Vimentin in cells. The targeting relationship prediction software predicts that CRNDE binds to miR-384. Luciferase reporting system was used to identify the targeting relationship. In SPC-A1 cells, siRNA CRNDE and miR-384 inhibitors were co-transfected, and the above methods were used to determine the reverse effect of miR-384 inhibitors on the invasion and migration of lung cancer SPC-A1 cells transfected with siRNA CRNDE.Comparison two groups was performed using t test, and comparison three groups was performed with one-way analysis of variance (ANOVA). Results The expression of CRNDE in lung cancer SPC-A1 cells transfected with siRNA CRNDE decreased (1.01±0.12 vs. 0.43±0.06), cell proliferation decreased (0.37±0.05 vs. 0.24±0.02, F=10.467, P<0.05), the number of invasive cells (141.25±15.23 vs. 101.02±9.84, F=9.420, P<0.05) and migrating cells (188.20±16.84 vs. 142.58±11.22, F=8.345, P<0.05) decreased, the expression levels of MMP-9, MMP-2 and Vimentin proteins in cells decreased, and E-cadherin protein level increased. There were complementary binding sites between CRNDE and miR-384. CRNDE targeted and negatively regulated the expression of miR-384. The miR-384 inhibitors reversed the effects of siRNA CRNDE on proliferation (0.26±0.03 vs. 0.35±0.04, t=3.118, P<0.05), migration (139.84±10.57 vs. 176.51±12.45, t=5.276, P<0.05), invasion (99.84±10.02 vs. 134.25±11.25, t=3.890, P<0.05) and expression of MMP-9, MMP-2, E-cadherin and Vimentin in lung cancer cells. Conclusion Down-regulation of lncRNA CRNDE targeting miR-384 inhibits invasion and migration of lung cancer SPC-A1 cells. Key words: Lung cancer; MicroRNA-384; Colorectal neoplasia differentially expressed targeting; Migration; Invasion

Inhibitory effect of irisin on vasular smooth muscle cell inflammation and proliferation by regulating autophagy through adenosine monophosphate-actived protein kinase/mammalian target of rapamycin pathway

Objective To explore the underlining mechanisms of irisin inhibiting inflammation and proliferation of vascular smooth muscle cells (VSMCs). Methods The cultured primary rat aortic VSMCs were pre-incubated with irisin and then treated with platelet derived growth factor-BB (PDGF-BB). Enzyme linked immunosorbent assay (ELISA) and methyl thiazol tetrazolium (MTT) assay were used to measure the VSMCs inflammation and proliferation, respectively. The protein expression and phosphorylation of adenosine monophosphate-actived protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway related molecule AMPK/p-AMPK (Thr172), acetyl-CoA carboxylase (ACC)/phosphorylated (p)-ACC (Thr308), mTOR/p-mTOR (Ser2448), ribosomal protein S6 kinase (p70SK6)/p-70SK6 (Thr389), and autophay related molecule microtubule-associated protein 1 light chain 3 (LC3) Ⅰ, LC3Ⅱ and nucleoporin p62 (P62) were detected by Western blotting. Results After treatment with PDGF-BB, VSMCs proliferation dramatically increased, and the inflammatory cytokines IL-1β (37.9±8.5 vs. 10.8±2.9, t=18.620, P<0.01), IL-6 (57.4±14.1 vs. 32.4±8.7, t=6.250, P<0.01), MCP-1 (158.3±39.1 vs. 57.2±12.5, t=14.280, P<0.01), ICAM-1 (371.8±77.3 vs. 160.4±39.8, t=10.850, P<0.01), tumor necrosis factor-α (TNF-α) (16.3±3.8 vs. 7.8±2.4, t=7.390, P<0.01) in the cell supernatant were significantly up-regulated. The protein expression levels of AMPK (0.39±0.11 vs. 0.83±0.18, t=14.250, P<0.01), p-AMPK (0.25±0.03 vs. 0.49±0.12, t=12.370, P<0.01), ACC (0.54±0.16 vs. 1.07±0.24, t=10.670, P<0.01), p-ACC (0.38±0.07 vs. 0.97±0.30, t=18.130, P<0.01), LC3Ⅰ (0.67±0.21 vs. 0.67±0.21, t=5.780, P<0.01) and LC3Ⅱ (0.42±0.13 vs. 0.93±0.38, t=6.820, P<0.01) were down-regulated, mTOR (2.57±0.53 vs. 1.23±0.25, t=21.350, P<0.01), and p-mTOR (1.35±0.0.37 vs. 0.57±0.12, t=8.870, P<0.01), p70SK6 (1.85±0.39 vs. 0.81±0.19, t=7.170, P<0.01), p-70SK6 (0.69±0.22 vs. 0.38±0.11, t=9.260, P<0.01) and P62 (1.24±0.38 vs. 0.58±0.14, t=5.270, P<0.01) were up-regulated. However, pre-incubation with irisin and then stimulation with PDGF-BB could significantly decrease VSMCs proliferation [(122.14±9.42)%, t=4.970, P<0.01], and significantly down-regulate the inflammatory cytokines IL-1β (17.38±4.10, t=6.740, P<0.01), IL-6 (34.9±8.9, t=2.760, P<0.05), MCP-1 (47.28±9.20, t=16.270, P<0.01), ICAM-1 (169.2±55.8, t=7.780, P<0.01) and TNF-α (6.9±2.0, t=8.180, P<0.01) in the cell supernatant. The protein expression levels of AMPK (0.72±0.26, t=2.670, P<0.05), p-AMPK (0.46±0.13, t=3.280, P<0.05), ACC (0.95±0.28, t=5.870, P<0.01), p-ACC (0.78±0.25, t=3.140, P<0.01), LC3Ⅰ (1.34±0.35, t=1.960, P<0.05) and LC3Ⅱ (1.03±0.19, t=7.260, P<0.01) were up-regulated, and mTOR (1.16±0.33, t=6.910, P<0.01), p-mTOR (0.66±0.18, t=6.830, P<0.01), p70SK6 (1.12±0.34, t=2.360, P<0.05), p-70SK6 (0.37±0.12, t=3.120, P<0.01) and P62 (0.68±0.18, t=3.360, P<0.05) were down-regulated. Conclusion Irisin could inhibit the inflammation and proliferation of VSMCs possibly by up-regulating autophagy through activation of the AMPK/mTOR signaling pathway. Key words: Irisin; Adenosine monophosphate-actived protein kinase/mammalian target of rapamycin signaling pathway; Autophagy; Vascualar smooth muscle cell proliferation; Vein graft stenosis

