Overexpression of N-terminal amino acids of p55γ regulatory subunit of PI3K inhibits cell adhesion of human gastric carcinoma MGC803 cells

AIM: To investigate the effect of the N-terminal 24-amino acid(N24) overexpression in p55γ re-gulatory subunit of phosphatidylinositiol 3-kinase(PI3K) on the cell adhesion of human gastric carcinoma cell MGC803.METHODS: MGC803 cells, which stably expressed GFP-N24 fusion protein and GFP alone, were generated by transfection with pEGFPN-24 plasmid and control plasmid pEGFP-C1, respectively. The morphological change of the cells was observed under inverted microscope. The expression of GFP-N24 fusion protein was detected by Western blot. The adhesion of the cells was determined by cell adhesion assay. The effects of GFP-N24 fusion protein on the expression of E-cadherin and β-catenin were analyzed by Western blot. The expression and secretion of matrix metalloproteinase 9(MMP9) and urokinase-type plasminogen activator(uPA) in the MGC803 cells were detected by gelatin zymography.RESULTS: MGC803/GFP-N24 cell line steadily expressed GFP-N24 fusion protein and MGC803/GFP cell line steadily expressing GFP were successfully established, but the expression of fusion protein GFP-N24 was lower than that of the control protein GFP. The morphological changes of the transfected cells from paving stone to fibroblast cell form after gene transfection, and the cytoplasm secretory granules were increased significantly. The cell adhesion to fibronectin and collagen decreased after GFP-N24 transfection. GFP-N24 fusion protein increased the expression of cell adhesion molecule E-cadherin and decreased the wnt signal pathway molecule β-catenin in the MGC803 cells. However, it did not affect the expression and secretion of tumor metastasis-related proteins MMP9 and uPA.CONCLUSION: Overexpression of N24p55γ inhibits cell adhesion by influencing the expression of E-cadherin and β-catenin in the MGC803 cells.     

Mechanical stretch induces translocation of high-mobility group box 1 protein in THP-1 cells

AIM: To investigate the translocation of high-mobility group box 1 protein(HMGB1) in THP-1 cells induced by mechanical stretch. METHODS: The vector that expressed the fusion protein of HMGB1 and enhanced green fluorescent protein(EGFP) was constructed. The cDNA of HMGB1 and EGFP was subcloned into hemagglutinin(HA)-tagged vector pcDNA3-HA by two-step method. THP-1 cells were transfected with pcDNA3-HMGB1-EGFP and exposed to cyclic mechanical stretch at 20 % elongation using Flexercell 4000T cell stretching unit. The translocation of HMGB1 in THP-1 cells was observed under fluorescence microscope. RESULTS: The recombinant plasmid was verified by enzyme digestion. The green fluorescence accumulated in the nuclei of the cells, indicating that the fusion protein was highly expressed in THP-1 cells and localized in the nuclei. Eighteen hours after mechanical stretch, the green fluorescence was observed in the cytoplasm. At the same time, the green fluorescence was still localized in the nuclei of the control cells treated without mechanical stretch. CONCLUSION: The HMGB1-EGFP fusion protein is successfully and effectively expressed in THP-1 cells. Mechanical stretch induces the translocation of HMGB1 protein from nucleus to cytoplasm.     

Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

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

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

Construction of fusion expression vector AR-GFP and its expression in Hek293 cells

AIM: To construct a hAR and GFP fusion gene vector and to observe the AR-GFP gene expression in Hek293 cells. METHODS: A recombined vector pcDNA3.1/myc-HisA-AR-GFP (pH-AG) was constructed by gene engineering technique. The recombined vector was transfected into Hek293 cells using calcium phosphate. RESULTS: AR-GFP fusion protein was successfully expressed in Hek293 cells without biologic activity, which was confirmed by fluorescence microscopy and Western blotting. CONCLUSION: The Hek293 cells transfected by AR-GFP fusion gene can express its protein successfully. However, it is not a cellular model for ARI screening.      

