Non-transfected Group Research Articles

Effects of B cell lymphoma/leukemia-2 associated X protein gene over-expression on expression of second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI in human glioblastoma U251 cells

Objective To explore the effects of B cell lymphoma/leukemia-2 associated X protein (bax) gene over-expression on the expression of second mitochondria-derived activator of caspase (Smac)/direct IAP-binding protein with low pI (DIABLO) in human glioma cell line.Methods The plasmid of expression vector pEGFP-bax was transfected into human glioblastoma U251 cells with lipofectamine2000,and meanwhile the non-transfected cells and the pEGFP-transfected U251 cells were taken as controls.Cellular bax and Smac/DIABLO expression was determined by Western boltting.Cellular distribution of Smac/DIABLO protein was evaluated by immunofluorescence (IF) assays and observed under inverted fluorescence microscope.Results The bax protein expression level in pEGFP-bax-transfected U251 cells was (0.82 ±0.12),significantly higher than in non-transfected group (0.35 ±0.05) and pEGFP-transfected control group (0.32 ± 0.09,P ＜ 0.05).The expression level of Smac/DIABLO protein in pEGFP-bax-transfected group was (1.09 ± 0.16),significantly higher than in non-transfected group (0.62 ± 0.10) and pEGFP-transfected control group (0.70 ± 0.20) (P ＜ 0.05).There was no significant difference in the expression of bax and Smac/DIABLO between non-transfected and pEGFP-transfected groups (P ＞0.05).IF revealed that Smac/DIABLO was mainly localized in the cytoplasm.Conclusion Transfection of extrinsic bax gene results in its over-expression in U251 cells,which could up-regulate the expression of Smac/DIABLO protein. Key words: Glioma; B cell lymphoma/leukemia-2 associated X protein; Second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI

Expression of p120-catenin in different bladder cancer cells and effect of p120-catenin to the prolifer-ation, invasion and adhesion of human bladder carcinoma cell line BIU-87 in vitro

Objective To investigate the expression of p 120-catenin in human bladder cell line BIU-87 and T24.And to explore the effect of down-regulation of p120-catenin on proliferation, invasion and adhesion of BIU-87 cell line. Methods The study was from Aug .2010 to May.2011.Real-time RT-PCR and Western blot were used to examine the expression of p 120-catenin in human bladder cell line BIU-87 and T24.There were three study groups：non-transfected group , the group transfected with p 120-catenin siRNA and the group transfected with control siRNA .p120-catenin siRNA was used to decrease the expression of p120-catenin.MTT assay and colony formation assay were used to study the BIU-87 cell growth and cell pro-liferation.The invasion of BIU-87 cells was measured by Transwell chamber assay .Cell adhesion artificial re-constituted basement membrane assay was used to examine the change of BIU-87 cell adhesion capacity . Results In both BIU-87 and T24 cells there were the expressions of p 120-catenin.But the expression in BIU-87 cells （0.11±0.39） was more than that in T24 cells （0.48±0.17）.The decreased of p120-catenin ex-pression could enhance the proliferation （182.7±13.4%） and the invasiveness （217.5±15.9） and decrease the adhesion capacity （57.3±6.4%） of BIU-87 cells. Conclusions There is higher expression level of p120-catenin in lower-grade malignant bladder cancer cells .The down-regulation of p120-catenin promotes the bladder cancer proliferation and invasiveness of bladder carcinoma cells , and inhibited the bladder canc-er cell adhesion . Key words: p120-catenin; BIU-87 cell; Proliferation; Invasion; Adhesion

Inhibitory effects of E2F transcription factor 1 small interfering RNA on apoptosis of mouse spermatogonial stem cells