MicroRNA-384 inhibits proliferation and invasion of pancreatic cancer cells by down-regulating pleiotropic growth factor expression

Objective To study the expression of microRNA (miRNA, miR)-384 in pancreatic cancer, para-carcinoma tissue and pancreatic cancer cell lines as well as the relationship between miR-384 and clinical pathology of pancreatic cancer, and the effects of miR-384 on proliferation and migration of pancreatic cancer cell lines. Methods The expression of miR-384 and pleiotropic growth factor (PTN) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in 67 pancreatic cancer tissues and corresponding adjacent tissues obtained by resection or aspiration biopsy. The expression of miR-384 and PTN mRNA and PTN protein in pancreatic cancer cell line PANC-1/MIAPaCa-2 was detected by RT-PCR and Western blotting before and after the transfection of adenovirus loaded with miR-384. The influence of the infection to the proliferation, migration and invasion of PANC-1/MIAPaCa-2 were detected by methyl thiazol tetrazolium (MTT) Assay and Transwell chamber. Results The relative expression of miR-384 was 0.003 7±0.000 9 and 0.006 5±0.002 2 in pancreatic cancer tissues and adjacent tissues respectively (t=6.177, P<0.01), and was associated with nerve invasion (χ2=4.750, P<0.05) and lymph node metastasis (χ2=13.400, P<0.01). While the pancreatic carcinoma tissues were divided into miR-384 low-expression group and high-expression group, the relative expression of PTN mRNA was 0.073±0.028 and 0.045±0.001, respectively, the differences of which were statistically significant (t=2.082, P<0.05). Compared with the negative control group, the adenovirus transfection group reveals that the expression of miR-384 in PANC-1 cells was increased 11.69 times (t=28.600, P<0.01), while the expression of PTN mRNA and PTN protein were decreased to 19.02% (t=31.200, P<0.01) and 38.00% (t=16.200, P<0.01) respectively, and the expression of miR-384 was increased 14.95 (t=21.300, P<0.01) times in MIAPaCa-2 cells while PTN mRNA and PTN protein decreased to 15.02% (t=13.300, P<0.01) and 13.74% (t=13.500, P<0.01) respectively. The proliferation, migration and invasion of the two cell lines were all decreased after transfection, which could be reversed by dealing with recombinant human PTN protein. Conclusion MiR-384 can inhibit tumor growth in pancreatic carcinoma, and the mechanism may be down-regulation of PTN expression by targeting PTN mRNA. Key words: Pancreatic cancer; Pleiotropic growth factor; Invasion and metastasis; RNA interference; MicroRNA-384

Effect of hypoxic treatment on gastric cancer cells and its mechanism

Objective To investigate the effect of hypoxia on proliferation, apoptosis, migration and invasion of gastric cancer SGC7901 cells and its possible mechanism. Methods SGC7901 cells were cultured in normoxic (normoxia) and hypoxic (hypoxia) groups respectively. Methyl thiazol tetrazolium (MTT), clonogenic assay, flow cytometry, cell scratch assay, Transwell assay, quantitative real time PCR (qPCR) and Western blotting The ability of gastric cancer SGC7901 cells to proliferate, to apoptosis, to migrate and to attack was tested experimentally. Results The Absorbance [0.77 compared to 0.98, t=-3.480, P<0.01], number of cells [188±13 compared to 237±20, t=-16.250, P<0.01], number of migrated cells [131±17 compared to 252±18, t=-19.880, P<0.01] and number of invasion cells [35±9 compared to 57±11, t=-2.490, P<0.05] between normoxia group and hypoxia was statistically significant. It indicated that the proliferation, migration and invasion ability of SGC7901 cells in hypoxia group were enhanced; The apoptosis rate between normoxia group and hypoxia was statistically significant [0.93±0.02 compared to 0.22±0.03, t=2.360, P<0.05]. It indicated that the apoptosis rate was slowed down in hypoxic group. The relative expression level of HIF-1α gene [1.02±0.31 compared to 1.68±0.43, t=-1.990, P<0.05] and the histone gray scale [21.73±3.53 compared to 56.92±2.43, t=-2.450, P<0.05] between normoxia group and hypoxia group was statistically significant. It indicated that the HIF-1α was increased expression of gastric cancer SGC7901 cells in hypoxia group; The histone gray scale of p-Akt between normoxia group and hypoxia was statistically significant [138.7±12.0 compared to 266.0±43.0, t=-25.570, P<0.01]. It indicated that the PI3K-Akt signaling pathway was activated. Conclusion Hypoxia may regulate the expression of HIF-1α by activating the PI3K-Akt signaling pathway and promote the proliferation, migration and invasion of gastric cancer SGC7901 cells. Key words: Hypoxia; Gastric cancer cell; Apoptosis; Invasion

Preparation and biosafety of a ink-jet all-core structure composite scaffold for bone tissue engineering