Injury effect of adenosine triphosphate on N9 microglia

AIM:To investigate the injury effect of adenosine triphosphate(ATP) on N9 microglia. METHODS:N9 microglia in logarithmic growth phase was randomly divided into 3 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treatment with ATP after cultured for 24 h. In KN-62 intervention group, after pretreatment with KN-62 for 30 min, ATP was added in the cells. The cell viability was assessed by XTT assay. Cellular morphological changes were observed under phase-contrast microscope. The cell cycle and apoptosis were detected by flow cytometry. The expression of P2X₇ receptor was examined by immunofluorescence staining. The protein levels of P2X₇ receptor were measured by Western blotting. The concentration of IL-1β in the culture supernatant was detected by ELISA. RESULTS:ATP at dose of 500 μmol/L and 1 mmol/L only caused small damage to the cell viability of N9 microglia. The cell viability was 88.5%±5.5% and 88.2%±8.4% after treated with ATP for 24 h,respectively. The cell viability dropped rapidly and cell shrinkage occurred when the concentration of ATP increased to 2 mmol/L or higher. With the extension of experiment time, the cell viability and cell density decreased further and cell shrinkage was getting worse. KN-62 intervention improved the viability of N9 microglia injured by ATP. The morphology and density of N9 microglia in KN-62 intervention group were much better than those in ATP group. ATP arrested N9 microglia at S phase and increased cell apoptosis significantly(P<0.01 vs control group). KN-62 intervention obviously relieved the cell cycle arrest and decreased the cell apoptosis caused by ATP(P<0.01). ATP and KN-62 intervention had no effect on the distribution of P2X₇ receptor. The protein levels of P2X₇ receptor had no significant difference among the 3 groups(P>0.05). ATP and KN-62 intervention had no effect on the release of IL-1β. CONCLUSION:High dose of ATP damages N9 microglia and its mechanism may be related to cell cycle arrest and apoptosis mediated by P2X₇ receptor but not to inflammatory response caused by microglia.     

F-box domain is involved in regulating the proliferation of MCF-7 cells by FBXO39 protein

AIM: To investigate the effect of F-box domain on the regulation of MCF-7 cell proliferation by FBXO39 protein. METHODS: The effect of F-box domain on the localization of FBXO39 protein in the MCF-7 cells was investigated. MCF-7 cell cDNA library was used as the template resource. The full-length cDNA sequence of FBXO39 was amplified by PCR method and subcloned into eukaryotic expression vector pEGFP-C2. The pEGFP-FBXO39ΔF (F-box domain deletion mutation) plasmid was successfully constructed with the template resource of pEGFP-FBXO39 plasmid. The recombinant plasmids were transfected into the MCF-7 cells, and then the expression of FBXO39 and FBXO39ΔF were determined by Western blot. The cellular localization of FBXO39 and FBXO39ΔF were observed by confocal microscopy. The localization of endogenous FBXO39 in the MCF-7 cells was detected by immunofluorescence staining. In addition, MTT and EdU assays were used to measure the cell proliferation, flow cytometry was used to measure the cell cycle distribution, and immunohistochemical staining was used to observe the expression of FBXO39 in the breast cancer and para-carcinoma tissues. RESULTS: The eukaryotic expression vector pEGFP-FBXO39 and pEGFP-FBXO39ΔF were constructed successfully. F-box domain had no effect on the cell localization of FBXO39. FBXO39 promoted MCF-7 cell proliferation but FBXO39ΔF did not. FBXO39 was highly expressed in the breast cancer tissues. CONCLUSION: F-box domain had no effect on the cellular localization of FBXO39 protein. However, it plays an important role in the biological function of FBXO39. FBXO39 may be related to breast cancer tumorigenesis.     

Effect of hemolysin protein HlyX of Leptospira interrogans serovar Lai on permeability of vascular endothelial cells

AIM: To clone and express the hemolysin gene hlyX of Leptospira interrogans serovar Lai and to investigate the effect of the expression product on the permeability of human umbilical vein endothelial cells (HUVECs).METHODS: The recombinant plasmid pET-hlyX was constructed by inserting the hlyX gene into prokaryotic expression vector pET32a(+), and transformed into E.coli BL21(DH3) to express the fusion protein Trx-HlyX with a His-tag.The fusion protein was purified using HisTrap affinity columns.The permeability of the monolayer HUVECs was measured by enzyme-linked immunosorbent assay for biotin-labeled albumin.Flow cytometry and Hoechst 33258 staining were applied to measure the apoptotic rate of HUVECs after incubation with Trx-HlyX.RESULTS: The recombinant plasmid pET-HlyX was successfully constructed and the fusion protein Trx-HlyX was highly expressed.Compared with the control cells, the purified recombinant protein Trx-HlyX significantly increased the permeability of transfected cells and promoted apoptosis of HUVECs (P<0.05).CONCLUSION: The recombinant plasmid pET-hlyX highly expresses the fusion protein Trx-HlyX.Purified protein Trx-HlyX influences the permeability and has cytotoxicity on HUVECs.     