Objective To observe inhibitory effects of E2F transcription factor 1 (E2F-1) small interfering RNA (siRNA) on apoptosis of mouse spermatogonial stem cells (SSCs).Methods Percoll density gradient centrifugation and flow cytometry was used to isolate,enrich and purify SSCs.Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were carried out to investigate the expression of E2F-1,and flow cytometry was ued to analyze the apoptosis rate.Results Compared to non-transfected group and control group,the expression of E2F-1 in experimental group was remarkably down-regulated at mRNA [2-(△△ct),0.32 vs.0.10 and 1.04,P ＜ 0.05] and protein levels (P ＜0.05).Tthe apoptosis rate in experimental group was reduced remarkably (7.69％ vs 23.07％ and 24.19％ ;P＜0.05).Conclusion E2F-1 siRNA could remarkably suppress the apoptosis of SSCs in vitro. Key words: RNA interference; Spermatogonial stem cells; E2F transcription factor 1 ; Apoptosis

HSPC117 is regulated by epigenetic modification and is involved in the migration of JEG-3 cells.

The human hematopoietic stem/progenitor cell 117 (HSPC117) protein is an essential component of protein complexes and has been identified to be involved in many important functions. However, how this gene expression is regulated and whether the HSPC117 gene affects cell migration is still unknown. The aim of this study was to identify whether HSPC117 mRNA expression is regulated by epigenetic modification and whether HSPC117 expression level affects the expression of matrix metalloproteinase 2 (MMP 2), matrix metalloproteinase 14 (MMP 14), and tissue inhibitor of metalloproteinases 2 (TIMP 2), and further affects human placenta choriocarcinoma cell (JEG-3) migration speed. In our epigenetic modification experiment, JEG-3 cells were cultured in medium with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), or both inhibitors. Then, the HSPC117 mRNA and protein expressions were assessed using real-time quantitative PCR (qPCR) and Western blot assay. The results showed that, compared to the control, HSPC117 mRNA expression was increased by TSA or 5-aza-dC. The highest HSPC117 expression level was found after treatment with both 5-aza-dC and TSA. Further, in order to investigate the effect of HSPC117 on MMP 2, MMP 14, and TIMP 2 mRNA expressions, pEGFP-C1-HSPC117 plasmids were transfected into JEG-3 cells to improve the expression of HSPC117 in the JEG-3 cells. Then, the mRNA expression levels of MMP 2, MMP 14, TIMP 2, and the speed of cell migration were assessed using the scratch wound assay. The results showed that over-expression of HSPC117 mRNA reduced MMP 2 and MMP 14 mRNA expression, while TIMP 2 mRNA expression was up-regulated. The scratch wound assay showed that the migration speed of JEG-3 cells was slower than the non-transfected group and the C1-transfected group. All of these results indicate that HSPC117 mRNA expression is regulated by epigenetic modification; over-expression of HSPC117 decreases MMP 2 and MMP 14 transcription, reduces cell migration speed, and increases TIMP 2 transcription.

Interleukin-35 gene modified mesenchymal stem cells induced the proliferation of regulatory T cells

Objective To investigate the feasibility of interleukin (IL)-35 gene transfection of mice bone marrow mesenchymal stem cells (BMSCs) and the effect of IL-35 gene modified MSCs (IL-35-MSCs) on the the proliferation of regulatory T cells (Tregs).Methods MSCs were cultured in vitro and transfected with IL-35 expressing plasmid by lipofectamine.The IL-35 expression of IL-35-MSCs was detected by enzyme linked immunosorbent assay (ELISA).The lymphocytes subsets after one-way mixed lymphocyte culture and in vivo transplantation were analyzed by flow cytometry to evaluate the effect of IL-35-MSCs on the proliferation of Tregs.Results The mice BMSCs can be isolated and cultured by the whole bone marrow adherent method.IL-35 is expressed in the MSCs supernatant and serum after IL-35 in vitro and in vivo transfection.Percentage of CD4 + CD25 + Tregs in mice treated with IL-35-MSCs increased significantly than control plasmid transfected MSCs and non-transfected MSCs groups(P ＜ 0.01).IL-35-MSCs up-regulated the CD4 + CD25 + Tregs level in the allogeneic mixed lymphocyte reaction system,lowered the percentage of CD4 + cells compared with the other two control groups (P ＜ 0.05).Conclusion IL-35-MSCs enhanced the proliferation of CD4 + CD25 + Tregs in vitro and in vivo compared to control MSCs.IL-35 and MSCs have synergic effects on the negative immune regulation and the induction of immune tolerance. Key words: Interleukin-35; Mesenchymal stem cells; Immune tolerance; Regulatory T cells