Objective To observe the hydrophilicity, biocompatibility and biosafety of ink-jet all-core structure composite scaffold. Methods The ink-jet all-core structure composite scaffold composed of poly (lactic-co-glycolic acid)/β-tricalciumphosphate (PLGA/β-TCP) skeleton ink-jet-wrapped with type I collagen was manufactured via rapid prototyping, and PLGA/β-TCP scaffold was chosen as the control group. Hydrophilicity was evaluated by water absorption rate. The proliferation ability of cells on the scaffold was determined by methyl thiazol tetrazolium (MTT) assay (thiazole blue). The scaffolds were implanted into rats, and blood routine, liver function and renal function tests were performed at 2, 4, 8, 12 weeks, to observe the changes of local wounds and liver and kidney functions in rats. Results For MTT assay, the values at 2nd, 6th, 10th, and 14th day in two groups were 0.14, 0.28, 0.62, 0.78, and 0.13, 0.19, 0.36, and 0.46, respectively. The hydrophilicity of the ink-jet all-core structure composite scaffold was significantly higher than that of the PLGA/TCP scaffold. there were no significant differences in blood routine, liver function and renal function between two groups (P>0.05). Conclusion The ink-jet all-core structure composite scaffold possesses admirable hydrophilicity, biocompatibility and biosafety. Key words: Ink-jet all-core structure composite scaffold; Hydrophilicity; Biosafety; Biocompatibility

Construction of short hairpin RNA interfering milk fat globule-epidermal growth factor 8 gene lentiviral vector and its effect on inhibition of breast cancer cells

Objective To construct the lentiviral vector of milk fat globule-epidermal growth factor 8 (MFG-E8) short hairpin RNA (shRNA), and stably transfected the HCC1973 cell line to detect its interference efficiency and influence in breast cancer cell HCC1973. Methods The knockout gene fragment was synthesized and inserted into the shRNA lentiviral vector PLKO.1 with a cherry protein reporter. The constructed recombinant plasmids were transfected into 293T cells respectively. The lentiviruses were transfected into HCC1973 cells and stably transfected PLKO.1-MFG-E8 shRNA recombinant lentiviruses were obtained. The expression of MFG-E8 was detected by real-time PCR and Western blotting. The cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay and Flow cytometry was used to detect the cell cycle and apoptosis, Transwell was used to detect the invasion and migration of cells. Statisticalanalysis of data using the statistical product and service solutions 17.0 software. Results The constructed vector of PLKO.1-MFG-E8 shRNA significantly reduced the expression of MFG-E8 gene and protein in breast cancer cell HCC1973, the lentivirus titer was 3×108 PFU/ml. The proliferation of breast cancer cells was significantly suppressed (t=4.426, P<0.05), G0/G1 phase cells increased significantly with a decrease of cells in S phase (t=2.264, P<0.05), the ability of cell invasion and migration was significantly reduced (t=4.802, P<0.05), after the lentiviral infection. Conclusion The expression of MFG-E8 gene and protein by shRNA interference can inhibit the malignant biological behavior of breast cancer cells effectively, which indicates that MFG-E8 is involved in the invasion, metastasis and apoptosis of breast cancer during the process of breast carcinogenesis. Key words: Milk fat globule-epidermal growth factor 8 short hairpin RNA; Cell cycle; Cell proliferation; Cell poptosis; Invasion and metastasis

Effect of long non-coding RNA LINC00311 on proliferation of osteoclasts in osteoporotic rats

Objective To explore the effect of long non-coding RNA (lncRNA) LINC00311 on the proliferation of osteoclasts in osteoporotic rats. Methods Osteoclasts were isolated from ovariectomized rat models of ovariectomy. These osteoclasts were then transfected into the LINC00331 vector (observation group), short hairpin RNA (shRNA)-LINC00331 (control group), and not transfected (blank group). Finally, Western blotting was used to detect the expression of Delta-like 3 (DLL3), Notch1, Notch2, Jagged1, hairy and enhancer of split-1 (Hes-1) and tartrate resistant acid phosphatase (TRAP). The cell relative survival rate was detected by methyl thiazol tetrazolium (MTT) method, and the cell apoptosis and cell cycle were detected by flow cytometry. Results The expression of Notch2 and TRAP in the observation group (302.23±45.23, 324.32±42.12) was significantly higher than that in the blank group, the expression level of DLL3, Notch1, Jagged1 and Hes-1, and the apoptosis rate was significantly lower than that of the blank group [(52.33±12.36)%, (72.32±5.36)%, (175.23±23.25)%, (72.36±21.02)%, (3.87±0.91)%]. The cell related indexes of the control group were opposite to those of the observation group (0.96±0.11). The expression of cell Notch2 and TRAP, the relative survival rate of cell survival in the control group were (36.63±12.12, 112.02±32.01, 0.31±0.05) significantly lower than that of the blank group [(198.35±25.41)%, (259.63±35.26)%, (298.36±45.56)%, (198.69±32.25)%, (17.95±2.04)%], and the expression level of DLL3, Notch1, Jagged1 and Hes-1 of the control group were significantly higher than that of the blank group . Conclusion LncRNA LINC00311 can inhibit osteoclast apoptosis and increase the proliferation ability of osteoclasts by down regulating Notch signaling pathway. Key words: Long non-coding RNA; Osteoporosis; Osteoclasts; Proliferation; Differentiation

Protective effect of verbascoside on brain glial cells with oxidative stress injury

