miR-155-specific siRNA enhances chemosensitivity of Burkitt lymphoma Raji cells to cytosine arabinoside by inducing apoptosis

AIM：To investigate the effect of miR-155-specific siRNA alone or in combination with cytosine arabinoside (Ara-C) on the growth and apoptosis of Burkitt lymphoma Raji cells. METHODS：miR-155-specific siRNA and/or Ara-C were used to treat the cells. Quantitative real-time polymerase chain reaction was used to detect the expression of miR-155. The growth of the cells was analyzed by CKK-8 assay. The cell apoptosis was determined by flow cytometry. RESULTS：The miR-155 expression level of the cells transfected with miR-155 siRNA was significantly lower than that in the 2 control groups. Ara-C or miR-155 siRNA alone inhibited the growth of Raji cells in a dose-depend manner. miR-155 siRNA combined with Ara-C produced more inhibition of cell proliferation (P<0.05). After treatment for 48 h, the apoptotic rate of Raji cells in miR-155 siRNA+Ara-C group ［(38.4±1.4)%］ was higher than that in Ara-C group ［(16.5±0.3)%］ and miR-155 siRNA group ［(14.6±0.3)%］, with statistically significant difference (P<0.05). The expression of caspase-3 in Ara-C+miR-155 siRNA group was increased significantly as compared with Ara-C group and miR-155 siRNA group. CONCLUSION：miR-155-specific siRNA enhances the chemosensitivity of Raji cells to Ara-C by inducing apoptosis through the caspase-3 pathway.     

Effect of CD151 on biological characteristics of human umbilical cord mesenchymal stem cells

AIM：To study the effect of CD151 on the biological characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs). METHODS：CD151 expression on hUC-MSCs was interfered by siRNA. The cells were divided into siRNA-CD151 group and negative control group (treated with siRNA-NC). The efficiency of interference after 72 h and the changes of other surface markers were detected by flow cytometry. The ability of differentiation was assessed by oil red O and von Kossa staining. The cell cycle was analyzed by flow cytometry. The mRNA expression of CD151, hepatocyte growth factor (HGF), transforming growth factor β1 (TGF-β1), cyclooxygenase 2 (COX-2) and indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs was detected by real-time PCR. The secretion of HGF by hUC-MSCs was measured by ELISA. RESULTS：The results of flow cytometry showed that the expression of CD151 (11.97±2.63 vs 95.66±1.56, P<0.01) and CD105 (93.66±0.21 vs 83.37±0.71, P<0.05) on hUC-MSCs in siRNA-CD151 group was lower than that in negative control group. The consistent results were also achieved by using the method of real-time PCR. Treatment with siRNA-CD151 down-regulated the progress of the cell cycle as the G1 phase increased and the S phase decreased. The mRNA expression levels of HGF and TGF-β1 in hUC-MSCs in siRNA-CD151 group were lower than those in negative control group, and opposite result of COX-2 mRNA expression was observed. The IDO mRNA in hUC-MSCs was unchanged with IFN-γ stimulation for 24 h. HGF concentration in siRNA-CD151 group was decreased as compared with negative control group. CONCLUSION：Interfering CD151 expression on hUC-MSCs doesn’t change other surface markers except CD105, and maintains the capacity of adipogenic differentiation. However, it changes the osteogenic differentiation, proliferation and the expression of immunomodulatory cytokines.     

Bone marrow-derived mesenchymal stem cells protect against hypoxia-induced injury and apoptosis of PC12 cells via up-regulation of erythropoietin expression