In vitro Study of Nucleostemin as a Potential Therapeutic Target in Human Breast Carcinoma SKBR-3 Cells

Although nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis and expression has been observed in some types of human cancer and stem cells, the molecular mechanisms involved in mediation of cell proliferation and cell cycling remains largely elusive. The aim of the present study was to evaluate NS as a potential target for gene therapy of human breast carcinoma by investigating NS gene expression and its effects on SKBR-3 cell proliferation and apoptosis. NS mRNA and protein were both found to be highly expressed in all detected cancer cell lines. The apoptotic rate of the pcDNA3.1-NS-Silencer group (12.1-15.4 ± 3.8%) was significantly higher than those of pcDNA3.1-NS (7.2-12.0 ± 1.7%) and non-transfection groups (4.1-6.5 ± 1.8%, P<0.01). MTT assays showed the knockdown of NS expression reduced the proliferation rate of SKBR-3 cells significantly. Matrigel invasion and wound healing assays indicated that the number of invading cells was significantly decreased in the pcDNA3.1-NS-siRNA group (P<0.01), but there were no significant difference between non-transfected and over-expression groups (P>0.05). Moreover, RNAi-mediated NS down- regulation induced SKBR-3 cell G1 phase arrest, inhibited cell proliferation, and promoted p53 pathway-mediated cell apoptosis in SKBR-3 cells. NS might thus be an important regulator in the G2/M check point of cell cycle, blocking SKBR-3 cell progression through the G1/S phase. On the whole, these results suggest NS might be a tumor suppressor and important therapeutic target in human cancers.

Construction and functional study of pGN, a mammary gland-specific expression plasmid.

The aim of this study was to construct a mammary gland-specific expression vector, pGN, and to validate its function in expressing growth hormone (GH) both in vitro and in vivo. First, the GH gene was amplified and inserted downstream of the b-casein 5'-arm. Next, the neo gene was cloned downstream of the b-casein 3'-arm as a selection marker. To analyze the bioactivity of the pGN plasmid, we expressed pGN in a Bcap-37 cell line and in the goat mammary gland. Quantitative PCR analysis revealed that the expression of GH mRNA in the pGN-transfected group was higher than that of the control group in Bcap-37 cells. Results of a radioimmunoassay and an enzyme-linked immunosorbent assay demonstrated that the pGN-transfected group expressed much more GH protein than the non-transfected group (P < 0.05). Upon injection of the pGN plasmid into the goat mammary gland, GH mRNA and growth hormone receptor mRNA expressions increased 2-fold. In vivo analyses revealed that GH protein expression was higher in the injected group than in the control group. Together, these results strongly demonstrated that the pGN plasmid was constructed correctly and exhibited favorable bioactivity in efficiently expressing GH both in vitro and in vivo.

Identification of Wnt/β‐catenin signaling pathway in dermal papilla cells of human scalp hair follicles: TCF4 regulates the proliferation and secretory activity of dermal papilla cell

It is clear that the dermal papilla cell (DPC), which is located at the bottom of the hair follicle, is a special mesenchymal component, and it plays a leading role in regulating hair follicle development and periodic regeneration. Recent studies showed that the Wnt signaling pathway through β-catenin (canonical Wnt signaling pathway) is an essential component in maintaining the hair-inducing activity of the dermal papilla and growth of hair papilla cells. However, the intrinsic pathways and regulating mechanism are largely unknown. In the previous work, we constructed a cDNA subtractive library of DPC and first found that the TCF4 gene, as a key factor of Wnt signaling pathway, was expressed as the upregulated gene of the hair follicle in low-passage DPC. This study was to explore the role of TCF4 in regulating the proliferation and secretory activity of DPC. We constructed a pcDNA3.0-TCF4 expression vector and transfected it into DPC to achieve stable expression by bangosome 2000. Furthermore, we used the method of chemosynthesis to synthesize three pairs of TCF4 siRNA and transfected them into DPC. Meanwhile, we compared the transfection group and non-transfection group. We first proposed that there was expression difference in TCF4 in DPC under different biological condition. This study may have a high impact on the molecular mechanism of follicular lesions and provide a new vision for the treatment of clinic diseases.

