Empty Plasmid Group Research Articles

Effect of microRNA-153 targeting phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway on proliferation in acute lymphoblastic leukemia cell

Objective To investigate the effect of microRNA (miRNA, miR)-153 on the proliferation and apoptosis of in human acute lymphoblastic leukemia (ALL) cell line Jurkat cells by phosphatidylinositol 3 kinase (PI3K)/serine-threonine protein kinase (Akt) pathway. Methods After miR-153 mimics (miR-153 mimics group) and miR-153 inhibitor (miR-153 inhibitor group) were transfected into Jurkat cells, cell proliferation, cycle and apoptosis. was detected; the expression of miR-153, PI3K and Akt mRNA and protein was measured; whether PI3K was the target gene of miR-153. was confirmed. Results Compared with the negative control group, the expression of miR-153 in the miR-153 mimics group was significantly increased by approximately 10-fold (t=39.596, P<0.01), and the expression of miR-153 was significantly decreased in the miR-153 inhibitor group (t=14.651, P<0.01). The activity of the cells in the miR-153 mimics group was significantly decreased (t=10.931, P<0.01), and the activity of the miR-153 inhibitor group was significantly increased (t=7.640, P<0.01). The expression of PI3K total protein, PI3K mRNA and p-Akt in the miR-153 mimics group was significantly decreased (t=7.161, P<0.01; t=6.684, P<0.01; t=10.769, P<0.01), and the expression of PI3K total protein, PI3K mRNA and p-Akt in the miR-153 inhibitor group was significantly increased (t=3.998, P<0.05; t=8.902, P<0.01; t=7.078, P<0.01). The proportion of G0/G1 phase cells increased significantly, while the S phase and G2/M phasecells decreased significantly (t=3.786, P<0.05; t=3.012, P<0.05; t=4.948, P<0.01) in the miR-153 mimics group; the G0/G1 phase cells decreased significantly, and the proportion of cells in S phase and G2/M phase increased significantly (t=5.356, P<0.01; t=3.055, P<0.05; t=3.893, P<0.05) in the miR-153 inhibitor group. The apoptosis rate of the miR-153 mimics group was significantly increased (t=4.673, P<0.05), while the apoptotic rate of the miR-153 inhibitor group was significant decline (t=5.618, P<0.01). Compared to the empty plasmid group, the relative luciferase activity of 3’untranslated regions (3’UTR) plasmid group was significantly reduced (t=7.925, P<0.01). Conclusion MiR-153 can directly target PI3K/Akt signaling pathway and regulate Jurkat cell proliferation and apoptosis. Key words: Acute lymphoblastic leukemia; MicroRNA-153; Phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway

The effect of miR-155 on HBV replication and PTEN expression in vivo

Objective: To construct the mmu-miR-155 eukaryotic overexpression vector pmR-155 and to investigate its effect on HBV replication and expression of PTEN in vivo. Methods: The mmu-mir-146a precursor gene fragment pre-mmu-mir-146a was amplified by PCR, then connected to the pmR-mCherry plasmid vector after double enzyme digestion, the accuracy of recombinant vector was verified by colony PCR、double enzyme digestion and sequencing; then the recombinant vector was transfected HBV transgene mice(Experimental Group)with hydrodynamics-based injection via vena caudalis, and pmR-mCherry plasmid、PBS were respectively transfected into the mice as Empty plasmid Group、Blank Group. The concentration of IFN-γ in the serum was detected by ELISA. The expression of SOCS1、PTEN mRNA in the liver was detected by qPCR at 30d post-transfectioned. The Western blot was performed to detect the changes in SOCS1、PTEN、HBX in the liver tissue at 30 d post-transfectioned. The results were analyzed with Student's t-test, or one-way analysis of variance and the least significant difference test. Results: the colony PCR、double enzyme digestion and sequencing verified that the gene was inserted into the pmR-mCherry vector. Compared with Blank Group, the expression of miR-155 in the Experimental Group was significantly increased(t = 8.90, P < 0.01); the concentration of IFN-γ in the Experimental Group was significantly increased(F = 26.58, P < 0.01); the mRNA(F(SOCS1 mRNA) = 19.72, P < 0.01; F(PTEN mRNA) = 7.38, P < 0.05) and protein(F(SOCS1) = 50.30, P < 0.01; F(PTEN) = 129.00, P < 0.01) expression of COCS1、PTEN was significantly decreased in the Experimental group and the protein of HBX was also significantly(F(HBX) = 77.97, P < 0.01). Conclusion: The pmR-155 eukaryotic overexpression vector is successfully constructed, this recombinant vector can express miR-155 stably; miR-155 can down-regulate cocs1、PTEN gene expression and up-regulate the expression of IFN-γ, it can inhibit the replication of HBV and a potential targets to treating hepatocellular carcinoma.

Effects of secreted frizzled-related proteins 2 and β-catenin on proliferation, migration and invasion of colorectal cancer cells

