Effect of mineralocorticoid receptor gene silencing by RNA interference on proliferation and apoptosis of murine macrophage cell line RAW 264.7

AIM: To establish stable knockdown of mineralocorticoid receptor (MR) expression through short hairpin RNA (shRNA)-mediated silencing in murine RAW 264.7 macrophages. METHODS: Stable MR silencing in RAW 264.7 cells was achieved by recombinant shRNA plasmid targeting murine MR gene via liposome-mediated transfection, followed by G418 selection. The efficacies of plasmid transfection and MR silencing in G418-resistant cells were verified by immunofluorescent microcopy and real-time PCR, respectively. Proliferative activity of MR-silencing cell line was analyzed by CCK-8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. RESULTS: MR gene expression was down-regulated by 70% compared with the negative control (NC) plasmid transfection. In addition, MR-silencing cells exhibited lower proliferative activity compared with NC and wide type RAW 264.7 cells (P<0.05), along with reduced proliferation index of 31.0%±1.3% (P<0.05), compared with the wide type cells (37.2%±0.5%) and the NC cells (37.5%±1.6%). In resting state, the apoptotic rate in wide type, NC and MR-silencing cells were 2.18%±0.36%, 6.65%±0.81% and 7.70%±1.34%, respectively, and no statistical difference was observed between NC and MR-silencing cells (P>0.05). CONCLUSION: MR gene silencing inhibits the proliferation of RAW 264.7 macrophages, but has no obvious effect on the apoptosis of the resting state cells.     

Brilliant blue G attenuates hippocampal injury of rats with acute carbon monoxide poisoning

AIM:To explore the role of ligand-gated ion channel purinergic P2X₇ receptor (P2X₇R) in acute carbon monoxide poisoning (ACMP)-induced brain injury, and to observe the effects of brilliant blue G (BBG) on ACMP-induced hippocampal injury of rats. METHODS:Male Sprague-Dawley rats (n=80) were divided into 4 groups including control group, ACMP group, ACMP +BBG group and BBG group. ACMP model was established by intraperitoneal injection of CO at 100 mL/kg. The degree of CO poisoning was determined by measuring the concentration of carboxyhemoglobin (HbCO) in venous blood. RT-qPCR was utilized to determine the mRNA level of P2X₇R. Wet-dry weight ratio was calculated to evaluate the edema in the hippocampus. ELISA was used to examine the concentrations of pro-inflammatory factors including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6. Morris water maze test was used to evaluate the learning and memory abilities. The morphological changes of the neurons located in hippocampal CA1 region were observed by HE staining. RESULTS:The rat survival rate in ACMP group (55%) was significantly reduced compared with control group (100%), while the mRNA expression of P2X₇R and the levels of pro-inflammatory factors including IL-1β, TNF-α and IL-6 in ACMP group were significantly increased (P<0.05). Moreover, ACMP administration increased the water content of hippocampal tissues and the escape latency of the rats, while decreased the exploration time in the target quadrant. All the effects induced by ACMP were reversed by BBG treatment. Additionally, ACMP treatment for 3 d resulted in cell disarrangement and morphologyical defect, and the nucleus was deeply dyed and condensed. However, BBG obviously alleviated the toxic effects of ACMP. CONCLUSION:P2X₇R is involved in ACMP-induced neuronal damage in hippocampus. BBG increases the survival rate, reduces the release of pro-inflammatory cytokines and the edema in the hippocampus, improves the learning and memory abilities, and attenuates neuronal damage in the CA1 region under ACMP.     

rhPGRN promotes cell proliferation by regulating autophagy and endoplasmic reticulum stress through MAPK/PI3K pathway