RelB-siRNA enhanced the radiosensitivity of murine prostate cancer cell line RM-1

To explore the transfecting effects of RelB small interfering RNA (RelB-siRNA) on murine prostate cancer cell line RM-1 sensitivity of radiotherapy. The RM-1 cells were divided into RelB-siRNA vector, empty vector, non-transfection and normal groups. After transfection, the first three groups were irradiated by X ray. Clone formation array method was used to measure the surviving fraction and the relevant parameter values after 0, 2, 4, 6, 8 Gy irradiation. Apoptotic rate was measured via Annexin V/PI flow cytometry after 6 Gy irradiation. The level of RelBmRNA was estimated by real-time polymerase chain reaction (PCR) after 6 Gy irradiation. And the RelB protein of cytoplasm and nucleus was detected by Western blot after 6 Gy irradiation. Clone formation array showed that RelB group at each dose point corresponding to the survival fraction were lower that the empty vector nd non-transfection groups. The D0, Dq and SF2 values of RelB group were 1.68, 0.60, 0.43, lower than those of empty vector group 1.92, 3.08, 0.89 and non-transfection group 1.93, 2.76, 0.84. Annexin V/PI flow cytometry demonstrated that the apoptotic rate of RelB group was 15.27% ± 1.62% and it was significantly higher than the non-transfection group 7.90% ± 1.50% and the empty vector group 8.40% ± 0.69% respectively (P < 0.01) .Real-time PCR indicated that RelB-mRNA amplification of RelB group was 1.18 ± 0.03 and it was significantly lower than the non-transfection group 2.10 ± 0.61 and the empty vector group 1.97 ± 0.66 respectively (P < 0.01) .Western blot suggested that cytoplasmic gray-scale ratio of RelB group was 0.50 ± 0.08 and it was significantly lower than the non-transfection 1.77 ± 0.19 and the empty group 1.52 ± 0.12 respectively (P < 0.01) . And the nuclear gray-scale radio of the RelB group was 0.18 ± 0.03 and it was significantly lower than the non-transfection group 0.61 ± 0.12 and the empty group 0.54 ± 0.13 respectively (P < 0.01) . The RelB protein inhibition rates of cytoplasmic and nuclear RelB-siRNA were 71.8% and 70.4% respectively. RelB-siRNA can effectively inhibit the RelB expression of murine prostate cancer cell line RM-1 and significantly increase its sensitivity to radiotherapy.

Inhibitory effects of indoleamine 2,3-Dioxygenase-transfected dendritic cells and Tryptophan catabolites on proliferation of CD4 + T lymphocytes