Objective To observe the effects of secreted frizzled-related proteins 2 (sFRP2) and β-catenin on proliferation, migration and invasion of colorectal cancer cells. Methods The pcDNA3.1 plasmid containing sFRP2 gene sequence was constructed to overexpress sFRP2 in HCT116 by transfection. The expression level of sFRP2 in HCT116 cells after transfection was detected by Western blotting. Cell counting kit-8 (CCK-8) assay was used to detect the effect of sFRP2 upregulation on the proliferation of colorectal cancer cells; Wound-healing assay was used to detect the effect of sFRP2 up-regulation on the migration of colorectal cancer cells; Transwell assay was used to detect the effect of sFRP2 up-regulation on the invasion ability of colorectal cancer cells. Results After plasmid transfection, the expression of sFRP2 and β-catenin significantly increased. The ratio of sFRP2 protein to β-actin was 0.62±0.03 vs. 0.21±0.02 (t=25.430, P=0.001), and the ratio of β-catenin to β-actin was 0.32±0.02 vs. 0.17±0.01 (t=15.000, P=0.001), and the difference was statistically significant. The absorbance values at 12, 24, 36, 48 and 60 h in sFRP2 transfection group, control group and empty plasmid group were measured to be 0.213±0.015 vs. 0.215±0.018 vs. 0.218±0.012, 0.345±0.053 vs. 0.351±0.041 vs. 0.362±0.038, 0.382±0.058 vs. 0.413±0.051 vs. 0.433±0.048, 0.441±0.079 vs. 0.503±0.033 vs. 0.534±0.053, and 0.482±0.034 vs. 0.594±0.045 vs. 0.628±0.062. As compared with the control group and the empty plasmid group, the cell proliferation rate in sFRP2 transfection group was significantly decreased (12 h: t=0.016, P=0.988 and t=0.041, P=0.970; 24 h: t=0.155, P=0.884 and t=0.452, P=0.675; 36 h: t=0.695, P=0.525 and t=1.173, P=0.306; 48 h: t=1.254, P=0.278 and t=1.693, P=0.166; 60 h: t=3.440, P=0.026 and t=3.576, P=0.023). The difference in the distances between the both sides of scratches at 0 h and 48 h in the blank group was used as control. The distance in the blank group, empty plasmid group, and sFRP2 transfection group was 1.00±0.00, 0.91±0.06, and 0.61±0.04, respectively. The distance in sFRP2 transfection group was farther than blank group and empty plasmid group, and the migration speed was significantly slower (t=16.890, P=0.001 and t=7.206, P=0.002); The number of transmembrane cells in the sFRP2 transfection group was (75.69±7.19) cells, significantly less than that in the control group [(195.39±8.68) cells, t=25.459, P=0.001], indicating the invasive ability of the cells was significantly decreased in the sFRP2 transfection group. Conclusion The up regulation of sFRP2 significantly inhibited the proliferation, migration and invasion of colorectal cancer cell line HCT116. Key words: Colorectal cancer; Secreted Frizzled-related proteins 2; β-catenin; Proliferation; Migration; Invasion

Influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2-vascular endothelial growth factor 165 in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells

Objective To observe the influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2 (BMP2)-vascular endothelial growth factor 165 (VEGF165) in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods The isolated rabbit BMSCs were used as the research objects, and the recombinant plasmid pIRES-BMP2-VEGF165 was transfected into cells in the presence of liposomes. Then, G418 was used to screen stable cell lines in vitro and mRNA and protein levels were detected for transfection efficiency. Stable cell lines were then cultured and the cells were divided into pIRES group, pIRES-BMP2-VEGF165 transfection group and osteogenic induction group. Western blotting was used to detect the expression of Collagen Ⅰ (ColⅠ) and osteocalcin (OCN) protein at 7th and 21st day after cultivation, respectively. After 14 days of cultivation, gomori modified calcium and cobalt staining was used to detect alkaline phosphatase activity. After 21 days of cultivation, calcified nodules of cells were detected by alizarin red method calcium staining. Results Compared with the empty plasmid pIRES group, the expression of BMP2 and VEGF165 was detected at the mRNA and protein levels of pIRES-BMP2-VEGF165 plasmid group. Compared with empty plasmid group and osteogenic induction group, the expression levels of ColⅠ and OCN protein in recombinant plasmid group were significantly increased (Col Ⅰ: 0.597±0.040 vs. 0.230±0.020, P=0.000; 0.597±0.040 vs. 0.457±0.035, P=0.005; OCN: 0.433±0.038 vs. 0.083±0.021, P=0.000; 0.433±0.038 vs. 0.350±0.036, P=0.046). Positive relative area (%) of alkaline phosphatase staining increased significantly (69.000±5.568 vs. 18.333±3.056, P=0.000; 69.000±5.568 vs. 43.333±5.508, P=0.002). Positive relative area (%) of calcified nodules increased markedly (37.747±3.763 vs. 1.587±0.972, P=0.000; 37.747±3.763 vs. 19.330±4.033, P=0.008). Conclusion Human recombinant eukaryotic expression plasmid pIRES-BMP2-VEGF165 was successfully transfected into BMSCs. As compared with osteogenic induction group, the recombinant plasmid pIRES-BMP2-VEGF165 was more effective in osteogenic differentiation of the cells. Key words: Bone mesenchymal stem cells; pIRES-bone morphogenetic proteins 2 -vascular endothelial growth factor 165 plasmid; Transfection in vitro; Bone morphogenetic proteins 2; Vascular endothelial growth factor 165

Exogenous Tissue Inhibitor of Metalloproteinase-2 Affects Matrix Metalloproteinase-2 Expression in Conjunctival Filtering Blebs and Bleb Scarring in Rats.

Objective To examine the effect of tissue inhibitor of metalloproteinase-2 (TIMP-2) on conjunctival filtering bleb scarring. Methods A model of conjunctival filtering bleb was established whereby rats were injected with saline, blank adenoviral vector, or adenoviral vector carrying TIMP-2 into the bleb. Filtration bleb formation and matrix metalloproteinase-2 (MMP-2) expression were examined. Results All operated eyes formed obvious elevated blebs on day 1. In the normal saline group, empty plasmid group, and gene transfection group maintenance time of filtrating blebs was 5–14, 5–14, and 6–16 days, and average survival time was 8.24, 8.16, and 9.44 days, respectively. MMP-2 expression increased slightly in the gene transfection group at 3 and 5 days after surgery, reached a peak after 14 days, and then gradually decreased. MMP-2 expression was weakly positive in the normal conjunctival epithelium, but was hardly detected in the lamina propria. Seven days after surgery, the epithelium and lamina propria of the conjunctival filtering bleb exhibited strong positive expression in the empty plasmid group but only weak expression in the adenovirus group. Conclusion Exogenous TIMP-2 interfered with local MMP-2 expression, delaying peak expression of MMP-2 and slowing the scarring of filtering blebs during wound healing of subconjunctival tissue.