AIM To extract and purify recombinant human progranulin (rhPGRN) and to examine its effect on the proliferation, autophagy and endoplasmic reticulum stress (ERS) in human chondrocyte C28I2 and mouse macrophage RAW264.7. METHODS The histidine-tagged protein was specifically affinity-purified using Ni-NTA Sefinose resin, and the concentration and purity of the target protein were verified by Coomassie blue staining, BCA method and Western blot. The effects of rhPGRN on the proliferation, autophagy and ERS in C28I2 and RAW264.7 cells were detected by cell counting, Western blot and RT-qPCR. RESULTS The highly purified biologically active human recombinant protein rhPGRN was successfully extracted from the cell line with stable PGRN transfection. rhPGRN promoted the proliferation of the C28I2 cells and RAW264.7 cells, up-regulated the mRNA expression of cell cycle-related molecules (PCNA, cyclin B1 and cyclin D1) and the protein expression of Ki67, and increased the phosphorylation levels of proliferation-related signaling molecules ERK and Akt. Treatment with ERK pathway inhibitor U0126 inhibited rhPGRN-promoted proliferation, autophagy and ERS in the cells. The rhPGRN-induced autophagy of the cells was also inhibited by PI3K/Akt pathway inhibitor 3-methyladenine. The rhPGRN-promoted protein expression of Ki67 was down-regulated by autophagy inhibitor bafilomycin A1 and ERS inhibitor 4-phenylbutyric acid. CONCLUSION These results not only established a method for stable extraction of biologically active high-concentration high-purity recombinant protein rhPGRN, but also confirmed that the biological effect of rhPGRN on promoting cell proliferation was achieved through regulating autophagy and ERS via MAPK and PI3K/Akt pathway.     

Effect of microtubule-destabilizing protein stathmin gene silencing on proliferation and apoptosis of nasopharyngeal carcinoma cell line 5-8F

AIM: To investigate the effects of stathmin gene silencing on nasopharyngeal carcinoma cell line 5-8F. METHODS: Double-strand siRNA targeting to stathmin gene was obtained by chemical synthesis and annealing, and was sub-cloned into the vector pGenesil-1.1. The plasmid was introduced into 5-8F cells by liposome-mediated transfection. The gene expression of stathmin, and the proliferation, morphology and apoptosis of the cells were analyzed by Western blotting, MTT assay and flow cytometry. RESULTS: The cell suppression rate in stathmin gene silencing group was (53.01?1.12)%, significantly higher than that in transfection reagent group and in negative control group. The cell apoptotic rate in stathmin gene silencing group was (8.75?0.67)%, also significantly higher than that in transfection reagent group and in negative control group (P<0.05). CONCLUSION: Silencing of stathmin gene in nasopharyngeal carcinoma cells inhibits the cell proliferation and induces cell apoptosis.     

Effects of intracellular expression of Mycobacterium tuberculosis CFP10-ESAT6 fusion protein on proliferation and apoptosis of mouse macrophages

AIM：To establish Mycobacterium tuberculosis culture filtrate protein 10 (CFP10)-early secretory antigenic target 6 (ESAT6) eukaryotic expression vector and investigate the effect of intracellular expression of CFP10-ESAT6 fusion protein on the proliferation and apoptosis of mouse macrophages (RAW264.7 cells). METHODS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was constructed by inserting CFP10-ESAT6 fusion gene into eukaryotic expression vector pEGFP-N1, and then transfected into RAW264.7 cells to express CFP10-ESAT6 fusion protein. The viability of RAW264.7 cells was measured by MTT assay. Flow cytometry was applied to measure the apoptotic rate and Toll-like receptor 2 (TLR2) expression in RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein or staurosporine. RESULTS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was successfully constructed and transfected into RAW264.7 cells. Compared with the control cells, intracellular expression of CFP10-ESAT6 fusion protein did not affect the viability of RAW264.7 cells, but could inhibit the apoptosis of RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein. Moreover, CFP10-ESAT6-expressing macrophages had markedly lower expression of TLR2 on the surface. CONCLUSION：Intracellular expression of CFP10-ESAT6 fusion protein has no cytotoxicity on mouse macrophages, but can inhibit the apoptosis of the macrophages treated with Mycobacterium tuberculosis 19 kD lipoprotein through down-regulating the expression of TLR2.     