Objective To demonstrate the inhibitory effect of indoleamine 2,3-dioxygenase (IDO)-transfected dendritic cells (DCs) and Tryptophan catabolites on proliferation of CD4 +T lymphocytes.Methods IDO cDNA was obtained and inserted into eukaryotic expression vector pEGFP-N1 to construct the recombinant expression plasmid pEGFP-N1-IDO.The recombinant plasmid was transfected into mouse DCs by GenePORTER 3000.IDO expression level was detected by using real-time quantitative polymerase chain reaction (Real-time PCR) and immunocytochemistry.The transfected DCs with or without Tryptophan catabolites and 1-mythotryptophan were co-cultured with allogeneic CD4 +T lymphocytes.Stimulation index (SI) was analysed by mixed lymphocyte reaction (MLR).Results pEGFP-N1-IDO was successfully generated and verified by sequencing.IDO mRNA expression level was significantly higher in pEGFP-N1-IDO-transfected DCs group (0.1550 ±0.0124) than that in DCs group (0.1197 ±0.0463) or pEGFP-N1-transfected DCs group (0.1152 ± 0.0260).Positive staining was found in the pEGFP-N1-transfected DCs by immunocytochemistry.SI in pEGFP-N1-IDO-transfected DCs group (2.8640 ± 0.1459) was significantly lower than that in non-transfected group (3.3040 ± 0.2394),pEGFP-N1-transfected DCs group (3.2340 ±0.1862) and 1-methyl-tryptophan (1-MT) addition group (3.1900 ±0.2906).The inhibitory concentrations of Tryptophan catabolites in pEGFP-N1-IDO-transfected DCs group were lower significantly than those in non-transfected group or pEGFP-N1-transfected DCs group.Conclusion pEGFP-N1-IDO-transfected DCs can express functional IDO.The pEGFP-N1-IDO-transfected DCs and tryptophan catabolites both can inhibit the proliferation of CD4 + T lymphocytes. Key words: Indoleamine 2,3 dioxygenase; Vector pEGFP-N1-IDO; Tryptophan catabolites; Dendritic cells

Effects of down-regulated TRAF6 gene expression on the proliferation and apoptosis in multiple myeloma cells

To investigate the down-regulated TRAF6 gene expression and its effects on proliferation and apoptosis in multiple myeloma (MM) cells. Detection of TRAF6 expression were conducted by RT-PCR and Western blot in MM cell lines of KM3, U266, RPMI8226 and primary cells from patients. RPMI8226 cell lines were transfected with siRNA of TRAF6. The efficiency of transfection was identified by using of fluorescence microscope, RT-PCR, and Western blot. The levels of proliferation were analyzed by CCK-8 method under the different concentrations of siRNA. Apoptosis rate were detected with Hoechst33258/PI double staining by flow cytometry. Apoptosis related proteins Bcl-2, BAX, and NF-κB signal pathway were observed before and after siRNA transfection by Western blot. The levels of TRAF6 mRNA and protein in MM cell lines, especially in primary myeloma cells, were significantly higher than those in controls. After transfected with 50 nmol/L siRNA in RPMI8226 cells, the relative level of TRAF6 mRNA (0.49±0.24) was significantly lower than that in non-transfected group (1.87±0.23) and idling group (1.74±0.35). The proliferation rate of siRNA transfected cells decreased with dose dependence (P<0.01). The apoptosis rates increased from 11.20% (before transfection) to 51.82% (after transfection), accompanied by down-regulated Bcl-2 protein, NF-κB signal pathway (p-p65 and p52), and up-regulated BAX protein. TRAF6 expression was high in myeloma cells. TRAF6 siRNA could inhibit proliferation of myeloma cells and induce apoptosis mediated by NF-κB classical and alternative pathway in myeloma cells.

Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament

Molecular mechanism of ossification of the posterior longitudinal ligament (OPLL) remains unclear. This study was to investigate different expressions of PERK between the spinal ligament fibroblasts from OPLL patients and non-OPLL patients, and demonstrate knockdown of PERK protein expression by RNA interference inhibiting expression of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) in the cells from OPLL patients. Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of OCN, ALP and COL I was detected in the cells from OPLL and non-OPLL patients by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of PERK was detected by Western blotting. And then, after 72h, when RNA interference against PERK was performed on the cells from OPLL patients, expression of the osteoblast-specific genes was compared again between the transfection group and non-transfection group. Spinal ligament fibroblasts were observed 7-10days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of OCN, ALP and COL I and protein expression of PERK in the cells from OPLL patients were significantly greater than those from non-OPLL patients. In addition, knockdown of PERK protein expression inhibited the mRNA expressions of OCN, ALP and COL I remarkably in the transfection group compared with the non-transfection group, at 72h after RNA interference targeting PERK was performed on the cells from OPLL patients. The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and PERK-mediated ER stress might be involved in development of OPLL.