Objective To investigate the protective effect of verbascoside on brain glial cells (HEB) with oxidative stress injured. Methods HEB cells were cultured by dulbeeeo modified eagle medium (DMEM) culture liquid containing 10% fetal bovine serum and randomly divided in to model group, verbascoside low, medium and high dose group, positive drug group and normal control group. Except normal control group, the other group were induced to oxidative stress injury by bing exposed to H2O2 at dose 100 mmol/L. 20, 40, 80 μg/L verbascoside were separately added into low, medium and high doses of verbascoside groups. Afer co-culturing for 4 h, the morphological changes of HEB were observed by light microscope; the survival rate of HEB was examined using methyl thiazol tetrazolium (MTT). The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in the HEB were detected with spectrophotometry. The levels of lactate dehydrogenase (LDH) in culture medium were determined by enzyme linked immunosorbent assay (ELISA). Statisticalanalysis of data using the statistical product and service solutions 17.0 software. Results Compared with the normal control group, HEB in model group shrank, more suspension cells were in the culture medium, Compared with the model group, HEB cells in each concentration group and positive drug group retracted less and the number of cells increased significantly. The cell survival rate of model group was (38±6)%. The cell survival rates of low, medium and high concentration of pistil glycoside were (45±2)%, (63±4)% and (83±7)%, respectively. The cell survival rate of positive drug group was (83±2)%. Compared with the model group, the cell viability of the three concentration groups and the positive drug group increased significantly (t=3.120, 3.920, 0.892, P<0.01). In the normal control group, SOD level was 1.83±0.64, MDA level was 6.25±0.47 and LDH concentration in cell culture medium was 1357.78±110.24. In model group, SOD level was 0.94±0.21, MDA level was 14.35±1.17 and LDH concentration in cell culture medium was 1 994.32±88.25. Compared with the normal control group, SOD level of HEB cells in model group decreased (t=4.289, P<0.01), MDA level increased (t=3.204, P<0.01), LDH concentration in cell culture medium increased (t=3.056, P<0.01). SOD levels were 1.56±0.41, 1.86±0.74, 1.78±0.47, MDA concentrations were 8.06±0.43, 6.84±0.91, 6.63±0.25, LDH concentrations in cell culture medium were 1 548.36±109.47, 1 384.39±107.95, 1 297.81.36±114.32, respectively. Compared with the model group, the SOD level of middle dose of piloside increased (t=2.891, P<0.05), MDA concentration decreased (t=2.942, P<0.05), LDH concentration in cell culture medium decreased (t=2.877, P<0.05), SOD level increased (t=3.128, 3.762, P<0.01), MDA concentration decreased (t=3.320, 3.265, P<0.01) and LDH concentration in cell culture medium decreased (t=3.708, 3.962, P<0.01) in high dose group and positive drug group. Conclusion Mulberry glycoside can protect HEB from oxidative stress, and the protective mechanism may be related to improving the antioxidant capacity of HEB. Key words: Verbascoside; Oxidative stress; Brain glial cells

Effect of microRNA-19b on proliferation, migration and invasion of pancreatic ductal adenocarcinoma

Objective To examine the expression of microRNA (miRNA, miR)-19b in pancreatic ductal adenocarcinoma (PDAC) cell lines, and the functional role of miR-19b in proliferation, migration and invasion of PDAC cells. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression level of miR-19b in human pancreatic ductal epithelial cell line (HPDE), human PDAC cell lines (PANC1, MiaPaCa2 and BxPC3) and huamn PDAC metastasis cell line (COLO357) and its fast-growing variant FG. The expression of miR-19b was inhibited or overexpressed in PDAC cell lines. Transwell assay and Matrigel assay were carried out to examine the migration and invasion capabities of PDAC cells with miR-19b silencing or overexpressing. Methyl thiazol tetrazolium (MTT) and colony formation assays were performed on PDAC cell lines with miR-19b silencing or overexpression. Western blotting was used to detect the expression of proliferation markers (p21 and p27) in PDAC cell lines after miR-19b was silenced or overexpressed. Results The results of RT-qPCR showed the expression level of miR-19b in human PDAC cell lines PANC1, MiaPaCa2, BxPC3 and metastatic cell line COLO357 and FG (198.45±21.22, 221.37±18.59, 182.51±13.48, 317.64±15.20, 331.34±17.18) was significantly higher than that in normal human pancreatic ductal HPDE (98.21±14.54, P<0.05). Transwell cell migration assay and Matrigel cell invasion assay showed that the migration and invasion ability of FG cells after miR-19b silencing [(22.54±2.87)%, (28.87±5.62)%] was significantly decreased as compared with the control group [(99.65±3.07)%, (98.73±4.38)%, P<0.01]. In contrast, the migration and invasion ability of BxPC3 cells overexpressing miR-19b [(317.53±10.21)%, (286.77±12.83)%] was significantly higher than that of the control group [(97.89±5.31)%, (98.25±7.57)%, P<0.01]. The colony formation and MTT assays showed that the proliferation ability of PDAC cells after miR-19b was silenced was significantly decreased as compared with the control group (P<0.01). The result of Western blotting showed that the expression of proliferation markers (p21 and p27) in PDAC was increased by (3.25±0.27) and (5.38±0.33) times respectively as compared with the control group after miR-19b silencing (P<0.01). When miR-19b was overexpressed, the expression of p21 and p27 decreased by (2.68±0.15) and (3.27±0.27) times respectively as compared with the control group (P<0.01). Conclusion MiR-19b could promote the migration and invasion of PDAC cells and increase the proliferation capability. Key words: Pancreatic carcinoma; MicroRNA-19b; Metastsis; Proliferation

Effect of ZNF593 gene on proliferation of glioma cells

Objective To investigate the expression level of ZNF593 in glioma cells and its effect on the proliferation of glioma cells. Methods The date from TCGA were analyzed to explore the expression of ZNF593 gene in glioma cells and its relation to prognosis. Glioma U251 cells were transfected with ZNF593 short hairpin RNA (shZNF593) and shRNA control (shCtrl) respectively. The mRNA expression of ZNF593 in the cells was detected by real-time quantitative polymerase chain reaction (Real-time PCR), and the protein level of ZNF593 was measured by Western blotting. Cell proliferation was detected by Celigo and methyl thiazol tetrazolium (MTT). Results The date from TCGA shows ZNF593 gene was highly expressed in glioma, and high expression of ZNF593 leads lower survival rate. After the ZNF593 gene was silenced, Real-time PCR showed that the expression abundance level of shZNF593 was 0.380, and that was 1.000 of shCtrl, the knockdown efficiency was 62%; Western blotting showed that the protein level of ZNF593 was decreased; Celigo Cell Counting indicated that the daily increase from the second day of shCtrl group were 1.971, 2.788, 3.827, 5.169, and those of shZNF593 group were 1.340, 1.641, 2.026, 2.095; The result of MTT in 490 nm from second day to fifth day of shCtrl group were 0.174, 0.316, 0.485, 0.620, 0.765, and those of shZNF593 group were 0.172, 0.235, 0.268, 0.316, 0.366 (P<0.05). Conclusion ZNF593 is significantly up-regulated in glioma, knock down ZNFF593 can suppress the proliferation of glioma cells. Key words: ZNF593; Glioma; Cell proliferation