Effect of Bmi-1 gene overexpression on proliferation of human gastric mucosal epithelial cell line GES-1

AIM: To investigate the effect of B lymphoma Moloney murine leukemia virus insertion region 1 ( Bmi-1 ) gene overexpression on the proliferation of a human normal gastric epithelial cell line GES-1. METHODS: The plasmid containing Bmi-1 gene or empty plasmid was transfected into GES-1 cells by retroviral mediation. The expression of Bmi-1 at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) analysis and Western blotting, respectively. The effect of Bmi-1 gene overexpression on the cell cycle of GES-1 cells was evaluated by flow cytometry. The proliferation of the stably transfected cells was measured by Cell Counting Kit-8. RESULTS: The results of qRT-PCR analysis and Western blotting demonstrated that stably transfected cell line was successfully established. The results of flow cytometry analysis showed that overexpression of Bmi-1 reduced the G₀/G₁ phase, arrested the cells in G₂/M phase and S phase. The growth curve showed that overexpression of Bmi-1 resulted in increased growth speed. CONCLUSION: Increase in Bmi-1 gene expression regulates the cell cycle and promotes the proliferation of GES-1 cells.     

Effect of SMYD3 over-expression on DNMT3B levels and proliferation ability in human cholangiocarcinoma cell line FRH0201

AIM: To explore the effect of SET and MYND domain-containing protein 3 (SMYD3) over-expression on the expression of DNA methyltransferase 3B (DNMT3B) and the proliferation ability in human cholangiocarcinoma cell line FRH0201. METHODS: Transient transfection of SMYD3 eukaryotic expression plasmid pEGFP-C3-SMYD3 into human cholangiocarcinoma cell line FRH0201 was performed. The expression of DNMT3B at mRNA and protein levels was detected by RT-PCR and Western blotting,respectively. Cell proliferation was examined by CCK-8 method and cell cycle situation was checked by flow cytometry. RESULTS: After transfected with SMYD3 eukaryotic expression plasmid pEGFP-C3-SMYD3, the over-expression of SMYD3 in FRH0201 cells was observed. Compared with the untransfected cells, the expression of DNMT3B was significantly increased (P<0.01), the proliferation rate was obviously accelerated (P<0.05) and the number of the cells in G₂/M phase was significantly increased (P<0.05) in FRH0201 cells transiently transfected with pEGFP-C3-SMYD3 plasmid. CONCLUSION: The transient transfection of pEGFP-C3-SMYD3 plasmid induces over-expression of DNMT3B and promotes the proliferation of human cholangiocarcinoma cell line FRH0201.     

Effects of silencing of COX-2 gene expression by siRNA on cell proliferation,cell cycles,apoptosis and tumorigenicity of human pancreatic cancer Capan-2 cells

AIM: To investigate the effects of silencing of cyclooxygenase-2 (COX-2) gene expression by siRNA on the proliferation, apoptosis, cell cycle and tumorigenicity of human pancreatic cancer Capan-2 cells.METHODS: The gene transfection was performed using Lipofectamine 2000 (Lipo). The proliferation, apoptosis and cell cycle of Capan-2 cells were tested by the methods of cell counting, microscopy and FCM. The mRNA expression of COX-2 was determined by RT-PCR and real-time PCR. The protein level of COX-2 was detected by Western blotting. The tumorigenicity of Capan-2 cells transfected with siRNA-COX-2 was determined using the model of nude mice. RESULTS: Transfection efficiency of 96.47% was obtained under the conditions that the transfection volume was 2 mL, concentration of Lipo was 5 μL and that of siRNA-COX-2 was 50 nmol/L. The best sequence of siRNA-COX-2 for silencing of COX-2 gene expression was siRNA006 with the silencing rate of up to 73% 24 h after tansfection. siRNA-COX-2 slowed down the growth of Capan-2 cells 48 h after transfection (P<0.05). At time points of 48 h and 72 h after transfection, the protein expression of COX-2 was down-regulated to 67% and 61% of the normal level, the proliferation inhibition rate was 35.48% and 56.32%, and the apoptotic rate was 2.03% and 3.27%, respectively. At time points of 24 h, 48 h and 72 h after transfection, the proportion of the cells in G₀/G₁ phrase was 58.03%, 63.31% and 65.66%, and that of the cells in S phase was 30.27%, 24.87% and 22.2%, respectively. The mean volume and weight of tumor tissues were remarkably decreased due to the transplantation of Capan-2 cells transfected with siRNA-COX-2.CONCLUSION: siRNA-COX-2 effectively silences the expression of COX-2 gene, inhibits the growth and decreases the tumorigenicity of Capan-2 cells.     