Enhanced sensitivity of breast cancer MDA-MB-231 cells to chemotherapy by silencing forkhead box C2 gene with RNA interference technology

Objective To observe the effects of forkhead box C2 gene (FOXC2) short hairpin RNA on docetaxel chemotherapy sensitivity of human breast cancer MDA-MB-231 cells.Methods MDA-MB-231 cells were transfected with recombinant expression plasmid FOXC2-small interfering RNA (siRNA) by lipofectamine 2000,and the expression of FOXC2 mRNA and protein was detected at 48 h after transfection.The morphological changes of apoptotic cells were observed under fluorescent microscope at 72 h after transfection.Methyl thiazol tetrazolium (MTT) assay was used to examine the inhibitory rate and the 50％ inhibitory concentration (IC50).Flow cytometry was applied to examine apoptosis and cell cycle at 72nd h after transfection.Results As compared with negative control plasmid and FOXC2-siRNA non-transfection group,the typical apoptotic morphology of MDA-MB-231 cells was observed,and the mRNA and protein expression levels of FOXC2 were obviously reduced in FOXC2-siRNA transfection group (P ＜ 0.05).Silencing FOXC2 increased sensitivity of MDA-MB-231 cells to docetaxel (P ＜ 0.05),and IC50 was decreased from (6.08 ±1.23) mg/L to (0.65 ±0.01) mg/L.Meanwhile,the apoptosis rate of MDA-MB-231 cells transfected with pFOXC2-shRNA was (20.01 ± 0.19)％,which was significantly higher than other three groups,and the proportion of cells in S phase and G0/G1 phase was decreased and that in G2/M phase was increased (P ＜0.05).Conclusion Down-regulation of FOXC2 expression by siRNA in breast cancer MDA-MB-231 cells could effectively induce apoptosis of MDA-MB-231 cells and increase their sensitivity to docetaxel. Key words: Breast cancer; Docetaxel; Apoptosis; Sensitizing effect

Effect of PC cell-derived growth factor RNA interference on biological behavior of esophageal squamous carcinoma cells

To investigate the effect of PC cell-derived growth factor (PCDGF) RNA interference on esophageal squamous carcinoma cells Eca-109 in vitro. The PCDGF-shRNA expression vector was transfected into the Eca-109 cells by liposome. After transfection, the mRNA and protein expressions of PCDGF were detected by RT-PCR and Western-blot respectively. Cell Counting Kit-8 (CCK-8) assay and Boyden chamber method were performed to measure the cell proliferation and invasion ability respectively. The expression levels of PCDGF mRNA and protein were both decreased in Eca-109 cells transfected with PCDGF-shRNA expression vector (transfection group). Twenty-four, 48 and 72 h after transfection, the cells proliferation in the transfection group was inhibited, and the inhibition rate was 20.4%, 21.1% and 20.9% respectively. The cell proliferation activity in the transfection group was significantly lower than that in the non-transfection group, liposome group and negative vector group (all P<0.05). The number of cell migration in the non-transfection group,negative vector group, liposome group and transfection group was 118.8±12.0, 100.8±9.0, 114.3±4.7, and 53.5±16.3 respectively. The differences were statistically significant between the transfection group and the other 3 groups (all P<0.05). PCDGF RNA interference can inhibit the proliferation and invasion abilities of esophageal squamous carcinoma cells in vitro. PCDGF gene may be the new target of gene therapy.

Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex

The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold 'bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3-7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from -78 to -2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na(+) channels from inactivation. Depolarization and the number of inactivated Na(+) channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

Overexpression of cytoglobin gene inhibits hypoxic injury to SH-SY5Y neuroblastoma cells.