Mechanism of insulin-like growth factor-binding protein-3 regulating the proliferation of glioblastoma through extracellular signal-regulated kinase 1/2 signaling pathway

Objective To study the mechanism of intracellular overexpression of insulin-like growth factor-binding protein-3 (IGFBP-3) and extracellular stimulation promoting proliferation of glioblastoma U87MG cells. Methods The pCMV/IGFBP-3 plasmid was constructed and transfected into U87MG cells. The cells were divided into untreated group (control), empty vector group (NC1) which was transfected with pcDNA3.1 (+ ), pCMV-myc empty vector group (NC2), pCMV-IGFBP-3 group (Ⅰ), pcDNA3.1 (+ )/pCMV-myc group (NC1+ NC2). The expression of IGFBP-3, extracellular signal-regulated kinase 1/2 (ERK1/2), and B cell lymphoma/leukemia-XL (bcl-XL) was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. The proliferative effect of IGFBP-3 cytokines on U87MG cells was analyzed by methyl thiazol tetrazolium (MTT) colorimetry. After stimulation by IGFBP-3 at different time periods, the total cell proteins were extracted from U87MG cells at the time of 0, 5 min, 10 min, 30 min, 1 h, 2 h, and then analyzed by Western blotting after quantitative analysis. The expression of ERK1/2 and bcl-XL proteins and change in ERK1/2 phosphorylation were recorded. Results The relative expression of ERK1, ERK2 and bcl-XL mRNA was (1.32±0.45), (1.19±0.14) and (2.83±0.56) respectively. The mRNA expression of ERK1 was increased (P 0.05), and that of bcl-XL was significantly increased (P<0.01). Western blotting showed that the gray level of ERK1/2 (178.86±4.89, 188.19±5.07) did not change, but its phosphorylation level was significantly increased, and the gray value of bcl-XL (151.85±5.71, 214.37±4.71) was significantly increased. The results of MTT colorimetric assay showed that with the increase of IGFBP-3 concentration, the cell line showed a significant proliferative effect, and the stimulatory effects of IGFBP-3 with 500 ng/ml was more significant (P<0.01). U87MG cells were stimulated and cultured. Western blotting assay showed that the expression level of ERK1/2 in U87MG cells stimulated and cultured at 0, 5, 10, 30 min, 1 h and2 h (163.18±4.45, 159.36±5.67, 161.27±3.23, 172.41±4.01, 177.41±2.42, 169.11±1.45) did not change; that of PERK1/2 (27.51±2.34, 52.25±3.56, 51.75±3.67, 57.55±5.01, 66.75±3.67, 47.49±2.76) increased gradually with time and decreased after 2 h, and that of bcl-XL (18.77±2.78, 46.14±3.29, 52.43±3.62, 82.87±2.71, 60.02±0.18, 56.94±2.16) increased first and then decreased with time, and then decreased at 1 h. Conclusion Overexpression of intracellular IGFBP-3 and extracellular stimulation of U87MG cells can increase the expression of bcl-XL which was the downstream regulatory gene of ERK1/2 and promote the proliferation of glioblastoma cells. Key words: Glioma; Insulin-like growth factor-binding protein-3; Extracellular signal-regulated kinase1/2; B cell lymphoma/leukemia-XL; Proliferation

Effect of microRNA-181 on proliferation and apoptosis of hepatocellular carcinoma cells via regulation of phosphatase and tensin ho-molog deleted from chromosome 10

Objective To explore effect of microRNA (miRNA, miR)-181b on proliferation and apoptosis of hepatocellular carcinoma cells via regulation of phosphatase and tensin ho-molog deleted from chromosome 10 (PTEN). Methods miR-181b inhibitor and miR-181b NC were transfected into HepG2 cell by liposome Lipofectamine™3000. The expression of miR-181b was detected by real-time quantitative polymerase chain reaction (Real-time PCR). Cell viability was measured by methyl thiazol tetrazolium (MTT) assay. Cell clone ability was detected by cell clone formation test. Cell apoptosis and cell cycle was detected by flow cytometry. The expression of PTEN protein and mRNA was measured by Western blotting and Real-time PCR. Luciferase reporter analysis was performed. Results The expression of miR-181b in miR-181b inhibitor group (0.23±0.02) was lower than that in miR-181b NC group (1.00±0.01, P<0.05). Cell viability in miR-181b inhibitor group (0.37±0.03) was lower than that in miR-181b NC group (0.59±0.06, P<0.05). Clone formation test showed cell clone number in miR-181b inhibitor group (37.64±3.70) was lower than that in miR-181b NC group (132.56±12.37, P<0.05). Cell early apoptotic rate and late apoptotic rate in miR-181b inhibitor group was higher than that in miR-181b NC group (P<0.05). At the same time, compared with miR-181b NC group, cell cycle was made arrested at G1 phase (P<0.05), the expression of PTEN protein and mRNA was up-regulated in miR-181b inhibitor group (P<0.05), miR-181b inhibitor targeted regulation the expression of PTEN. Conclusion miR-181b inhibitor could inhibit HepG2 cell proliferation and induce cell apoptosis by targeting up-regulation expression of PTEN. Key words: Hepatocellular carcinoma; MicroRNA-181b; Phosphatase and tensin ho-molog deleted from chromosome 10; Proliferation; Apoptosis