ox-LDL increases endothelial lipase in RAW264.7 macrophages

AIM: To investigate the effect of oxidized low-density lipoprotein (ox-LDL) on the expression of endothelial lipase (EL) in murine RAW264.7 macrophages. METHODS: RAW264.7 cells were incubated with ox-LDL at concentrations of 0~100 mg/L for 24 h.On the other hand, the cells were incubated with or without PDTC (NF-κB inhibitor) for 30 min and then with ox-LDL (50 mg/L) for 24 h. The expression of EL and p65 was detected by Western blotting. RESULTS: The level of EL was significantly increased after ox-LDL incubation in RAW264.7 cells (P<0.05). NF-κB was activated by ox-LDL at concentration of 50 mg/L for 15~30 min in RAW264.7 cells. The increase in EL induced by ox-LDL was markedly inhibited by a NF-κB inhibitor PDTC (P<0.05). CONCLUSION: ox-LDL significantly increases the expression of EL in RAW264.7 macrophages, which is possibly related to NF-κB activation.     

Effect of progesterone on SH-SY5Y cells injured by ATP

AIM: To investigate the neuroprotective effect of progesterone against adenosine triphosphate (ATP)-injured human neuroblastoma SH-SY5Y cells.METHODS: The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations. The cell viability was measured by CCK-8 assay. The membrane permeability was detected using fluorescent dye YO-PRO-1. Cytosolic Ca²⁺ concentration was measured with fluorescent dye Fluo-3/AM. The expression of purinergic P2X₇ receptor was assessed by Western blot.RESULTS: The viability of the SH-SY5Y cells was significantly decreased (P<0.05) and YO-PRO-1 uptake was obviously increased (P<0.05) in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1, 3, 5 and 7 mmol/L for 2 h. The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3, 10 and 30 nmol/L for 30 min (P<0.05) as compared with ATP group. YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced (P<0.05) by progesterone (30 nmol/L) or P2X₇ receptor antagonist KN-62 (500 nmol/L) pretreatment for 30 min, and no obvious difference between treatments with progesterone and KN-62 was observed. Cytosolic Ca²⁺ fluorescence intensity in normal group was a little, but that in ATP group was increased (P<0.05). Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca²⁺ induced by ATP (P<0.05). However, no obvious difference between treatments with progesterone and KN-62 was found. The expression of P2X₇ receptor in ATP group was significantly higher than that in control group (P<0.05), and progesterone inhibited ATP-induced P2X₇ receptor expression (P<0.05).CONCLUSION: Progesterone inhibits P2X₇ receptor expression, membrane pore formation, intracellular Ca²⁺ increase and cell death induced by ATP, so progesterone may protect SH-SY5Y cells against ATP-induced injuries.     

Effects of human xeroderma pigmentosum group D gene on proliferation of human vascular smooth muscle cells induced by interleukin-6

AIM: To investigate the effects of human xeroderma pigmentosum group D (XPD) gene on the proliferation of human vascular smooth muscle cells (VSMCs) induced by interleukin-6 (IL-6). METHODS: Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into VSMCs by liposome, and then these cells were incubated with IL-6 at 1×10⁵ U/L for 48 h. The cells were divided into 6 groups: blank control group; pEGFP-N2 group; pEGFP-N2/XPD group; IL-6 group; IL-6 + pEGFP-N2 group; IL-6 + pEGFP-N2/XPD group. The expression of green fluorescent protein was observed under fluorescence microscope. The cell growth was detected by MTT method. The cell cycle and apoptosis rate were examined by flow cytometre. The expression levels of XPD, Bcl-2, Bax and wild type P53 (wt-P53) were detected by RT-PCR and Western blotting.RESULTS: Green fluorescence was observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating successful transfection MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth, and reduced the positive effects of IL-6 on VSMCs growth. Flow cytometry results showed that the transfection of pEGFP-N2/XPD increased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, decreased the cell numbers in S phase, and reduced the effects that IL-6 decreased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, and increased the cell numbers in S phase. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, Bax and wt-P53, decreased the expression of Bcl-2, and reduced the effects that IL-6 decreased the expression of Bax and wt-P53, and increased the expression of Bcl-2. CONCLUSION: XPD gene inhibits VSMCs proliferation, promotes VSMCs apoptosis, and reduces the effects that IL-6 promotes VSMCs proliferation and inhibits VSMCs apoptosis. Therefore, XPD gene is likely to be potential molecular target for treatment of atherosclerosis.     