The regulation of hypoxia inducible factors on glucose transporter 1, pyruvate kinase M2 and vascular endothelial growth factor in BGC-823 cells

Objective To compare the expression of glucose transporter 1 (GLUT1), pyruvate kinase M2 (PKM2), vascular endothelial growth factor (VEGF) in BGC-823 cells after transfected with small interfering RNA of hypoxia-inducible factors (HIF-1α, HIF-2α, HIF-1β) and investigate the effect of hypoxia inducible factors (HIFs) on the regulation of GLUT1, PKM2 and VEGF in gastric cancer. Methods Small RNA interference (RNAi) was used to silence BGC-823 cells. The mRNA and protein expression levels of HIFs, GLUT1, PKM2 and VEGF were detected by quantitative real-time reverse transcription polymerase chain reaction (PCR) and Western blotting. Results The mRNA (0.372±0.097) and protein (0.351±0.041) expression of HIF-1α was lower than in empty plasmid group (0.980±0.115 and 0.520±0.030) and blank control group (1.000±0.000 and 0.478±0.062) after infection with RNAi, the differences were all statistically significant (F=50.358, P=0.000; F=10.931, P=0.010). The mRNA (0.391±0.091) and protein (0.293±0.031) expression of HIF-2α was lower than in empty plasmid group (0.980±0.115 and 0.520±0.030) and blank control group (1.000±0.000 and 0.430±0.056) after infection with RNAi, the differences were all statistically significant (F=72.416, P=0.000; F=9.016, P=0.016). The mRNA (0.383±0.091) and protein (0.323±0.038) expression of HIF-1β was lower than in empty plasmid group (0.987±0.100 and 0.445±0.059) and blank control group (1.000±0.000 and 0.457±0.040) after infection with RNAi. The differences were all statistically significant (F=78.273, P=0.000; F=7.636, P=0.022). The mRNA (GLUT1: 0.721±0.067, 0.833±0.059, 0.493±0.067; VEGF: 0.711±0.057, 0.826±0.054, 0.793±0.068) and protein (GLUT1: 0.263±0.072, 0.303±0.042, 0.198±0.057; VEGF: 0.391±0.049, 0.451±0.054, 0.473±0.086) expression of GLUT1 and VEGF was all decreased after silencing HIFs (HIF-1α, HIF-1β, HIF-2α) when compared with empty plasmid group (mRNA: 1.046±0.094, 1.039±0.092; protein: 0.391±0.064, 0.690±0.066) and blank control group (mRNA: 1.000±0.000, 1.000±0.000; protein: 0.438±0.055, 0.707±0.040) and the differences were all statistically significant (VEGF mRNA: F=35.232, P=0.000; GLUT1 mRNA: F=15.364, P=0.000; VEGF protein: F=8.150, P=0.003, GLUT1 protein: F=17.109, P=0.000). The levels of VEGF decreased the most obvious when transfection with HIF-2α siRNA, while the levels of GLUT1 decreased the most obvious when transfection with HIF-1α siRNA. The mRNA (0.611±0.036) and protein (0.351±0.047) expression of PKM2 was all decreased after silencing HIF-1α when compared with empty plasmid group (1.037±0.197 and 0.731±0.057) and blank control group (1.000±0.000 and 0.737±0.043), the differences were all statistically significant, (F=12.480, P=0.007; F=60.403, P=0.000). Whlie only mRNA (0.793±0.067) decreased after silencing HIF-2α, the differences were statistically significant (F=12.480, P=0.007). The protein changed a little (0.675±0.030, F=1.211, P=0.373), and both mRNA (0.980±0.115) and protein (0.641±0.076) had no significant change after transfection with HIF-1β (F=0.144, P=0.869; F=2.404, P=0.171). Conclusion The results suggest that HIF-1α, HIF-2α and HIF-1β are all involved in regulating GLUT1 and VEGF. HIF-1α pathway plays a major role in regulating PKM2 and HIF-2α may regulate PKM2 in transcription level. Key words: Gastric cancer; Hypoxia-inducible factors; Pyruvate kinase M2; Glucose transporter 1; Vascular endothelial growth factor

Regulation of Krüppel-like factor 6 via activating transcription factor 4 pathway to apoptosis of human lens epithelial cells

Objective To investigate the regulating effects of Kruppel-like factor 6 (KLF6) on the apoptosis of human lens epithelial cells (HLECs) by activating transcription factor 4 (ATF4) pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis. Methods HLECs (HLE-B3) were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group, and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B (UVB) for 200 seconds, The morphological changes of the cells were observed by hematoxylin & eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 (siKLF6) and pSilencer plasmid, respectively, and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group; pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6+ pSilencer group; pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4+ pEGFP-C2 group; pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6+ siATF4 group, and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay. Results Cultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis, karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group, the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB+ ATF4 transfected group and UVB+ pEGFP-C2 plasmid group, respectively, with a significant difference between them (t=23.13, P<0.01). The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group, and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group (all at P<0.01). ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37±0.11, which was significantly higher than 0.31±0.11 in the normal control group (t=8.034, P=0.001); the apoptotic rate of the cells was increased in the KLF6+ pSilencer group and decreased in the siATF4+ pEGFP-C2 group in comparison with the empty plasmid group (P<0.01, P=0.02). In addition, the apoptotic rate in the KLF6+ siATF4 group was remarkably lower than that in the KLF6+ pSilencer group (P<0.01). Conclusions KLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating pathway of KLF6 to apoptosis. Key words: Kruppel-like factor 6; Kruppel-like transcription factors/metabolism; Activating transcription factor 4; Epithelial cells, lens; Cell line, crystalline; Humans; Apoptosis