A plasmid for cytoglobin expression, pAcGFP1-C1-cytoglobin, was transfected into SH-SY5Y cells. Cobalt chloride was used to establish a model of hypoxia. Western blotting indicated that cytoglobin was overexpressed and there was low expression of hypoxia-inducible factor-1α in SH-SY5Y cells after transfection. Following cobalt chloride-induced hypoxia, cytoglobin and hypoxia-inducible factor-1α expression gradually increased in SH-SY5Y cells. Flow cytometry showed that with increasing duration of hypoxia, the proportion of normal cells significantly diminished in the transfected and non-transfected groups. The proportion of cells in the early stages of apoptosis increased. However, the proportion of apoptotic cells was significantly lower in the transfected group compared with the non-transfected group. These results demonstrate that cytoglobin and hypoxia-inducible factor-1α are strongly up-regulated by hypoxia, and that there is a strong relationship between hypoxia-inducible factor-1α and cytoglobin during hypoxic injury.

Induction of chondrogenesis of adipose-derived stem cells by novel recombinant TGF-β3 fusion protein

A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs). The recombinant pIRES-EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups: experimental group (MMP group), negative control group (no MMP) and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen (ColII) was detected by using Alcian blue staining and immunohistochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and sequencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differentiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.

Effect of microRNA-32 on the proliferation and migration of gastric cancer cells

Objective To explore the effect of microRNA-32 (miRNA-32)on the biological behaviors of gastric cancer cell and its mechanism.Methods Gastric cancer cell line SGC-7901 cells were transiently transfected with miRNA-32 analogue,miRNA-32 inhibitor and empty plasmid vectors by lipofectamine and divided into analogue transfection group,inhibitor transfection group,empty plasmid transfection group and non-transfection group.The expression of green fluorescent protein was observed under fluorescent microscopy.The expression of miRNA-32 at mRNA level was detected by quantificational real-time polymerase chain reaction.The cell proliferation was evaluated by CCK-8 assay.The cell migration ability was measured by scratch test and Transwell chamber assays.The data were analyzed by one-way ANOVA.Results Compared with empty plasmid transfection group and non-transfection group,the expression of miRNA-32 mRNA of miRNA-32 analogue transfection group (relative quantitative value:2.327) was significantly up-regulated and that of miRNA-32 inhibitor transfection group (relative quantitative value:0.402) was significantly down regulated (F=11.238,P＜0.05).The width of scratch of miRNA-32 analogue transfection group was (61.39± 2.21) μm at 24 hours; miRNA-32 inhibitor transfection group was (29.97±0.66) μm.The migration distance of inhibitor transfection group was far than that of analogue transfection group (F=9.371,P＜0.05).After transfection for 48 hours,the cell number of migrated cells of analogue transfection group was significantly less than that of non transfection group,which was 16.93±4.63 and 93.93± 7.09,respectively (F=6.853,P＜0.05).After transfection for 48 hours and 72 hours,the cell growth inhibiting rate of miRNA 32 analogue transfection group was (43.474 ± 18.636)％ and (45.050±23.764)％,respectively,the cell growth was significantly inhibited (F=7.986 and 8.635,P=0.028 and 0.032).Conclusion The cell growth and migration ability of human gastric cancer cell line SGC-7901 are obviously inhibited through upregulating the expression of miRNA-32. Key words: Stomach neoplasms; Cell proliferation; Cell movement; MicroRNAs

Annexin A1 protein regulates the expression of PMVEC cytoskeletal proteins in CBDL rat serum-induced pulmonary microvascular remodeling