Effects of p21-activated protein kinase 2 down-regulation on proliferation and apoptosis of human breast cancer cells

AIM：To study the effect of p21-activated protein kinase 2 (PAK2) knockdown by RNA interference on the proliferation and apoptosis of human breast cancer cells. METHODS：The short hairpin RNA (shRNA) targeting PAK2 gene was designed and used for packing lentivirus in 293T cells.Human breast cancer MCF-7 cells were infected by the virus particles and PAK2 knockdown stable cell line was established by puromycin selection. The knockdown efficiency was assessed by Western blotting. The proliferation ability of MCF-7 cells was evaluated by CellTiter 96 AQueous and anchorage-independent growth assays. The cell apoptosis induced by staurosporine was detected by flow cytometry. RESULTS：The protein level of PAK2 was significantly suppressed after silencing of PAK2 gene in MCF-7 cells (P<0.01). Furthermore, knockdown of PAK2 caused remarkable inhibition of the cell proliferation and colony formation (P<0.01). Staurosporine induced more apoptosis in the PAK2 knockdown cells compared with the control cells (P<0.01). CONCLUSION：Knockdown of PAK2 inhibits the proliferation of MCF-7 cells and increases the sensitivity of chemotherapeutic drug-induced cell apoptosis, suggesting that PAK2 might be a new therapeutic target in breast cancer treatment.     

Effects of Smo gene silencing on cell activity and apoptosis of human cervical carcinoma HeLa cells

AIM: To investigate the effect of Hedgehog (Hh) signaling pathway on the viability and apoptosis of cervical carcinoma cells by shRNA technique to knock down Smoothened (Smo) gene. METHODS: Smo shRNA was used to transfect the cervical carcinoma HeLa cells. The expression of Smo and Gli1 at mRNA and protein levels in the HeLa cells was determined by RT-PCR and Western blot, respectively. The effect of Smo gene silencing on the growth of the cells was measured by MTT assay. The apoptosis and cell cycle were determined by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression of Smo and Gli1 were evenly reduced obviously after transfected with Smo shRNA for 72 h (P<0.05). The viability of HeLa cells transfected with Smo shRNA was significantly inhibited. The percentages of the cells in G₀/G₁ phase and early apoptosis rate were obviously higher in Smo shRNA transfection group than those in control group. CONCLUSION: Smo gene silencing effectively inhibits the cell growth and induces the apoptosis of human cervical carcinoma cells.     

Effects of fibulin-3 expression on malignant pleural mesothelioma cells

AIM: To observe the effects of over-expression and silencing of fibulin-3 on malignant pleural mesothelioma (MPM) cell line SMC-1, and to search for a new method for the treatment of MPM. METHODS: The vectors for fibulin-3 over-expression and short hairpin RNA (shRNA) were constructed, and control vector, fibulin-3 over-expression vector (Exp), over-expression control vector (Exp-NC), interference vector (shRNA1, shRNA2, shRNA3 and shRNA4), and interference control vector (shRNA-NC) were transfected into the SMC-1 cells. The cell cycle distribution and apoptosis were analyzed by flow cytometry. The expression of fibulin-3 before and after interference was determined by real-time PCR and Western blot. RESULTS: Compared with control group, the number of the SMC-1 cells in G₂ phase in Exp group was increased (P<0.05), while that in shRNA2 group was decreased (P<0.05). The results of apoptosis analyzed by flow cytometry showed that compared with control group, the apoptotic rate in Exp group was decreased (P<0.05), while that in shRNA2 group was increased (P<0.05). The mRNA and protein expression levels of fibulin-3 and mesothelin in Exp group were up-regulated, while those in shRNA group were down-regulated (P<0.05).CONCLUSION: The vectors for over-expression and silencing of fibulin-3 are successfully constructed, proving that transfection of Exp and shRNA effectively changes fibulin-3 expression and the SMC-1 cell growth. Fibulin-3 may be a target molecule for MPM treatment.     