Protection effects of glutathione 2 on human lens epithelial cells against ultraviolet radiation-induced apoptosis

Objective To observe the damage of human lens epithelial cells (LECs) induced by ultraviolet (UV)B radiation and the expression changes of glutaredoxin 2 (Grx2) in the cells, and to investigate the protective effects of Grx2 on human LECs against UVB-induced apoptosis. Methods Human LECs (HLE-B3) were cultured and exposed to different energy of UVB (0, 10, 30, 50 mJ/cm2) with the wavelength of 297 nm.The morphology of the cells was examined under the optical microscope 2, 4, 8, 12 and 16 hours after irradiation of UVB.The survival rate of the cells was evaluated with cell counting kit 8 (CCK8). The apoptosis rate of the cells was detected by TUNEL assay.Real-time quantitative PCR and Western blot were employed to detect the expressions of Grx2 mRNA and Grx2 protein in the cells, respectively.The cells were transfected with pcDNA3.1-Grx2 plasmid by lipofectamine 2000 as the Grx2 transfected group, and pcDNA3.1 plasmid was transfected into cells as the empty plasmid group.The cells were irradiated by 50 mJ/cm2 for 4 hours, and the apoptosis rate of human LECs was detected by TUNEL assay. Results Cultured cells grew well with the green fluorescence for Grx2 expression in the non-UVB exposure group, and shrinkage and death of the cells were found after UVB irradiation.The survival rate of the cells was gradually reduced after irradiation of 10, 30, 50 mJ/cm2 UVB as the increase of UVB energy and lapse of time.The expression level of Grx2 mRNA were 2.53±0.48 and 3.53±0.14 in the 10 mJ/cm2 UVB group and 30 mJ/cm2 UVB group 4 hours after irradiation, which were significantly higher than 1.01±0.08 and 1.00±0.09 in the non-UVB exposure group (all at P<0.05). The expression level of Grx2 mRNA in the cells was 15.30±3.01 at 1 hour after irradiation in the 50 mJ/cm2 UVB group, which was significantly higher than 1.00±0.07 in the non-UVB exposure group (P<0.05). The expression levels of Grx2 protein showed the same tendency to Grx2 mRNA.The apoptosis rate of the cells in the Grx2 transfected group was (15.34±1.71)%, and that in the empty plasmid group was (22.11±2.46)% at 4 hours after 50 mJ/cm2 UVB irradiation, with a significant difference between them (t=3.189, P<0.05). Conclusions UVB irradiation induced damage of human LECs in dose- and time-dependent manner.However, the expression of Grx2 in human LECs is up-regulated transiently after exposure of UVB, which has a protective effect on the cells against the UVB-induced apoptosis. Key words: Ultraviolet rays/adverse effects; Epithelial cells; Lens, crystalline; Glutaredoxin; Apoptosis/radiation effects; Cells, cultured; Humans

PTPN12 Affects Nasopharyngeal Carcinoma Cell Proliferation and Migration Through Regulating EGFR.

Nasopharyngeal carcinoma (NPC) shows the leading morbidity in otorhinolaryngological malignant tumor. It is a common malignancy in China with obvious reginal distribution. NPC is a polygenic disease that is affected by numerous factors. Protein tyrosine phosphatase nonreceptor type 12 (PTPN12) regulates multiple tumor proliferation and development, including breast cancer and colon cancer. However, the role of PTPN12 in NPC occurrence and development has not been elucidated. NPC cell line CNE2 was cultured in vitro and divided into three groups, including control, empty plasmid, and PTPN12 groups. PTPN12 mRNA and protein expressions were tested by real-time polymerase chain reaction and Western blot. CNE2 cell proliferation was detected by MTT assay. Cell migration was determined by wound healing assay. Cell apoptosis was evaluated by caspase 3 activity detection. Epidermal growth factor receptor (EGFR) expression was assessed by Western blot. PTPN12 plasmid transfection increased PTPN12 mRNA and protein expressions, suppressed cell proliferation and migration, reduced EGFR level, and enhanced caspase 3 activity compared with control and empty plasmid groups (p < 0.05). PTPN12 regulates NPC proliferation and migration through negative regulating EGFR. It could be treated as a molecular target for NPC diagnosis and prognosis analysis.

Calcitonin gene-related peptide-transfected bone marrow mesenchymal stem cells combined with autologous bone grafting to treat osteonecrosis of the femoral head in rabbit model