AIM： To investigate the effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) on cobalt chloride (CoCl2)-induced injury and apoptosis of PC12 cells. METHODS：PC12 cells were divided into control group, CoCl2 group, BM-MSCs-siCTL+CoCl2 group and BM-MSCs-siEPO+CoCl2 group. The viability of the PC12 cells was measured by MTT assay. Flow cytometry and Hoechst 33258 staining were used to determine the apoptotic rate and the changes of chromatin distribution in PC12 cells. The expression of erythropoietin (EPO) in BM-MSCs was measured by RT-PCR and Western blotting. The mRNA expression of Bcl-2 and Bax in PC12 cells was detected by RT-PCR. Caspase-9 and caspase-3 assay kits were used to detect the activity of caspase-9 and caspase-3. RESULTS：The viability of PC12 cells treated with CoCl2 for 24 h and 48 h decreased to （43.0±6.4）% and （33.8±5.7）%, respectively, while 1∶15 ratio of BM-MSCs co-culture increased the cell viability to （77.9±3.8）% and （75.2±9.7）%,respectively. The expression of EPO in BM-MSCs was up-regulated after treated with 0.6 mmol/L CoCl2 for 24 h and 48 h, while EPO siRNA significantly abrogated the EPO expression in BM-MSCs. BM-MSCs-siCTL co-culture significantly inhibited the apoptosis of PC12 cells induced by CoCl2. However, EPO siRNA the protective effect of BM-MSCs. Compared with CoCl2 treatment group, BM-MSCs co-culture induced remarkable increase in the expression of Bcl-2 and decrease in the expression of Bax in PC12 cells, which was reversed by EPO siRNA. BM-MSCs-siCTL co-culture remarkably abrogated the CoCl2 induced up-regulation of caspase-9 and -3, while BM-MSCs-siEPO co-culture significantly reversed the down-regulation of caspase-9 and -3 induced by BM-MSCs-siCTL co-culture. CONCLUSION：BM-MSCs protect PC12 cells from apoptosis induced by CoCl2. The protective effect of BM-MSCs might be executed by up-regulating the expression of EPO.     

Effects of DHEA and G6PD antisense oligodeoxynucleotides on Raji cells

AIM:To investigate the suppressive effects of dehydroepiandrosterone (DHEA) and glucose-6-phosphate dehydrogenase (G6PD) antisense oligodeoxynucleotides on Raji cells. METHODS:Raji cell line was cultured in vitro in the presence of DHEA at different concentrations ranged from 0.05 μmol/L to 500 μmol/L or G6PD antisense oligodeoxynucleotides. The viability and proliferation of the cells pretreated with dehydroepiandrosterone or G6PD antisense oligodeoxynucleotides were evaluated. Meanwhile, intracellular activities and mRNA expression of G6PD were analyzed. RESULTS:DHEA and G6PD antisense oligodeoxynucleotides does not influence the viability of cells in culture. Raji cells treated with DHEA at concentration of 50 μmol/L or 500 μmol/L for 72 h or with 10.0 μmol/L G6PD antisense oligodeoxynucleotides for 48 h had significant lower cell numbers compared with control (P<0.01). Raji cells treated with DHEA at concentration more than 5.0 μmol/L for 72 h had significant decreased G6PD activities (P<0.01) but no change in mRNA expression levels was observed. With 10.0 μmol/L G6PD antisense oligodeoxynucleotides pretreatment for 48 h, the G6PD mRNA expression levels and activities were significantly decreased (P<0.01). CONCLUSION:DHEA or G6PD antisense oligodeoxynucleotides at specific concentration have suppressive effects on G6PD activities and proliferation in Raji cells to a certain extent, but the suppressive mechanisms are different.     

Effects of curcumin analogue T83 on apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM：To compare the effect of T83 (a 4-arylidene curcumin analogue) on the apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance. METHODS：The effects of T83 on the viability, apoptosis, mitochondrial membrane potential (MMP), expression of procaspase-3/procaspase-9/Cyt-C proteins and relative PTEN/Akt/p27 mRNA expression in CNE-2R cells and CNE-2 cells were detected and compared by the methods of MTT assay, Hoechst staining, flow cytometry, Western blotting and qRT-PCR. RESULTS：T83 inhibited the viability of CNE-2R cells with the IC50 of 0.9,0.4 and 0.2 μmol/L for 24 h, 48 h and 72 h,respectively, which was more effective than that inhibiting the viability of CNE-2 cells with the IC50 of 1.8,0.5 and 0.4 μmol/L, respectively. After treated with T83 for 48 h, chromatin condensation, margination and splitting into a massive structure were observed in CNE-2R cells and CNE-2 cells,and the late apoptotic rate of CNE-2R cells was increased from 1.57% to 27.26%, which was higher than that of CNE-2 cells (1.74% to 8.15%). After treated with T83 for 36 h, the MMP in CNE-2R cells decreased by 87.71% and that decreased by 50.47% in CNE-2 cells. After treated with T83 for 48 h, the protein levels of procaspase-3 and procaspase-9 were decreased, and the protein level of Cyt-C was increased, which were more susceptible in CNE-2R cells than those in CNE-2 cells. After treated with T83 for 24 h, the relative mRNA expression of PTEN and p27 was significantly up-regulated, and the mRNA expression of Akt was down-regulated, which were more susceptible in CNE-2R cells than those in CNE-2 cells. CONCLUSION：Compared with CNE-2 cells, the inhibitory effect of T83 on the viability of CNE-2R cells is more specific by starting the mitochondrial apoptotic pathway, which is due to the inhibition of PTEN/Akt/p27 signaling pathway.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

miR-126 regulates expression of vascular endothelial growth factor in EA.hy926 cells