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.     

Effect of IGFBP7 on proliferation of human breast cancer cell line MCF-7

AIM: To investigate the mechanism that insulin-like growth factor binding protein 7 (IGFBP7) inhibits proliferation of human breast cancer cell line MCF-7. METHODS: Plasmid pCMV6-IGFBP7 or empty plasmid was transfected into MCF-7 cells. The expression of IGFBP7 in MCF-7 cells after transfection was detected by Western blotting. The effects of IGFBP7 on the colony-forming efficiency and the cell cycle were studied by soft agar colony formation assay and flow cytometry,respectively. The effects of IGFBP7 on the expression of ERK1/2, p-ERK1/2, cyclin D1, CDK4, cyclin E, CDK2, p21^CIP1/WAF1, p27^KIP1, p53, Rb and p-Rb in MCF-7 cells were detected by Western blotting. RESULTS: Only the transfectant of pCMV6-IGFBP7 expressed IGFBP7. IGFBP7 remarkably reduced colony-forming efficiency (P<0.01) and G₀/G₁ arrest (P<0.01), inhibited phosphorylation of ERK1/2 (P<0.01), down-regulated cyclin D1 and cyclin E (P<0.01), up-regulated p27^KIP1, p21^CIP1/WAF1 and p53 (P<0.01), and inhibited phosphorylation of Rb (P<0.01) in MCF-7 cells. PD98059, an inhibitor of MEK1 and MEK2, imitated part of the tumor-suppressing activity of IGFBP7. CONCLUSION: IGFBP7 inhibits the proliferation of human breast cancer cell line MCF-7 by down-regulating cyclin D1 and cyclin E, up-regulating p27^KIP1, p21^CIP1/WAF1 and p53 and inhibiting phosphorylation of Rb. ERK1/2 signaling pathway might be involved in the regulation of cyclin D1 and p27^KIP1 by IGFBP7.     

Roles of maspin in biological behaviors of PC-3 prostate cancer cells

AIM: To investigate the roles of maspin in the biological behaviors of prostate cancer cells. METHODS: Specific shRNA targeting maspin gene was designed. The plasmid targeting maspin gene was constructed and lentiviral expression system was used for transfection. qRT-PCR and Western blotting were performed to identify the stable maspin-shRNA-transfected PC-3 cells. The expression of apoptosis-related genes was analyzed by qRT-PCR. Dynamic observation of cell growth and doubling time were conducted by an xCELLigence system. The cell death upon proteasome inhibitor treatment was determined by flow cytometry analysis. The expression levels of RelA and RelB were detected by Western blotting. RESULTS: The recombinant plasmid containing maspin-shRNA was successfully constructed. Limited dilution was performed to obtain monoclonal PC-3-siMaspin cells. The doubling time of PC-3-siMaspin cells was 26.83 h while that of PC-3-control cells was 37.95 h. The mRNA expression of bcl-2 and A20 in PC-3-siMaspin cells was increased, while that of bax and bim was down-regulated. The cell death rates of PC-3-control cells and PC-3-siMaspin cells after treated with MG-132 were 27.1% ?5.6% and 7.5% ?2.3% at 8 h, 24.2% ?3.7% and 8.2% ?2.5% at 24 h, and 28.7% ?3.7% and 7.6%?2.5% at 36 h after treatment,respectively. RelA expression was decreased in PC-3-control cells treated with MG-132 while that in PC-3-siMaspin cells stayed unchanged. CONCLUSION: Maspin expression is increased in androgen-independent prostate cancer PC-3 cells. Maspin silencing significantly reduces the doubling time and accelerates the cell growth. Maspin silencing markedly reduces the sensitivity of PC-3 cells to proteasome inhibitor, which may be linked to the abolishment of RelA degradation.     