Objective To investigate the histopathological changes of calcitonin peptide-transfected bone marrow mesenchymal stem cells (BMSCs) combined with bone transplantation in the treatment of osteonecrosis of the femoral head (ONFH) in rabbit model. Methods The osteonecrosis models on one side femoral head of rabbits were established by liquid nitrogen. Forty-eight rabbit ONFH models were randomly divided into 3 groups. In human calcitonin gene-related peptide α (hCGRPα)-transfected BMSCs group (A), the BMSCs transfected with hCGRPα in combination with autologous bone graft were implanted into the femoral head. In empty plasmid group (B), the BMSCs transfected with empty plasmid (only pLNCX2 plasmid) and autologous bone graft were implanted. In irrelated sequence group (C), BMSCs transfected with irrelated gene [only pLNCX2-green fluorescent protein (GFP)] and autologous bone graft were implanted. Animals were sacrificed at 3rd and 6th month respectively to observe the histopathological changes in the femoral heads of rabbits. Results At 3rd month, the bone trabecular area fraction respectively was (40.89±1.12)%, (36.12±1.17)%, (36.33±1.53)% and (42.11±1.91)% in groups A, B and C and normal femoral head. The percentage of empty osteocyte lacunae respectively was (11.34±1.58)%, (16.33±1.45)%, (17.21±1.23)% and (10.47±1.41)% in groups A, B and C and normal femoral head. In group A, there were proliferation and growth of a large number of osteocytes, more mature bone trabeculae and higher area fraction, less empty osteocyte lacunae, and stronger biomechanical strength in the femoral head, which were nearly close to those in the normal control group and obviously superior to those in groups B and C. In groups B and C, there were thin and irregular bone trabeculae, more empty osteocyte lacunae, lower area fraction and imperfect bone repair. At 6th month, the bone trabeculae were mature, densification and satiation, and neatly arranged rules were seen in the femoral head of group A. The area fraction of bone trabeculae and the number of empty bone lacunae in group A were superior to those in groups B and C. Conclusion The implantation of hCGRPα-transfected BMSCs combined with autologous bone graft into the femoral head of ONFH animal models can significantly promote cells proliferation, accelerate osteogenesis, enhance bone tissue repair, and obtain good effect in the treatment of ONFH. Key words: Calcitonin gene related peptide; Bone marrow mesenchymal stem cells; Osteonecrosis of the femoral head necrosis; Model, animal

Liver kinase B1 gene enhances radiosensitivity of lung cancer H460 cells: an in vivo study

Objective To investigate the effect of liver kinase B1(LKB1) on the radiosensitivity of subcutaneous xenograft tumor of lung cancer H460 cells in nude mice. Methods Human lung cancer H460 cells were implanted into female nude mice (BALB/c-nu) to establish a subcutaneous xenograft tumor model of lung cancer. A total of 24 female nude mice in which the model was successfully established were equally and randomly divided into four groups: pEGFP-Ctrl plasmid (empty vector plasmid) group, irradiation (IR)+ pEGFP-Ctrl plasmid group, pEGFP-LKB1 plasmid (overexpressing LKB1) group, and IR+ pEGFP-LKB1 plasmid group. The growth of xenograft tumors was observed and the tumor inhibition rate and enhancement factor (EF) were calculated. The expression of LKB1 in each group was measured by immunohistochemistry and Western blot to analyze the relationship between LKB1 and radiosensitivity. Results Compared with the pEGFP-Ctrl plasmid group, the IR+ pEGFP-Ctrl plasmid group, pEGFP-LKB1 plasmid group, and IR+ pEGFP-LKB1 plasmid group showed varying degrees of inhibition of tumor growth, particularly in the IR+ pEGFP-LKB1 plasmid group, and the tumor inhibition rates were 31.30%, 14.78%, and 43.48%, respectively. The EF of LKB1 in the IR+ pEGFP-LKB1 plasmid group was 1.18.The immunohistochemistry and Western blot showed that LKB1 could be effectively expressed in the pEGFP-LKB1 plasmid group and IR+ pEGFP-LKB1 plasmid group, but not in the other two groups. Conclusions The subcutaneous xenograft tumor model of human lung cancer H460 cells has been successfully established in nude mice. LKB1 has a radiosensitizing effect on the subcutaneous xenograft tumor of lung cancer H460 cells in nude mice. Key words: Lung cancer cell line; Liver kinase B1; Radiosensitivity; Xenograft

Role of Yes-associated protein 1 in angiotensinⅡ-induced pulmonary fibrosis in rats

Objective: To explore the mechanism of Yes-associated protein 1 (Yap1) in angiotensinⅡ(AngⅡ)-induced pulmonary fibrosis. Methods:In vivo, 18 male Wistar rats were randomly divided into three equal groups with 6 rats in each group, including control group, bleomycin-treated group (BLM), and BLM+ AngⅡ group. 28 days later, the lung tissues in all groups were harvested for the HE and Masson staining as well as the immunohistochemical (IHC) staining for Yap1. In vitro, the isolated fibroblasts were treated with 10(-7) mmol/L AngⅡor the AngⅡ-targeted inhibitor irbesartan for the scheduled time for mRNA and protein expressions of Yap1, PDZ-binding motif (TAZ), and collagen Ⅰusing PCR and Western blot, as well as the translocation test from the nucleus to the cytoplasm of Yap1 and TAZ. Subsequently, the fibroblasts were assigned into 4 groups: the empty plasmid (vector) group, the vector+ AngⅡ group, the Yap1 shRNA group, and the Yap1 shRNA+ AngⅡ group. Western blot was used to detect the relative expressions of Yap1, TAZ, Smad3 and collagen Ⅰ. The CCK-8 and EdU assays were performed to determine the proliferative capacity. Results:In vivo, severe lung fibrosis and increased Yap1 expression of IHC staining were found in BLM group. Additionally, more severe lung fibrosis and higher Yap1 expression were detected in the BLM+ AngⅡ group than the BLM group (both P<0.05). In vitro, both the mRNA and protein relative expressions of Yap1, TAZ and collagenⅠ were markedly higher in AngⅡ-treated groups than the control group (all P<0.05). Meanwhile, the relative expression of phosphorylated Yap1 reached its peak at 2 h after AngⅡ stimulation. In the protein translocation tests, after treated with AngⅡ for 24 h, the relative protein levels of Yap1 and TAZ in the nucleus of the AngⅡ group were significantly higher than those in the control group (0.382±0.007 vs 0.031±0.001, 1.097±0.030 vs 0.357±0.015). However, the relative protein expressions in the cytoplasm of the AngⅡ group were obviously less than that in the control group (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015). Compared with the AngⅡ group, the expressions of Yap1 and TAZ in the AngⅡ+ irbesartan group were higher in cytoplasm (0.598±0.060 vs 0.323±0.058, 1.495±0.052 vs 0.858±0.059), while lower in the nucleus (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015) (all P<0.05). Furthermore, the relative protein expressions of Yap1, TAZ, Smad3 and collagenⅠin Yap1 shRNA+ AngⅡ group were distinctly lower than the vector+ AngⅡ group (all P<0.05). In the cell proliferation tests, the absorbance and the percentage of EdU positive cells of vector+ AngⅡ group exceeded that of vector group (both P<0.05). However, the absorbance and the percentage of EdU positive cells in the Yap1 shRNA+ AngⅡgroup were less than the vector+ AngⅡ group (both P<0.05). Conclusion: AngiotensinⅡ promoted the collagen synthesis and cell proliferation in primary lung fibroblasts by increasing the Yap1 activity, leading to the progress of fibrosis.