Effect of fibroblast growth factor receptor 2 on osteogenic differentiation of mouse bone marrow mesenchymal stem cells

Objective To observe the effect of fibroblast growth factor receptor 2 (FGFR2) on the ability of bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts. Methods The FGFR2 gene of mouse BMSCs was overexpressed, and the proliferation ability at 24, 48 and 72 h was measured by methyl thiazol tetrazolium (MTT) assay. After the FGFR2 gene was overexpressed, the osteogenic differentiation and mineralization ability of the cells was examined by alizarin red staining and alkaline phosphatase (ALP) staining, and Western blotting was used to detect the expression of osteocalcin (OCN), osteopontin (OPN), runt related transcription factor-2 (Runx2), ALP and bone morphogenetic protein-2 (BMP-2) protein related genes of osteogenic differentiation after 14 days of osteogenesis induced differentiation. A mouse model of castrated osteoporosis was established, and the effect of up-regulation of FGFR2 on osteoporosis in mice was studied. The number of bone trabeculae and bone volume fraction were evaluated by mircoCT. Results The proliferation of BMSCs at 24, 48 and 72 h was promoted after up-regulation of FGFR2. The number of calcium nodules in BMSCs (95.7±8.9) in the FGFR2-up-regulated group was significantly greater than that in the control group (26.4±3.9). The results of ALP staining showed that the osteogenic differentiation ability in the FGFR2-up-regulated group was stronger than that in the control group. The quantitative analysis of ALP activity showed that the ALP activity in the FGFR2-up-regulated group [(6.33±1.33) U/L]was significantly stronger than that in the control group [(2.54±0.75) U/L]. The expression levels of OCN, OPN, UNX2, ALP and BMP-2 proteins in the FGFR2-up-regulated group were higher than those in the control group. The trabecular number (Tb.N) of bone trabecula in the FGFR2 up-regulated group [(2.37±0.26)/mm]was greater than that in the control group [(1.54±0.34)/mm], and the tibial bone volume fraction (BV/TV) in the FGFR2-up-regulated group [(9.97±1.77)%] was higher than that in the control group [(5.91±0.98)%]. Conclusion Upregulation of FGFR2 can promote the proliferation and osteogenic differentiation of mouse BMSCs, which may promote the recovery of osteoporosis. Key words: Fibroblast growth factor receptor 2; Bone marrow mesenchymal stem cells; Osteogenic differentiation; Osteoporosis

In vitro and in vivo effects of RNA interference targeting c-Myc gene on gastric cancer SGC7901 cells

Objective To observe the effect of RNA interference to c-Myc on proliferation of gastric cancer SGC7901 cells in vitro and in vivo. Methods The c-Myc gene of gastric cancer SGC7901 was silenced by RNA interference technique. The expression of c-Myc gene of SGC7901 was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). The proliferation of gastric cancer SGC7901 was detected by methyl thiazol tetrazolium (MTT) assay and in vivo tumorigenesis. Results After the silencing of c-Myc gene by RNA interference technology, the proliferation rate and tumorigenic growth rate of gastric cancer SGC7901 decreased significantly (on day 7, compared with the blank group, the tumor inhibition rate of small interfering RNA (siRNA)-scramble group was 98.78% (P>0.05), while that of the experimental group was 49.96% (P<0.05). Conclusion After the c-Myc interference, gastric cancer SGC7901 in vitro and in vivo proliferation rate decreased. Key words: Gastric carcinoma; RNA interference; c-Myc; Proliferation

Expression of special AT rich sequence binding protein-1 in lung adenocarcinoma and its effect on proliferation, invasion and apoptosis of lung adenocarcinoma cells

Objective The study aimed to explore the relationship between the expression of special AT rich sequence binding protein-1 (SATB1) and clinical characteristics in lung adenocarcinoma and its role in the proliferation, invasion and apoptosis of lung adenocarcinoma cell line A549. Methods Tissue sections were stained by immunohistochemistry streptavid-in-peroxidase (SP) method. The green fluorescent protein expression vector of SATB1-small interfering RNA (siRNA) was constructed and transfected into lung adenocarcinoma cell line A549. The effect of transfection was observed by fluor escence microscope. The silencing efficiency was measured by Western blotting. Cell proliferation, invasion and migration activity were detected by methyl thiazol tetrazolium (MTT), Transwell and wound-healing assay. Apoptosis rate was measured by flow cytometry. MTT, transwell and wound-healing assay were adopted to study cell proliferation, invasion and migration activity, and the apoptosis rate was tested by flow cytometry. Results SATB1 was highly expressed in human lung adenocarcinoma tissues (positive rate 61%), and was lowly expressed in adjacent tissues (positive rate 20%), and the expression level was negatively correlated with the degree of differentiation (P<0.01). After transfection with SATB1-siRNA, the transfection efficiency was about 70%, and the expression of SATB1 in A549 cells was decreased (P<0.01). Compared with the negative control group, the cell proliferation, invasion and migration ability were significantly decreased (P<0.01). The apoptotic rate was increased (P<0.01). Conclusion SATB1 is closely related to the pathogenesis and development of lung adenocarcinoma, which may provide important clues for finding potential therapeutic targets and prognosis of lung adenocarcinoma. Key words: Lung adenocarcinoma; Special AT rich sequence binding protein-1; Cell proliferation

Expression of lysine-specific demethylase 1 in hepatocellular carcinoma tissues and its effect on cell proliferation and invasion