BackgroundHepatopulmonary syndrome (HPS) is characterized by advanced liver disease, hypoxemia and intrapulmonary vascular dilatation (IPVD). The pathogenesis of HPS is not completely understood. Recent findings have established the role of proliferation and phenotype differentiation of pulmonary microvascular endothelial cells (PMVECs) in IPVD of HPS; the change in cytoskeletal proteins and their molecular mechanism play an essential role in the proliferation, phenotype modulation and differentiation of PMVECs. However, little is known about the relevance of cytoskeletal protein expression and its molecular mechanism in IPVD of HPS. In addition, ANX A1 protein has been identified as a key regulator in some important signaling pathways, which influences cytoskeletal remodeling in many diseases, such as lung cancer, liver cancer, etc.MethodsPMVECs were cultured from the normal rats and then divided into three groups(Ad-ANXA1-transfected group, a non-transfected group, and an adenovirus empty vector group) and incubated by nomal rat serum or hepatopulmonary syndrome rat serum respectively. mRNA level was evaluated by real time reverse transcription polymerase chain reaction, and protein expression was detected by western blot. Cell proliferation was determined by the MTT and thymidine incorporation assay.ResultsIn this study, we found that the serum from a common bile duct ligation(CBDL) Rat model decreased the expression levels of the ANX A1 mRNA and protein by at least two-fold in human PMVECs. We also found the expression of cytoskeletal proteins (Destrin, a1-actin, and a1-tubulin) in PMVECs significantly increased. After stimulating ANX A1 over-expression in PMVECs by adenovirus-mediated ANX A1 (Ad-ANXA1) transfection, we found the expression levels of cytoskeletal proteins were significantly suppressed in PMVECs at all time points. Additionally, we report here that serum from a CBDL Rat model increases the proliferation of PMVECs by nearly two-fold and that over-expression of Ad-ANXA1 significantly inhibits HPS-rat-serum-induced PMVEC proliferation (p <0.05). These findings suggest that the ANX A1 down-regulation of PMVEC proliferation in the presence of HPS-rat-serum may be the major cause of aberrant dysregulation of cytoskeletal proteins (Destrin, a1-actin, and a1-tubulin) and may, therefore, play a fundamental role in the proliferation and phenotype differentiation of PMVECs in the PVD of HPS.ConclusionFinally, the fact that transfection with Ad-ANXA1 results in inhibition of the aberrant dysregulation of cytoskeletal proteins and proliferation of PMVECs suggests a potential therapeutic effect on PVD of HPS.

Survivin Status Affects Prognosis and Chemosensitivity in Epithelial Ovarian Cancer

The objective of this study was to explore the clinical significance of survivin expression in epithelial ovarian cancer (EOC) and the effect of survivin small hairpin RNA (shRNA) on survivin expression, apoptosis, and chemosensitivity in the human ovarian cancer cell line OVCAR3. A retrospective review of 90 consecutive EOC patients with a median follow-up time of 51 months was conducted. Survivin expression was examined by immunohistochemistry. OVCAR3 cells were transfected in vitro with survivin shRNA. Survivin mRNA expression levels were detected using reverse transcription-polymerase chain reaction. Flow cytometry was applied to determine survivin protein expression levels and cell apoptotic rates. The MTT method was used to examine the effects of survivin shRNA on chemosensitivity in OVCAR3 cells. Positive cytoplasmic expression of survivin was associated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage, nonmucinous type, high grade, and recurrence. Positive survivin expression was also associated with platinum resistance (r = 0.306, P = 0.003). Statistical results indicated that FIGO stage (hazard rate = 1.649, P = 0.047) and cytoplasmic expression of survivin (hazard rate = 1.734, P = 0.010) were independent prognostic factors. Survivin mRNA and protein levels were lower in OVCAR3S (ovarian cancer cells transfected with a survivin recombinant vector) cells at 24 hours after transfection as compared with controls. The flow cytometric analysis revealed that survivin shRNA induced accumulation of cells in the G0/Gl phase, with a decrease in G2/M phase cells following 24 hours of culture as compared with a nontransfected group (P < 0.01). Furthermore, survivin shRNA increased the sensitivity of OVCAR3 cells to paclitaxel 15-fold (P < 0.05), whereas it had no significant effect on cisplatin (P > 0.05). In addition to FIGO stage, cytoplasmic survivin protein expression is an independent molecular marker for predicting EOC prognosis. Sequence-specific shRNA targeting survivin can effectively suppress survivin expression, enhance apoptosis, and increase the sensitivity of ovarian cancer cells to paclitaxel but not to cisplatin.