黄瓜动蛋白特异肽段的原核表达和多克隆抗体制备

 以黄瓜果实总RNA为模板,通过RT-PCR获得了动蛋白基因CsKF1和CsKF2的cDNA序列,进行测序,构建T-easy载体。SMART在线预测CsKF1和CsKF2均含有动蛋白（Kinesin）约340个氨基酸残基的马达区保守序列（moter domain）。体外表达蛋白的马达区和非马达区,将带有His标签的CsKF1-N、CsKF2-C、CsKF1-M、CsKF2-M融合蛋白通过E. coli BL21（DE3）表达,摸索不同诱导条件下目的蛋白的表达和纯化。其中His-CsKF1-N、His-CsKF2-C、His-CsKF1-M原核表达蛋白的分子量分别为25、30和40 kD,均以0.1 mmol · L^-1 IPTG,22 ℃ 6 h诱导表达效果最好。His-CsKF2-M原核表达蛋白的分子量约38 kD,以18 ℃ 8 h诱导表达效果最好。融合蛋白His-CsKF1-N和His-CsKF2-C经过亲和纯化后作为抗原,通过5轮免疫注射兔子后,取心脏血作为抗血清,通过AminoLink Plus kit试剂盒亲和纯化得到多克隆抗体anti-CsKF1-N和anti-CsKF2-C。经过Western blot分析表明多克隆抗体具有特异性,可与抗原特异结合。     

Expression of fusion protein anti-HER2-ScFv-GFP in insect cells and binding to surface of breast cancer cells

AIM: To express a recombinant fusion protein anti-human epidermal growth factor receptor-2 single-chain variable fragment with green fluorescent protein (anti-HER2-ScFv-GFP) using the insect cells-Bac-to-Bac baculovirus expression system and to analyze the binding function of this fusion protein with HER2 on the surface of the breast cancer cells. METHODS: Human anti-HER2-ScFv gene from mice was fused with GFP gene. To obtain the recombinant plasmid pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP, we inserted it into Bac-to-Bac baculovirus expression plasmid pFAST Bac-to-Bac HT A. The identified recombinant plasmid was transferred into Escherichia coli DH10Bac to allow the generation of a recombinant bacmid. After transfected the recombinant virus bacmid into the insect cells Tn-5B1-4, the recombinant virus was collected to infect Tn-5B1-4. SDS-PAGE and Western blotting analysis were used to verify the expression product in Tn-5B1-4. The fusion protein was purified with Ni²⁺-NTA affinity chromatography. The purified fusion protein was bound to the surface of HER2-positive breast cancer cells SKBR3 and HER2-negative breast cancer cells MCF7. The binding effects on the surface of breast cancer cells were observed under laser confocal microscope. RESULTS: The fusion gene anti-HER2-ScFv-GFP was successfully constructed with the length of 1 539 bp. The green fluorescence was also observed in Tn-5B1-4 cells infected with the recombinant virus under fluorescent microscope. A 60 kD protein was examined and confirmed by SDS-PAGE and Western blotting. Under laser confocal microscope, strong green fluorescence was observed on the surface of the HER2-positive breast cancer cells SKBR3. However, no green fluorescence was observed on the surface of HER2-negative breast cancer cell MCF7. Obvious green fluorescence on the surface of HER2-positive breast cancer cell SKBR3 was also found after the cells were eluted with 1×PBS. CONCLUSION: The fusion protein anti-HER2-ScFv-GFP was successfully expressed in insect cells Tn-5B1-4, and can firmly bind to the surface of breast cancer cells SKBR3 and emit the green fluorescent light.     