STAT3 influences the characteristics of stem cells in cervical carcinoma.

The purpose of the study was to investigate the role of the signal transducer and activator of transcription 3 (STAT3), signal transduction protein in regulating the biological characteristics of stem cells in cervical carcinoma. Overexpressed plasmid of STAT3 was constructed and used to transfect SiHa into cervical carcinoma cells. STAT3-targeted specific siRNA was designed and produced. The effects of STAT3 upregulation (or inhibition) on the expression of NANOG, OCT4 and SOX2 markers of stem cells were measured, using western blot analysis and RT-qPCR. In addition, the tumor sphere experiment was also conducted to detect the formation of tumor spheres after the intervention of expression of STAT3 and the expression of STAT3, NANOG, OCT4 and SOX2 was detected in 35 cases of cervical carcinoma tissues and 31 cases of normal cervical tissues using immunohistochemistry. We determined whether the STAT3 overexpression plasmid was successfully constructed using enzyme digestion, PCR for bacterium solution, western blot analysis and RT-qPCR and found that the plasmid met the requirements of subsequent procedures. Compared with the empty plasmid group and STAT3 low expression group, the mRNA and protein expression of markers of stem cells, OCT4, SOX2 and NANOG were significantly elevated in the STAT3 overexpression group with statistically significant differences (P<0.05), the formation ratio of tumor spheres in the STAT3 overexpression group was also significantly higher than those in the other two groups (P<0.05). The positive expression of STAT3, OCT4, NANOG and SOX2 in the cervical squamous carcinoma group was also markedly higher than that in the chronic cervicitis group (P<0.05). This study led us to a conclusion that STAT3 can regulate the characteristics of stem cells in cervical carcinoma, and STAT3, NANOG, OCT4 and SOX2 are highly expressed in cervical squamous carcinoma, thus able to promote the progression of cervical carcinoma.

RACK1 Silencing Induces Cell Apoptosis and Inhibits Cell Proliferation in Hepatocellular Carcinoma MHCC97-H Cells.

This study aimed to explore the effects of RACK1 gene silencing on the apoptosis and proliferation of hepatocellular carcinoma (HCC) MHCC97-H cells. After transfecting MHCC97-H cells with siRNA, RACK1 gene silencing model was established. The cells were divided into blank group, siRNA group and empty plasmid group, respectively. The mRNA and protein expressions of RACK1, cyclin D1 and BAX were determined by qRT-PCR and Western blotting. CCK-8 assay, flow cytometry and FITC-Annexin V/PI staining were used to determine cell viability, cell cycle and cell apoptosis, respectively. The results of qRT-PCR and Western blotting suggested that when compared with the blank group and the empty plasmid group, the mRNA and protein expressions of RACK1 and Cyclin D1 decreased significantly while the mRNA and protein BAX expressions increased substantially in the siRNA group (all P<0.05). The results of CCK-8 assay revealed that the siRNA group exhibited significantly lower cell viability when compared with the blank group and the empty plasmid group (both P<0.05); and the cell viability in the siRNA group decreased gradually with the increase of time. The results of flow cytometry and FITC-Annexin V/PI staining indicated that when compared with the blank group and the empty plasmid group, the proportion of cells in S phase was markedly lower and the apoptosis rate was significantly higher in the siRNA group (both P<0.05). Our study suggests that inhibition of RACK1 could suppress cell proliferation and induce apoptosis in HCC MHCC97-H cells.

Effect of silencing sperm associated antigen 9 gene specially by RNA interference on human prostate carcinoma PC-3 cells

Objective To investigate the effect of silencing sperm associated antigen 9 (SPAG9) gene specially by RNA interference on human prostate carcinoma PC-3 cells, and the possible mechanism. Methods The plasmid vector expressing SPAG9 short hairpin RNA (shRNA) was constructed and transfected into PC-3 cells by lipofectamine 2000. Cells were collected and assayed at 48 h after transfection. The SPAG9 mRNA and protein expression level was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Cell proliferation was tested by cell counting kit-8 (CCK-8) assay. Cell apoptosis was detected by fluorescence-activated cell sorter (FACS). The invasion ability of cells was examined by Transwell assay. To explore its possible mechanism, the expression of matrix metalloproteinase (MMP)-2, vascular endothelial growth factor (VEGF), Vimentin and E-cadherin proteins was measured by Western blotting after SPAG9 gene knockdown for 48 h. Results The recombinant plasmid pGPU6-green fluorescent protein (GFP)-SPAG9 expressing SPAG9 shRNA had been constructed successfully. RT-PCR and Western blotting showed the expression level of SPAG9 mRNA and proteinin in pGPU6-GFP-SPAG9 group was 1.96±0.16 and 0.39±0.05 respectively, lower than empty plasmid group (3.23±0.18 and 1.13±0.04), blank control group (3.08±0.12 and 1.25±0.05), respectively, the differences were both statistically significant (P=0.000). The absorbance (A) value was 31.9±2.6 in pGPU6-GFP-SPAG9 group, weaken than that in empty plasmid group (47.8±2.8, P=0.023), blank control group (48.1±2.8, P=0.016), the differences were statistically significant. FACS analysis showed that the apoptosis rate was (12.12±0.69)% in pGPU6-GFP-SPAG9 group, more than empty plasmid group [(6.43±0.31)%, P=0.020], blank control group [(5.19±0.33)%, P=0.014], the differences were statistically significant. Western blotting showed the expression level of MMP-2, VEGF, and Vimentin in pGPU6-GFP-SPAG9 group was lower than in the control groups, however, the average expression level of E-cadherin in pGPU6-GFP-SPAG9 group was higher than in the control groups, the differences were all statistically significant (P=0.000). Conclusion Silencing SPAG9 gene can inhibit SPAG9 gene expression, suppress proliferation, induce cell apoptosis of PC-3 cells by decreasing MMP-2, VEGF and Vimentin expression level, and increasing E-cadherin expression level. Key words: Prostate cancer; Sperm associated antigen 9 gene; RNA interference