AIM：To explore the effects of miR-126 on the expression of vascular endothelial growth factor (VEGF) in vascular endothelial cell line EA.hy926. METHODS：EA.hy926 cells were cultured in vitro and transfected with miR-126 mimics or miR-126 inhibitor by cation-mediated transfection method. The total RNA was extracted from the culture cells 36 h after transfection of miR-126 mimics or miR-126 inhibitor. The expression of VEGF at mRNA and protein levels was detected by real-time PCR and Western blotting. RESULTS：Thirty-six hours after transfection of miR-126 inhibitor at concentration of 50 nmol/L, the expression of VEGF at mRNA and protein levels increased significantly as compared with the negative control (P<001). However, transfection of miR-126 mimics at concentration of 50 nmol/L for 36 h significantly decreased the expression of VEGF at mRNA and protein levels as compared with the negative control. CONCLUSION：miR-126 inhibits the expression of VEGF. VEGF may be one of the target genes regulated by miR-126.     

CD147 affects autophagy of prostate cancer PC-3 cells

AIM：To study the autophagy of prostate cancer PC-3 cells induced by CD147 in vitro. ME-THODS：The method of amino acid starvation to induce autophagy was used. The expression of CD147was detected by Western blotting. To study the functional effects of CD147 on autophagy in prostate cancer PC-3 cells, the down-regulation of CD147expression was induced by the technique of RNAi. The conversion of autophagic marker protein LC3-I to LC3-II was determined by Western blotting. The cell death after starvation-induced autophagy was analyzed by trypan blue exclusion assay. RESULTS：The CD147 expression gradually increased in starvation-induced autophagy. The down-regulation of CD147 significantly increased the expression of autophagy-related protein LC3-II compared with control group. Meanwhile, the cell death rates increased from (19.3±3.1)% and (22.3±3.5)% in control groups to (38.4±3.1)% in silencing the expression of CD147in the PC-3 cells (P<0.05). CONCLUSION：CD147 inhibits starvation-induced autophgy and autophagy death in the prostate cancer PC-3 cells.     

Emodin inhibits proliferation and blocks cell cycle of human breast cancer MCF-7 cells

AIM：To investigate the effects of emodin on the proliferation of human breast cancer MCF-7 cells and its mechanisms. METHODS：MTT assay was used to observe the viability of MCF-7 cells. The cell cycle distribution and apoptosis of MCF-7 cells was analyzed by flow cytometry. The membrane surface morphology and three-dimensional ultrastructure of MCF-7 cells were observed under atomic force microscope (AFM). RESULTS：MTT assay showed that emodin could inhibit MCF-7 cell proliferation in a dose-dependent manner. Flow cytometric analysis demonstrated that emodin induced cell cycle arrest at G0/G1 phase. Annexin V/PI double staining confirmed that emodin had no effect on cell apoptosis. AFM images revealed that the cell nuclear area was full and the surface of cell membrane was flat and smooth in control group. Compared with control group, the cell nuclear area collapsed and shrank in emodin group at 48 h. The cell membrane ultrastructure showed that the particles in emo-din group had an intensive distribution. The height of cell nuclear area was decreased, and the surface average roughness (Ra) and root mean square roughness (Rq) were elevated in emo-din group compared with control group. CONCLUSION: Emodin has cytotoxicity on MCF-7 cells via cell cycle arrest at G0/G1 phase and ultrastructural changes.     