Effect of lentiviral vector of microRNA-9-1 on differentiation of mouse bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of microRNA-9 in inducing bone marrow mesenchymal stem cell(MSCs) differentiation into neurons.METHODS: The lentiviral vector of microRNA-9-1(microRNA-9-1-LV) was constructed and transfected into mouse MSCs. The cells were divided into non-transfected group, transfected group(transfected with microRNA-9-1-LV) and negative control group(transfected with FU-RNAi-NC-LV). MSCs were treated with β-mercaptoethanol(β-ME) as an inducer for triggering the cells to differentiate into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The mRNA expression of microtublin-associated protein 2(MAP-2) was detected by RT-PCR. The expression of neuron-specific markers,neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP), were measured by immunocytochemical method. The viability of MSCs was determined by MTT method. RESULTS: The results of PCR confirmed successful construction of mouse microRNA-9-1-LV. The virus titer was 1×10¹² TU/L(TU, transduction unit). The best transfection efficiency(up to 91.3%±4.2%) and survival rate appeared when multiply of infection(MOI)was 20 and on 4th day. β-ME induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group. The expression levels of NSE and MAP-2 in transfected cells were higher than those in the cells of other group(P<0.05).CONCLUSION: MicroRNA-9-1-LV has high transfection efficiency in mouse MSCs. Higher differentiation rate from mouse MSCs to neurons is induced by β-ME after the cells are transfected with microRNA-9-1-LV.     

Effect of lung cancer cells transfected with interleukin 18 gene on the immunological activity of dendritic cells

AIM: To study the effect of interleukin 18 gene transfected lung cancer cells on the phenotype and immunological activity of dendritic cells (DC). METHODS: A secretive IL-18 expression vector containing IL-12 P40 signal sequence was constructed and transfected into NCI-H460 lung cancer cells. DC induced from human peripheral blood were divided into 4 groups (NT, PV, GT and PD). DC were stimulated by non-transfected NCI-H460 cells, pure vector transfected NCI-H460 cells and IL-18 transfected NCI-H460 cells respectively for group NT, PV, GT, and non-stimulated DC for group PD. CD54, CD80, CD83 and CD86 on DC in the 4 groups were detected with flow cytometry. T cell proliferation stimulated by DC in the 4 groups was assayed with MTT method. IL-12 release in cultured DC supernatant was measured by ELISA. RESULTS: Sequencing result of the secretive IL-18 was correct. The transfected cells expressed IL-18 fusion gene and 18 kD IL-18 protein. DC in GT group expressed more surface molecules than those in other 3 groups. T cell proliferation and IL-12 secretion in GT group were higher than those in other 3 groups. CONCLUSION: IL-18 gene transfected NCI-H460 cell increases surface molecule expressions on DC. It also enhances immunological activity and IL-12 secretion in DC.     

Construction of pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/ζ eukaryotic expression vector and expression in NIH 3T3 cells

AIM: To construct a recombinant eukaryotic expression vector pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/ζ and detect its expression in NIH 3T3 cells.METHODS: CD28-ζ cDNA was amplified from the plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/IgGFc/CD80 gene.The recombinant plasmids were transfected into NIH 3T3 cells,and resistant clones were obtained by G418 selection.The gene expression of the fusion protein was determined by RT-PCR and FACS.RESULTS: The recombinant eukaryotic vector was constructed successfully,determined by PCR and enzyme digestion analysis.The target gene was amplified from NIH 3T3 cells transfected with the vectors by RT-PCR.The FACS showed that recombinant protein was expressed in NIH 3T3 cells.CONCLUSION: Construction of pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/ζ expression vector and its expression in NIH 3T3 cells lay the foundation for further research of generation of modified T lymphocytes to CD20 positive lymphoma.     

Effect of overexpression of p16 on anion exchange function of anion exchanger 1 at HeLa cell lines

AIM: To study the effect of the overexpression of p16 on an anion exchange function of band 3 in HeLa cells. METHODS: The expression of p16 and band 3 in HeLa cells was detected by immunohistochemistry (IHC). The p16 cDNA was subcloned to plasmids pEGFP-C1 by PCR and identified by restriction enzyme digestion and sequencing, and then, the recombinant pEGFP-C1-p16 plasmids were transiently transfected into HeLa cells. The expression of fusion protein in HeLa cells was detected by fluorescence microscope. 6-methoxy-N-(3-sulfopropyl)-quinolinium(SPQ)fluorescent probes were used to detect the anion exchange function of band 3. RESULTS: P16 and band 3 were expressed in HeLa cells. The amplificated p16 cDNA sequence was the same as the report sequence. The transfective efficacy of pEGFP-C1-p16 was above 60%. The anion exchange function increased after the transfection of pEGFP-C1-p16 plasmids. CONCLUSION: p16 facilitates the anion exchange function of band 3 in HeLa cells.     