MicroRNA-145 inhibits the activation of the mTOR signaling pathway to suppress the proliferation and invasion of invasive pituitary adenoma cells by targeting AKT3 in vivo and in vitro.

PurposeThis study was designed to explore how miR-145 regulates the mTOR signaling pathway in invasive pituitary adenoma (IPA) by targeting AKT3.MethodsA total of 71 cases of IPA tissues and 66 cases of non-IPA tissues were obtained in this study. In vitro, the IPA cells were assigned into blank control, empty plasmid, miR-145 mimic, miR-145 inhibitor, miR-145 mimic + rapamycin, miR-145 inhibitor + rapamycin and rapamycin groups. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to detect the protein expressions of PI3K, AKT3, mTOR mRNA and the mRNA expression of miR-145 both in vivo and in vitro. Additionally, the S6K and RPS6 mRNA and protein expressions as well as the relative phosphorylation levels were determined in vitro. MTT assay, flow cytometry and transwell assay were used to testify the cell proliferation, apoptosis and invasion ability, respectively.ResultsThe IPA tissues exhibited significantly lower expression of miR-145 but higher PI3K, AKT3 and mTOR mRNA and protein expressions when compared with the non-IPA tissues. Compared with the blank control and empty plasmid groups, the miR-145 mimic group showed significantly decreased PI3K, AKT3, mTOR, S6K and RPS6 mRNA and protein expressions as well as phosphorylation levels; besides, the IPA cell proliferation, migration and invasion ability were strongly inhibited, accompanied with the increased number of apoptotic cells. In the miR-145 inhibitor group, the PI3K, AKT3, mTOR, S6K and RPS6 mRNA and protein expressions as well as the phosphorylation levels were significantly increased; cell proliferation, migration and invasion ability were remarkably elevated, accompanied with reduced apoptotic cell number.ConclusionThe study demonstrates that miR-145 inhibits the mTOR signaling pathway to suppress the IPA cell proliferation and invasion and promotes its apoptosis by targeting AKT3.

ELF5 in epithelial ovarian carcinoma tissues and biological behavior in ovarian carcinoma cells.

The expression of E74-like factor 5 (ELF5) in epithelial ovarian carcinoma tissues and its effects on biological behavior in ovarian carcinoma cells were assessed in search for a new approach for gene treatment of epithelial ovarian carcinoma. RT-PCR technology was applied to detect the expression of ELF5 mRNA in epithelial ovarian carcinoma (n=49), borderline ovarian epithelial tumor (n=19), benign ovarian epithelial tumor (n=31) and normal ovarian tissues (n=40). Then, we transfected recombinant plasmid pcDNA3.1-ELF5+EGFP into human ovarian carcinoma SKOV3 cells (recombinant plasmid group) in vitro and screened out stably transfected cells to conduct multiplication culture. Western blot analysis was performed to detect the expression of ELF5 protein in the different groups. Flow cytometry was employed to detect cell apoptosis and cycles. ELF5 mRNA in epithelial ovarian carcinoma and borderline ovarian epithelial tumor tissues were significantly lower (P<0.05) than those in benign ovarian epithelial tumor and normal ovarian tissues. ELF5 protein expression in the cells of recombinant plasmid group was significantly higher compared with empty plasmid and blank control groups. The capacity of cell reproductive recombinant plasmid group at each time point decreased (P<0.05). Flow cytometry detection showed that 67.03% of cells in recombinant plasmid group was blocked in G0/G1 phase (P<0.05), compared with empty plasmid group (37.17%) and blank control group (38.24%). Apoptotic rate of recombinant plasmid group was significantly lower (31.4±1.9%; P<0.05), compared with that of empty plasmid group (9.1±2.2%) and blank control group (8.7±1.5%), and the differences were statistically significant. In conclusion, ELF5 interfered with cell cycle of human ovarian carcinoma SKOV3 cells and promoted apoptosis of human ovarian carcinoma SKOV3 cells inhibiting their growth and invasive capacity; and thus providing a new approach to gene treatment of ovarian carcinoma.