Bone marrow-derived mesenchymal stem cells regulate the function of Th17/ Treg in peripheral blood of severe asthmatic children

AIM：To investigate the regulatory function of bone marrow-derived mesenchymal stem cells (MSCs) on T helper 17 cells (Th17) and regulatory T cells (Treg) in peripheral blood of severe asthmatic children. METHODS：MSCs were isolated, cultured and identified in vitro. MSCs digested with mitomycin were cocultured with T lymphocytes (TLC) at different ratios (1∶1, 1∶2, 1∶10 and 1∶20) from severe asthmatic children for 72 h. The proliferation of TLC was measured by CCK-8 method. In the coculture system of the 1∶2 ratio and the single TLC system, the supernatant levels of interleukin-17 (IL-17) and transforming growth factor-β (TGF-β) were measured by ELISA. The mRNA expression of retinoic acid-related orphan nuclear receptor C (RORC) and forkhead box protein 3 (Foxp3) in TLC was detected by qRT-PCR. RESULTS：After cocultured with MSCs, the proliferation of TLC decreased significantly in a dose-dependent manner (P<0.05). It also showed decreases in IL-17 (3 799±441 vs 4 890 ±373, P<0.05) and RORC mRNA level (1.21±0.14 vs 3.85±0.48, P<0.05), while an increase in TGF-β level (209±32 vs 117±26, P<0.05) was observed. No influence on the mRNA expression of Foxp3 was found (P>0.05). CONCLUSION：MSCs suppresses Th17 polarization of naive peripheral blood CD4+ T cells and matures Th17 cells secreting IL-17, which may effectively revise Th17/Treg imbalance of asthma.     

Effect of cladribine on growth inhibition and autocrining cytokines in human umbilical vein endothelial cell line EA.hy926

AIM: To study the effects of cladribine on growth and secretion activity of human umbilical vein endothelial cell line EA.hy926, and to investigate the mechanism of its anti-tumor effect by inhibiting endothelial cells. METHODS: The effects of cladribine at different concentrations on the cell viability were detected by CCK-8 assay. Apoptosis and cell cycle distribution were examined by flow cytometry. The protein expression levels were determined by Western blot. The levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) secreted by EA.hy926 cells with cladribine treatment for 48 h were analyzed by ELISA. The nitric oxide (NO) production was measured by Gries method. RESULTS: Cladribine at 0.4~1 μmol/L inhibited the viability of EA.hy926 cells in time-and dose-dependent manners. The IC₅₀ was about 3.644 μmol/L. The results showed 43.74% cells in S phase when the concentration of cladribine was 0.4 μmol/L, and 77.23% cells in S phase when the concentration of cladribine was 1 μmol/L. The apoptosis was not induced by cladribine at 0.4~10 μmol/L. The protein expression of Bax and caspase-3 did not change. The expression of p21 increased and the p53 decreased (P<0.05). The levels of TNF-α and TGF-β1 secreted by EA.hy926 cells increased after cladribine treatment for 48 h. The levels of VEGF and NO decreased. CONCLUSION: Cladribine obviously inhibits the viability of EA.hy926 cells. The mechanism is related to the cell cycle arrest. Cladribine promotes the secretion of TNF-α and TGF-β1 by EA.hy926 cells and inhibits the secretion of VEGF and NO.     

MicroRNA expression in hepatocytes with hydrogen peroxide-induced oxidative stress-injury and alleviating effect of mesenchymal stem cell-conditioned medium

AIM: To evaluate the changes of microRNA (miRNA) in hepatocytes during hydrogen peroxide-induced oxidative stress injury, and to observe the alleviating effect of mesenchymal stem cell-conditioned medium (MSC-CM) in this progress. METHODS: The hepatocyte oxidative stress injury model was established using hydrogen peroxide and human normal liver cell line L02. MSC-CM was prepared using centrifugation and filter. The effects of MSC-CM on hepatocyte injury were evaluated by apoptosis analysis, cell viability detection, cell cycle, and mitochondrial membrane potential (MMP). Twenty-one differentially expressed miRNAs were selected by gene chip hybridization, in which miR-143, miR-145, miR-301a and let-7a were confirmed by RT-qPCR. Bioinformatics software was utilized to predict target proteins of these miRNAs, and then the proteins were verified by Western blot.RESULTS: MSC-CM markedly attenuated hydrogen peroxide-induced oxidative stress injury by reducing apoptosis, promoting cell viability and regulating cell cycle. The expression of miR-143, miR-145, miR-301a and let-7a, indentified by RT-qPCR, increased under the condition of oxidative stress injury, while decreased after MSC-CM treatment. The expression of miR-143 predicted target proteins, HK2 and ADRB1, decreased under the hydrogen peroxide-exposure, while increased after MSC-CM treatment, which is consistent with the regulatory trend of miR-143. CONCLUSION: MSC-CM might attenuate hydrogen peroxide induced oxidative stress injury via inhibiting apoptosis and regulating some miRNA expression.     