Effect of miR-25 on apoptosis of H9c2 cells induced by hypoxia/reoxygenation

AIM:To investigate the role and mechanism of microRNA-25 (miR-25) in apoptosis of H9c2 cells induced by hypoxia/reoxygenation. METHODS:The H9c2 cells with over-expression of miR-25 were treated with hypo-xia/reoxygenation. Real-time PCR was used to detect the expression of miR-25 and high mobility group box-1 (HMGB1) mRNA. Western blot was performed to examine the protein expression levels of HMGB1, Bcl-2 and cleaved caspase-3. Flow cytometry was used to analyze the proportion of apoptotic cells and the cell cycle. Dual-luciferase reporter assay was used to confirm that HMGB1 was the target gene of miR-25 in the H9c2 cells. Moreover, the H9c2 cells transfected with HMGB1-shRNA were subjected to hypoxia/reoxygenation to verify whether HMGB1 participated in the regulation of apoptosis of H9c2 cells. RESULTS:Over-expression of miR-25 significantly reduced the protein expression levels of HMGB1 and cleaved caspase-3, and increased the expression of Bcl-2 and the entrance into S phase in H9c2 cells induced by hypoxia/reoxygenation (P<0.01). The result of dual-luciferase reporter assay showed that compared with the control group, transfection with HMGB1-3' UTR-psi-CHECK2+miR-25 mimic strongly inhibited the luciferase activity (P<0.05). After the H9c2 cells transfected with HMGB1-shRNA was treated with hypoxia/reoxygenation, the expression of Bcl-2 was up-regulated, the expression of cleaved caspase-3 was down-regulated, and the cells in S phase were increased (P<0.05). CONCLUSION:miR-25 reduces apoptosis of H9c2 cells induced by hypoxia/reoxygenation, and its mechanism may be related with the inhibition of HMGB1 expression via interacting with its 3'-UTR.     

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

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

Dominant negative human epidermal growth factor receptor induces G0/G1 arrest in human gastric cancer cells

AIM：To explore the effect of dominant negative epidermal growth factor receptor (DNEGFR) on the cell cycle of human gastric cancer cells. METHODS：Two human gastric cancer cell lines were used in the study. The cells were divided into 6 groups, including untreated SGC-7901 cells (US group), SGC-7901 cells stably transfected with pEGFP-N1 (ES group), SGC-7901 cells stably transfected with pEGFPN1-DNEGFR (DS group), untreated NCI-N87 cells (UN group), NCI-N87 cells stably transfected with pEGFP-N1 (EN group), and NCI-N87 cells stably transfected with pEGFPN1-DNEGFR (DN group). The cell cycle was determined by flow cytometry. The protein levels of cyclin-dependent kinase 2 (CDK2), cyclin D1, phosphorylated glycogen synthase kinase 3 beta at Ser9 ［p-GSK-3β (Ser9)］, p21 and p27 were detected by Western blotting. RESULTS：Transfection of the human gastric cancer cells with pEGFPN1-DNEGFR led to G0/G1 arrest, and down-regulated CDK2, cyclin D1, p-GSK-3β (Ser9) and up-regulated p21 and p27 as well. CONCLUSION：DNEGFR down-regulates cyclin D1 by activating GSK-3β, down-regulates CDK2, and up-regulates p21 and p27, which induce G0/G1 arrest in human gastric cancer cells in the end.     