Effect of silencing hHBrk1 on proliferation and migration of lung carcinoma cells

AIM: To observe the effect of hHBrk1 gene on proliferation and migration of lung carcinoma cells. METHODS: Recombinant plasmids harboring 19-nt-long small interfering RNA (siRNA) were constructed and tested to selectively downregulate hHBrk1 gene in human lung cancer 95D cell line in vitro by stable transfection with Lipofectamine 2000. The mRNA level of the cells transfected with siRNA plasmids were monitored by Northern blotting and RT-PCR. Growth curve and flow cytometry were applied to determine the cell proliferation and cell cycle. Ability of cell migration was measured by Trans-well system. RESULTS: hHBrk1 gene was silenced by targeting siRNA, and stable silencing cell model was constructed. No difference in proliferation and clone formation between hHBrk1 silencing cells and control cells was observed. The ability of migration was decreased in hHBrk1 silencing cells as compared with control cells. CONCLUSION: hHBrk1 may play an important role in migration of the lung cancer cells.     

Effect of human xeroderma pigmentosum D on expression of Mdm2 and Mdm4 in HepG2 cells

AIM：To investigate the effects of human xeroderma pigmentosum D (XPD) on the expression of murine double minute 2 (Mdm2) and murine double minute 4 (Mdm4) in human hepatoma cells. METHODS：Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into HepG2 cells using liposome, and the cells were divided into blank control group, pEGFP-N2 group and pEGFP-N2/XPD group. The cell growth was detected by MTT assay. The cell cycle and apoptotic rate were examined by flow cytometry. The mRNA and protein expression levels of XPD, Mdm2, Mdm4 and P53 were determined by RT-PCR and Western blotting. RESULTS：The results of MTT assay showed that the cell growth was inhibited by the transfection of pEGFP-N2/XPD. The results of flow cytometry showed that the transfection of pEGFP-N2/XPD increased the cell number in G 1 phase, decreased the cell number in S phase and increased the apoptotic rate of HepG2 cells. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, decreased the expression of Mdm2 and Mdm4, and increased the expression of P53. CONCLUSION：XPD down-regulates Mdm2 and Mdm4 expression and up-regulates P53 expression in hepatoma cells. Moreover, the proliferation of hepatoma cells can be inhibited and the apoptosis can be induced by XPD.     

LRRK2/NF-κB signaling modulates inflammatory responses in BCG-infected RAW264.7 macrophages

AIM: To investigate the biological function and potential mechanism of leucine-rich repeat kinase 2 (LRRK2) in RAW264.7 macrophages during Mycobacterium tuberculosis infection. METHODS: The bacillus Calmette-Guerin (BCG)-infected RAW264.7 cell model was established. Colony-forming unit (CFU) analysis was used to determine the mycobacterial viability. The releases of interleukin (IL)-1β, IL-6 and interferon-γ (IFN-γ) in the RAW264.7 cells were detected by ELISA. qPCR and Western blot were used to measure the mRNA and protein expression levels, respectively. RESULTS: LRRK2 was robustly enhanced in the RAW264.7 cells in response to BCG infection. Additionally, silencing of LRRK2 suppressed intracellular growth of mycobacteria during BCG challenge. Moreover, silencing of LRRK2 dramatically attenuated the accumulation of inflammatory cytokines IL-1β, IL-6 and IFN-γ induced by BCG infection. More importantly, LRRK2 modulated BCG-induced inflammatory responses by positively regulating the nuclear factor-κB (NF-κB) signaling pathway. CONCLUSION: LRRK2/NF-κB signaling pathway positively modulates inflammatory responses during BCG infection, which may provide a better understanding of the pathogenesis of tuberculosis and useful information for developing potential therapeutic interventions against the disease.