Objective To investigate the expression of lysine-specific demethylase 1 (LSD1) in hepatocellular carcinoma tissues, and to explore the effects of down-regulation of LSD1 gene expression on proliferation and invasion of human hepatoma HepG2 cells. Methods A total of 91 cases of hepatocellular carcinoma undergoing elective surgery in our hospital and Henan Cancer Hospital were selected. Immunohistochemistry was used to detect the expression of LSD1 protein in hepatocellular carcinoma tissues and adjacent tissues. Human hepatoma HepG2 cells were cultured and divided into LSD1 interference sequence group, negative control group and blank group. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of LSD1 gene in cells. Methyl thiazol tetrazolium (MTT) assay was used to detect cell proliferation. Transwell chamber was used to detect cell invasion. Western blotting was used to detect the expression of LSD1, Snail, E-cadherin and N-cadherin proteins in cells. Results The positive expression rate of LSD1 protein in hepatocellular carcinoma tissues was 81.32%, which was significantly higher than that in adjacent tissues (35.16%, P<0.01). The expression of LSD1 protein in hepatocellular carcinoma tissues was related to the degree of differentiation, TNM staging and vascular invasion (P<0.05). The relative expression level of LSD1 mRNA in LSD1 interference sequence group was 1.19±0.10, which was significantly lower than that in the negative control group and blank group (2.29±0.14 and 2.34±0.11, P<0.05). The absorbance (A) values at 24, 48, 72 and 96 h in the LSD1 interference sequence group were 0.23±0.05, 0.32±0.08, 0.38±0.08 and 0.52±0.03, respectively, which were significantly lower than those in the negative control group (0.38±0.08, 0.49±0.05, 0.67±0.04 and 0.77±0.10) and blank group (0.33±0.03, 0.52±0.09, 0.70±0.08 and 0.78±0.07, P<0.05). The number of invasive cells in the LSD1 interference sequence group was (86.3±3.9), which was significantly less than that in the negative control group (110.6±9.4) and the blank group (117.7±7.0, P<0.05). The relative expression levels of LSD1, Snail, and N-cadherin proteins in the LSD1 interference sequence group were (0.25±0.03, 0.30±0.02 and 0.32±0.03), which were significantly lower than those in the negative control group (0.77±0.06, 0.76±0.10 and 0.67±0.04) and blank group (0.84±0.14, 0.74±0.07 and 0.69±0.06, P<0.05), and the relative expression level of E-cadherin protein (0.71±0.05) was significantly higher than in the negative control group (0.37±0.02) and blank group (0.34±0.04, P<0.05). Conclusion LSD1 protein was highly expressed in hepatocellular carcinoma tissues, and might play an important role in the proliferation and invasion of human hepatoma HepG2 cells through epithelial-mesenchymal transition. Key words: Carcinoma, hepatocellular; Lysine-specific demethylase 1; Proliferation; Invasion; Epithelial-mesenchymal transition

MicroRNA-34a promotes hypertension-induced vascular injury by targeting B-lymphocytoma-2

Objective To investigate the level of microRNA (miRNA, miR)-34a in serum of hypertensive patients and its effect on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) by targeting B cell lymphoma/leukemia-2 (bcl-2), and clarify the underlying mechanism of hypertension-induced vascular injury. Methods Real-time quantitative polymerase chain reaction (qPCR) was used to detect the level of miR-34a in serum of 98 hypertensive patients and 65 healthy controls; the potential targets of miR-34a was predicted by TargetScan; the interaction between miR-34a and bcl-2 was measured by dual luciferase reporter assay, western blot and qPCR; the effect of miR-34a on proliferation and apoptosis of HUVECs was detected by methyl thiazol tetrazolium (MTT) and flow cytometry. Results The level of miR-34a in serum of 98 hypertensive patients (2.432±0.201) was significantly higher than that of 65 healthy controls (0.996±0.112, P 0.05). The protein and mRNA level of bcl-2 in HUVECs after overexpressing miR-34a (0.403±0.041, 0.206±0.018) decreased significantly than that in Blank group (1.000±0.098, 1.000±0.067) and miR-NC group (0.996±0.087, 1.012±0.049). The proliferation of HUVECs after overexpressing miR-34a (48 h: 0.269±0.021, 72 h: 0.324±0.028) decreased significantly than that of the Blank group (48 h: 0.324±0.025, 72 h: 0.458±0.044) and miR-NC group (48 h: 0.335±0.028, 72 h: 0.469±0.051, P<0.05), and apoptosis (23.54±2.65) was significantly higher than that of Blank (6.45±0.42) and miR-NC (7.32±0.54, P<0.05). Conclusion MiR-34a is overexpressed in serum of hypertensive patients. Overexpressing miR-34a can inhibit proliferation of HUVECs by targeting bcl-2 expression, which may be one of the potential mechanisms of vascular injury in hypertensive patients and provide experimental basis for using miR-34a as a new target to diagnose and treat hypertension in the future. Key words: MicroRNA-34a; B cell lymphoma/leukemia-2; Hypertension; Vascular injury

Functions of p38 mitogen-activated protein kinase alpha and beta in human pancreatic cancer cells

Objective To elucidate possible functions of p38α and p38β in human pancreatic cancer. Methods Expression of p38α, p38β, p38γ and p38δ were determined by immunoblot in 7 cultured human pancreatic cancer cell lines. To selectively inhibit p38α or p38β, cell clones were established in a stable manner expression either p38α short hairpin RNA (shRNA) or p38β shRNA. Proliferation, single cell movement, in vitro migration, colony forming ability as well as in vivo tumorigenicity were determined by methyl thiazol tetrazolium (MTT) assay, video-time laps microscopy, boyden-chamber, soft-agar assay, tumor formation and growth in nude mice respectively. Results All pancreatic cancer cells showed p38 isoforms protein expression at various levels. Selective inhibition of p38α or p38β in Mia Paca-2 and Panc-1 cells revealed that both kinases enhance cell proliferation and anchorage-independent growth. Single cell movement and cell invasion were markedly reduced in p38α shRNA expressing clones, but not altered in p38β shRNA expressing clones. Inhibition of p38α enhanced in vivo tumorigenicity profoundly in Mia Paca-2 and Panc-1 cells. In Mia Paca-2 cell, the in vivo tumor formation rate of two p38β shRNA expressing clones is 18.3% and 50.0% respectively, which decreased significantly comparing to the wild type cell and null vector transfection cell. The tumor volume decreased significantly either (P<0.05). In Panc-1 cell, the in vivo tumor formation rate of two p38β shRNA expressing clones is 25.0% and 12.5% respectively. Conclusion p38 mitogen activated protein kinases (p38MAPKs) are expressed in pancreatic cancer cells and the isoforms may exert different functions. Key words: p38 mitogen-activated protein kinase; Pancreatic cancer; p38α; p38β