Biological characteristics of primarily cultured microvascular endothelial cells in mouse myocardium

AIM：To investigate an effective and stable method to isolate myocardial microvascular endothelial cells (MMVECs) from the mouse heart and to observe their biological characteristics.METHODS：The cells from the mouse heart were isolated by collagenase II digestion followed by different speed adhesion. Then the cells were cultured on the dish coated with polylysine in endothelial cell-specific culture medium. The biological characteristics were observed by trypan blue staining. The cell growth curve and the cell proliferation were also evaluated by MTT assay at different passages. The expression of DiI-ac-LDL, FITC-UEA-1, specific markers of endothelial cells and the expression of CD31, vWF and CD34 were determined by immunofluoresence staining. To evaluate the function of the MMVECs, in vitro tube formation was evaluated under microscope.RESULTS：Two days after the enzyme digestion, the MMVECs formed small and isolated clusters. The MMVECs grew quickly in monolayer with the characteristics of endothelial cell shape at 4~5 d. The cells became confluent and cobblestone-like which were ready for passage at 7~8 d. After passage, the viability of the cells was more than 95%. The first and the third generation of the cells presented an S-shape growth curve. The cell proliferation of the first to the third generation was quick and slowed down after the fifth passage. MMVECs were highly positive for DiI-ac-LDL and FITC-UEA-1 ［(89.2±3.5)%］, indicating the cells were MMVECs. The other relative antigen expression (CD31, vWF and CD34) on the MMVECs was (56.7±3.7)%, (78.5±2.6)% and (67.8±4.2)%, respectively. The MMVECs formed the tubes in vitro after cultured for 6～12 h.CONCLUSION：We can obtain high-purity MMVECs using the combination of collagenase II digestion, the different speed adhesion process and the endothelial cell-specific culture medium for effective and reliable MMVECs isolation and culture.     

Overexpression of TAP1 up-regulates HLA-Ι in human glioma U251 cells

AIM：To investigate the relationship between the overexpression of transporter associated with antigen processing 1 (TAP1) and the human leukocyte antigen I (HLA-I).METHODS：The full length of TAP1 gene was obtained from the cDNA library. The lentiviral vector pSIN-EF2-IRES-GFP-puro was digested by BamH I and EcoR I, and the full length of TAP1 gene was inserted into the vector by T4 DNA ligase. Subsequently, the recombinant plasmid was transformed into Escherichia coli DH5α cells and the correct transformant was selected. The recombinant plasmid and the Lenti-X HTX packaging mixture were co-transfected into 293T cells, and the virus particle was acquired. Human glioma U251 cells were transfected with the lentivirus. The expression of TAP1 and HLA-I was determined by real-time fluorescence quantitative PCR, Western blotting and flow cytometric analysis.RESULTS：TAP1 gene was successfully transfected into the U251 cells and stably expressed in the cell line. The expression of TAP1 in U251 cells at mRNA and protein levels increased by (8.73±1.07) and (11.71±0.83) folds, respectively. As a result, the mRNA expression of HLA-A, HLA-B, HLA-C (heavy chain) and β2-microglobulin (light chain) was up-regulated by (3.51±0.36), (4.78±0.85), (2.94±0.28) and (3.23±0.24) folds, respectively. The protein expression of HLA-I also increased to (3.14±0.53) fold. The surface expression of HLA-I on the U251 cells transfected with TAP1 gene was largely enhanced as well.CONCLUSION：Overexpression of TAP1 up-regulates the expression of HLA-I. TAP1 plays an important role in HLA-I processing pathway.     

Ligustilide inhibits angiogenesis and epithelial-mesenchymal transition in human hemangioendothelial cells by reducing VEGF levels