MicroRNA-9 inhibits epithelial-mesenchymal transition in gastric cancer SGC-7901 cells by NRP1

AIM:To investigate the inhibitory effect of microRNA-9(miR-9) on epithelial-mesenchymal transition (EMT) in the gastric cancer SGC-7901 cells and its mechanism.METHODS:The gastric cancer cell line SGC-7901 was transfected with miR-9 mimics or negative control mimic (NCM),as miR-9 or NCM group,respectively.The SGC-7901 cells without transfection were used as control group.The expression level of miR-9 in each group was detected by RT-qPCR.The migration and invasion abilities of the SGC-7901 cells in the 3 groups were detected by Transwell assay.The protein expression of N-cadherin,E-cadherin,α-catenin and neuropilin-1(NRP1) was determined by Western blot.Antagonistic effect of NRP1 over-expression on miR-9 inhibition of EMT was detected by Western blot.The relationship between miR-9 and NRP1 was analyzed by dual luciferase assay.RESULTS:The expression level of miR-9 in miR-9 group was significantly up-regulated,which was 538 times higher than that in control group (P<0.05).The number of migratory cells in miR-9 group was significantly lower than that in control group (P<0.05).Compared with control group,the protein expression of N-cadherin and NRP1 in miR-9 group was significantly decreased,while the protein expression of E-cadherin and α-catenin protein was significantly increased.Over-expression of NRP1 resulted in the increase in the protein expression of N-cadherin in the gastric cancer cells of miR-9 group,and the decrease in the protein expression of E-cadherin and α-catenin significantly.The result of dual luciferase assay showed that NRP1 was a downstream target gene of miR-9(P<0.05).CONCLUSION:miR-9 may inhibit the expression of EMT-related proteins through the downstream target gene NRP1,thus inhibiting the EMT of gastric cancer SGC-7901 cells.     

Clinical significance of miRNA-326 expression in gastric cancer and effect of up-regulation of its expression on viability and apoptosis of gastric cancer cells

AIM: To investigate the clinical significance of microRNA-326 (miRNA-326) expression in gastric carcinoma and the effect of up-regulation of its expression on the viability and apoptosis of gastric cancer cells. METHODS: The expression of miRNA-326 in 55 tissue samples of gastric cancer was detected by RT-qPCR, and the relationship between the expression and the clinicopathological features was analyzed. The expression of miRNA-326 in gastric cancer BGC-823 cells was detected by RT-qPCR. The BGC-823 cells were transfected by liposome method, and randomly divided into normal control group (untransfected), mimic-NC group (transfected with negative control mimic) and miRNA-326 mimic group (transfected with miRNA-326 mimic). After up-regulation of miRNA-326 expression, the cell viability was measured by CCK-8 assay, and the apoptosis of the cells was analyzed by flow cytometry. The protein levels of matrix metalloprotein 9 (MMP-9), p21, cyclin D1, Bcl-2 and cleaved caspase-3 were determined by Western blot, and the mRNA expression of cyclin D1 was detected by RT-qPCR. Whether CCND1 (the gene of cyclin D1) was the target gene of miRNA-326 was evaluated by dual-luciferase reporter assay. RESULTS: The expression of miRNA-326 in the gastric cancer tissues was significantly lower than that in the adjacent tissues (P<0.05). The miRNA-326 expression had a significant correlation with the tumor size, lymph node metastasis, differentiation, and clinical stages (P<0.05), but it had no correlation with the age and sex of the patients. Moreover, the expression of miRNA-326 was also closely related to the survival rate of the patients (P<0.05). The expression of miRNA-326 in the BGC-823 cells was significantly lower than that in the normal gastric mucosa GES-1 cells (P<0.05). Compared with normal control group, the expression of miRNA-326 in mimic-NC group did not change significantly, while that in miRNA-326 mimic group was increased significantly (P<0.05). Compared with normal control group, the cell viability in miRNA-326 mimic group was significantly decreased, and the apoptosis was increased (P<0.05). In addition, compared with normal control group, the protein levels of MMP-9, cyclin D1 and Bcl-2, and the mRNA expression of cyclin D1 in miRNA-326 mimic group were decreased, while the protein levels of p21 and cleaved caspase-3 were increased (P<0.05). However, no significant difference of above protein and mRNA levels between mimic-NC group and normal control group was observed. Compared with mimic-NC+miR-326 mimic group, the activity of luciferase in the cells transfected with pmiR-CCND1-WT plasmid was significantly decreased (P<0.05), but that in the cells transfected with pmiR-CCND1-Mut plasmid did not change significantly. CONCLUSION: The expression level of miRNA-326 in gastric cancer tissues is low, and it may promote cell viability and inhibit cell apoptosis by targeting CCND1.