Decreased chemosensitivity of platinum in human colon cancer cells by overexpression of Golgi phosphorylation protein 3 and possible mechanisms

Objective To investigate the influence of overexpression of Golgi phosphorylation protein 3 (GOLPH3) gene on the chemosensitivity of Cisplatin in human colon cancer HT29 cell line. Methods HT29 cells were divided into four groups: control group, small interfering RNA (siRNA) transfection group, experimental group 1 (10 μmol/L Cisplatin), and experimental group 2 [small interfering RNA (siRNA)-GOLPH3+ 10 μmol/L Cisplatin]. The silencing effect of GOLPH3 gene was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The tumor cell proliferation and formation ability of HT29 cells were measured respectively by methyl thiazol tetrazolium (MTT) assay and tumorsphere formation assay. Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) flow cytometry was used to detect the apoptosis of each group. The protein expression levels of GOLPH3, permeability glycoprotein (P-gp) and β-catenin in HT29 cells were detected by Western blotting. Sixteen nude mice were randomly divided into 4 groups (4 mice in each group): control group, transfection group, experimental group 1 and experimental group 2. The corresponding colon cancer cells were injected subcutaneously into the axillary tissue. The tumor formation in nude mice was observed within 30 days. Results The relative expression level of GOLPH3 mRNA and protein in the transfection group was significantly lower than in the control group (0.162±0.062 vs. 1.002±0.223; 0.099±0.112 vs. 1.003±0.094) (P<0.01). The cell apoptosis rate in the transfection group was significantly higher than in the control group [(15.520±2.921)% vs. (1.843±1.298)%, P<0.01]. Under cisplatin treatment, the A value (0.746±0.085) and the tumorsphere number (212.800±30.380) in the experimental group 1 were significantly higher than those in the experimental group 2 (0.236±0.071 and 30.750±14.500) (P<0.01). Annexin V-FITC/PI flow cytometry showed that the apoptosis rate of the experimental group 1 was significantly lower than that of the experimental group 2 [(23.890±6.363)% vs. (59.400±2.392)%, P<0.01]. The volume of the axillary subcutaneous tumor in the siRNA transfection group, the experimental group 1 and the experimental group 2 was significantly lower than that in the control group [(1.738±0.121), (1.573±0.224), (0.625±0.189) mm3 vs. (2.083±0.110) mm3,P<0.05 for all]. In the experimental group 2, the tumor volume of the nude mice was significantly less than that in the experimental group 1 in the same period after 15 days (P<0.01). Compared to the control group, the expression levels of GOLPH3, β-catenin and P-gp in the experimental group 1 were significantly upregulated (1.000±0.173 vs. 2.734±0.440, 1.000±0.078 vs. 2.472±0.444, and 1.014±0.346 vs. 4.269±0.454) (P<0.01), and the expression levels of these proteins in the experimental group 2 were significantly down-regulated in the experimental group 1 (0.249±0.084 vs. 2.734±0.440, 0.993±0.052 vs. 2.472±0.444, and 1.842±0.383 vs. 4.269±0.454) (P<0.01). Conclusion The overexpression of GOLPH3 can decrease the chemosensitivity of Cisplatin in colon cancer HT29 cells by activating Wnt/β-catenin signal pathway. Key words: Golgi phosphorylation protein 3; Colon cancer cell; Wnt/β-catenin signal pathway; Chemosensitivity; Cisplatin

MicroRNA-133b inhibits colorectal cancer cell proliferation through targeting matrix metalloproteinase-9

Objective To investigate the molecular mechanism by which microRNA (miRNA, miR)-133b inhibits colorectal cancer cell proliferation through targeting matrix metalloproteinase-9 (MMP-9). Methods To examine the expression level of MMP-9 in clinic tissue specimens using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR); to achieve MMP-9 knockdown or overexpression using RNA interference or recombinant overexpressing plasmid, and then monitor the cell proliferation of colorectal cancer cell, HCT116, in response to MMP-9 knockdown or overexpression using methyl thiazol tetrazolium (MTT) and colony formation assays; to predict the upstream candidate miRNA which might regulate MMP-9 expression and to validate the prediction using FQ-PCR and luciferase reporter gene assays; to achieve miR-133b and MMP-9 overexpression in HCT116 cells, and then monitor the cell proliferation of colorectal cancer cells. Results The expression of MMP-9 significantly increased in colorectal tissues, compared with the paired adjacent normal tissues (3.17±0.79 vs. 1.00±0.25, P 0.05). After overexpression of miR-133b, the cell proliferation ability was significantly lower than control group by MTT (1.34±0.04 vs. 1.54±0.06, P<0.01) and cloning efficiency (8.67±3.06 vs. 17.67±2.52, P<0.05), respectively. Furthermore, we con-overexpression of miR-133b and MMP-9, the cell proliferation ability was higher than miR-133b overexpression group by MTT (1.66±0.05 vs. 1.34±0.04, P<0.01) and cloning efficiency (22.00±4.00 vs. 8.67±3.05, P<0.05). Conclusion MiR-133b could inhibit colorectal cancer cell proliferation through direct targeting and downregulating MMP-9. Key words: Colorectal cancer; MicroRNA-133b; Matrix metalloproteinase-9; Cell proliferation