AIM To investigate the effect of ligustilide on human hemangioendothelial cells (HemECs) and to analyze its mechanism. METHODS The effect of ligustilide at different concentrations on the viability of HemECs was measured by CCK-8 assay. The HemECs were divided into control group and ligustilide （10, 25 and 50 μmol/L） treatment groups, and the proliferation of HemECs was detected by EdU staining. The effects of ligustilide on the angiogenesis of HemECs was tested by microtubule formation experiment. The protein expression of vascular endothelial growth factor (VEGF) and epithelial-mesenchymal transition (EMT)-related markers in HemECs cells was determined by Western blot. RESULTS There was no significant difference in the viability of the cells treated with ligustilide at the concentrations between 0.1~50 μmol/L compared with control cells. Compared with control group, ligustilide at 25 and 50 μmol/L significantly reduced the number of EdU-positive cells and microtubule-like structures (P<0.05), reduced the protein expression level of VEGF (P<0.05), increased the protein expression of E-cadherin, and decreased the protein expression of vimentin and β-catenin (P<0.05). Compared with control group, the expression of VEGF and vimentin was significantly up-regulated, and the protein expression of E-cadherin was significantly down-regulated in VEGF overexpression group (P<0.05). Compared with VEGF overexpression group, the expression of VEGF and vimentin in 50 μmol/L ligustilide-treated VEGF-overexpressing cells were significantly reduced (P<0.05), and the protein expression of E-cadherin was significantly increased (P<0.05). CONCLUSION Ligulide inhibits the proliferation of HemECs, and also inhibits the angiogenesis and EMT process of HemECs by reducing the level of VEGF.     

Effects of gemcitabine on inhibiting proliferation and inducing apoptosis of CD34+CD38- myeloid leukemia stem cells

AIM：To investigate the effects of gemcitabine (GEM), a novel analog of deoxycytidine and nucleoside reductase inhibitor similar to cytarabine (Ara-C) in structure, on the proliferation and apoptosis of myeloid leukemic stem cells (LSCs), CD34+CD38-KG1a cells. METHODS：The expression of CD34 and CD38 on the surface of acute myeloid leukemia KG1a cells was detected by flow cytometry. The effects of GEM at various concentrations for 24 h and sustained medication for 14 d and 21 d on the proliferation and colony-forming ability of KG1a cells were analyzed by soft agar colony-forming experiment. The changes of the cell cycle of KG1a cells treated with various concentrations of GEM were tested by flow cytometry. The apoptosis of KG1a cells was determined by flow cytometry with the staining of Annexin V-FITC and propidium iodide (PI). RESULTS：The percentage of CD34+CD38- cells in acute myeloid leukemia KG1a cells was (98.02±0.72)%.Treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell cycle distribution of the KG1a cells, whereas KG1a cells treated with 0.5 mg/L GEM for 24 h were arrested at G0/G1 phase. After treatment with 0.1 mg/L and 0.5 mg/L GEM for 24 h, the colony numbers at 14 d and 21 d were lower than that in saline control group. No difference of the colony numbers between the cells treated with normal saline and 0.05 mg/L GEM for 14 d and 21 d was observed. After sustained medication with 0.05 mg/L, 0.1 mg/L and 0.5 mg/L GEM and Ara-C for 14 d and 21 d, the colony numbers decreased as compared to saline control group. Treatment with 0.5 mg/L GEM for 24 h increased the apoptotic rate of KG1a cells compared with saline control group, while treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell apoptosis. CONCLUSION：GEM inhibits the proliferation and colony-forming ability, arrests the cell cycle at G0/G1 phase and induces apoptosis of CD34+CD38- acute myeloid leukemia cells.     

Exogenous ATP induces formation of membrane pore in PC12 cells

AIM：To investigate the formation of membrane pore in PC12 cells induced by exogenous adenosine triphosphate (ATP) and to identify the key molecular targets. METHODS：PC12 cells were treated with different concentrations of ATP to establish the injury model. The morphological change was observed under an inverted phase-contrast microscope. The viability of the PC12 cells was measured by CCK-8 assay. Fluorescent dye YO-PRO-1 was used to detect the membrane permeability. The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was assessed by real-time PCR and Western blotting. RESULTS：After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous, and the number of adherent cells decreased gradually in a dose-dependent manner. The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with control group (P<0.05). YO-PRO-1 uptake in the PC12 cells exposed to ATP (0, 1, 3 and 5 mmol/L) for 15 min, 30 min and 60 min increased in a dose-dependent and time-dependent manner. The cell viability increased and the intracellular fluorescence intensity induced by ATP were significantly antagonized in brilliant blue G (a P2X7 receptor inhibitor) pretreatment group (P<0.05), whereas it did not change in carbenoxolone (a Panx1 inhibitor) pretreatment group (P>0.05). The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P<0.05), but the expression of Panx1 was not changed (P>0.05) when PC12 cells were exposed to ATP for 3 h. CONCLUSION：Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P2X7 receptor in PC12 cells.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.