Effects of PKM2 Gene Silencing on the Proliferation and Apoptosis of Colorectal Cancer LS-147T and SW620 Cells

Background/Aims: This paper aims to explore the effects of pyruvate kinase (PK) M2 gene silencing on the proliferation and apoptosis of colorectal cancer (CRC) LS-147T and SW620 cells. Methods: CRC LS-147T and SW620 cells highly expressing PKM2 were randomly selected by quantitative real-time polymerase chain reaction (qRT-PCR) and then assigned into the blank (no transfection), PKM2-shRNA (transfection with shRNA) and empty plasmid (transfection with empty plasmid) groups. Immunofluorescence was applied to detect PKM2 protein expression. qRT-PCR and Western blotting were conducted to assess mRNA and protein expression of PKM2, p53 and p21. The cell counting kit-8 (CCK-8) assay was used to assess cell proliferation. Flow cytometry was used to assess the cell cycle and apoptosis rate, and a senescence-associated β-galactosidase staining kit was used to assess cell senescence. Results: PKM2 exhibited high mRNA expression among CRC LS-147T and SW620 cells with remarkable protein expression noted in the cytoplasm and nucleus. The PKM2-shRNA group exhibited reduced PKM2 mRNA and protein expression, whereas p53 and p21 expression was increased compared with the blank and empty plasmid groups. Cell proliferation in PKM2-shRNA cells decreased significantly compared with the blank group and empty plasmid groups. The PKM2-shRNA group exhibited more cells in the G₁ phase and fewer cells in the G₂/M phase compared with the blank and empty plasmid groups. In addition, the PKM2-shRNA group exhibited significantly increased apoptosis rates and β-galactosidase activity compared with the blank and empty plasmid groups. Conclusion: Our study demonstrates that PKM2 gene silencing suppresses proliferation and promotes apoptosis in LS-147T and SW620 cells.

The Th1/Th2-associated cytokines in the animal model of Graves ophthalmopathy

Background Graves opthalmopathy (GO) is an autoimmune disease, and its pathogenesis is not clear.Recent reports found that the imbalance of Th1/Th2 type immune response may be a main cause of GO. Objective This study was to establish an animal model of GO by immuning female BALB/c mice with recombinant plasmid of human thyroid-stimulating hormone receptor (hTSHR) A-subunit encapsulated with liposome induces. Methods Thirty-two female BALB/c mice aged 6-8 weeks were randomized to recombinant plasmid group, blank control group, empty plasmid group and liposome group.Recombinant plasmid pcDNA3.1+ /hTSHR289 containing 30 μg liposome was firstly injected into pretibial muscle and then into peritoneal cavity to construct the GO models at 0, 3, 6 weeks respectively in the recombinant plasmid group, and pcDNA3.1+ and liposome was used in the same way in the empty plasmid group and liposome group, respectively.No any intervene was performed in the blank control group.Heart blood of the mice was collected before injection and 4 days and 17 weeks after injection for the detection of serum interleukin (IL)-2, IL-4 and IL-2/IL4.The mice in the recombinant plasmid group were sacrificed at the end of 17 weeks of initial injection, and thyroids and orbital tissues were extracted for the regular histopathological examination.The expressions of CD20 for B cell specific markers (B) and CD3e for T cell specific markers (T) were detected by immunochemistry and B∶T>1.0 was Th2 response, and B∶T<1.0 was Th1 response. Results Serum IL-2 levels in the recombinant plasmid group were significantly lower than those after injection of the 2nd time and 3rd time in the blank control group, empty plasmid group and liposome group (all at P 0.05). The serum IL-2 levels after injection of the 2nd and 3rd time were lower than those before injection and after injection of the 1st time in the recombinant plasmid group (before injection: P=0.009, 0.019; after injection of the 1st time: P=0.002, 0.004), and the serum IL-2 level raised at the sacrifice of the mice, which was significantly higher than that after injection of the 3rd time( [44.31±1.77]pg/ml vs. [42.43±2.29]pg/ml) (P=0.031). Serum IL-4 levels and IL4/IL2 values after injection of the 2nd time and 3rd time were significantly higher than those before injection and after injetion of the 1st time in the recombinant plasmid group, blank control group, empty plasmid group and liposome group, and in the sacrifice of the mice, the serum IL-4 level was higher than that after injection of the 3rd time in the recombinant plasmid group (all at P 1.0. Conclusions Immunization syngeneic BALB/c mice with liposome encapsulated thyrotropin receptor extracellular domain recombinant plasmid is an effective way to establish a GO animal model.The Th1 type response appears in initial 17 weeks following the first immunization, but the immune balance shifts toward a Th2 type after the second immunization. Key words: Graves ophthalmopathy; Th1/Th2 balance; Interleukin-2; Interleukin-4; Disease models, animal; Inbred BALB/c mice

Tumor susceptibility gene 101 impact on the invasion and metastasis of liver cancer cell proliferation

Objective To study of the effects of tumor susceptibility gene 101 (TSG101) on proliferation, invasion and metastasis of liver cancer cells. Methods To establish stable expression of TSG101 liver cancer cell lines, then cells can be divided into transfection group, the empty plasmid group, blank control group, through the cell counting kit-8 (CCK-8) multiplication experiment observation group cell proliferation; and by Transwell chambers attacking invasion ability and migration experiment ability to observe each cell metastasis. Results CCK-8 multiplication experiment found TSG101 group and 36 h after cell proliferation rate (1.93±0.45) significantly higher than the blank control group (1.28±0.51) and the empty plasmid group (1.31±0.29), and the difference was statistically significant (P 0.05); (2) the transwell Chambers cell invasion experiment: TSG101 group cells through number (125.0±10.3) significantly higher than the blank control group (83.0±8.7) and the empty plasmid group (85.0±9.2), and the difference was statistically significant (P<0.05). Blank control group compared with the empty plasmid group had no statistical significance; (3) transwell Chambers cell migration experiment: TSG101 group penetrate the cell number (88.0±7.0) is higher than the blank control group (59.0±6.8) and the empty plasmid group (53.0±7.7), and the difference was statistically significant (P<0.05). Blank control group compared with empty plasmid groups has no statistical significance. Conclusion High expression of TSG101 protein of liver cancer cell proliferation, invasion and metastasis ability are enhanced. Key words: Tumor susceptibility gene101; Hepatocellular carcinoma; Proliferation; Invasion; Metastasis