Breeding of APPSWE transgenic mice and gene identification of filial generation

AIM: To explore the ideal breeding way of APPSWE transgenic mouse (Tg2576) and identify filial generation mice with APPSWE gene for laying a foundation of AD research. METHODS: (1) Three different copulation ways were adopted to observe the survival rate and positive rate with APPSWE gene of filial generation mice. (2) The genome DNA was extracted from the tails of filial generation mice and PCR method was employed to amplify the APPSWE gene fragment. The gene type was observed by electrophoresis.(3) PCR product was inserted in pGEM-T vector to detect its gene sequence. RESULTS: The male Tg2576 mice mated with female Tg2576, and four litters were reproduced, all of them died during two days. The male Tg2576 mice mated with female C57BL, and the survival rate of their filial generation was 81% and positive rate with APPSWE gene was 43.3%. The male C57BL mice mated with the female Tg2576,and the survival rate and positive rate with APPSWE gene of filial generation mice were 82.4% and 21.9%, respectively. By Wilcoxon's Rank Sum Test, there were no significant different between the survival rates of filial generation mice by two breeding way (P＞0.05), but there were significant different between the positive rates with APPSWE gene (P＜0.05). Electrophoresis result showed the molecular weight of PCR product was 428 bp and was in accordance with that of APPSWE gene fragment. By gene sequencing, gene sequence of PCR product was verified as same as APPSWE gene double mutant. CONCLUSION: It is ideal way of breeding filial generation mice with APPSWE gene that male Tg2576 mouse mate with female C57BL. PCR technique can identify the filial generation with APPSWE gene precisely, and offer an ideal animal model for further research on AD.     

Proctective effect of microRNA-30a on regulating beclin-1 expression in hypoxia-reoxygenated neonatal rat cardiomyocytes

AIM: To explore the potential mechanism of microRNA-30a (miR-30a) overexpression in neonatal rat cardiomyocytes during hypoxia/reoxygenation (H/R). METHODS: The miR-30a overexpression was induced in primary neonatal rat cardiomyocytes by lentivirus transfection. The cardiomyocytes were divided into 5 groups: normal group, H/R group, LV-GFP+H/R group, LV-GFP-miR-30a+H/R group and 3-methyladenine(3-MA)+H/R group. The expression level of miR-30a after lentivirus transfection and H/R was determined by real-time PCR, while the protein levels of LC3 and Beclin-1 after H/R and lentivirus transfection were detected by Western blotting. The cardiomyocyte death after H/R were measured by TUNEL and PI staining. RESULTS: Compared with LV-GFP group, significant down-regulation of Beclin-1 protein level was observed in cardiomyocytes with miR-30a overexpression, while the protein levels of Beclin-1 and LC3 in the cardiomyocytes with miR-30a overexpression were down-regulated after H/R, and apoptosis of these cells were significantly decreased after H/R. CONCLUSION: The protein level of Beclin-1 is down-regulated in cardiomyocytes with miR-30a overexpression. Inhibition of autophagy decreases the cardiomyocyte death after H/R.     

The construction of lentivirus-mediated RNAi vector containing cytochrome C oxidase

AIM: To construct a recombinant lentivirus RNAi vector carrying cytochrome C oxidase gene to obtain the titer of the lentiviral stock for investigation of the expression in the eukaryotic cell and the affection of the COX gene silencing in the eukaryotic cells. METHODS: According to the DNA of the cytochrome C oxidase gene, we designed and synthesized complementary single-strand DNA oligos, annealed the single-stranded oligos to generate a ds oligo, cloned the ds oligo into pENTR/U6 to obtain an entry clone; An LR recombination reaction was performed between the pENTR/U6 entry construct and pLenti6/BLOCK-iT-Dest to generate expression construct, the 293FT cell line was cotransfected with pLenti6/BLOCK-iT expression construct, and the viral packaging mix, viral supernatant was harvested to determine the titer. RESULTS: The DNA sequence of interest clone to the vector was constructd to generate an entry clone and an expression clone successfully, which were proved by sequence determination. A vector producing cell line 293FT was established， and the titer for transfection was obtained. Western blotting analysis demonstrated that COX shRNA expression construction could suppress the expression of MTCOX-I. CONCLUSION: A lentivirus RNAi vector containing cytochrome C oxidase gene was successfully constructed.     

Overexpression of SHP-1 mediated by lentivirus restrains atherosclerotic progression in mice

AIM: To investigate the role of SH2-domain-containing protein-tyrosine phosphatase-1 (SHP-1) lentivirus in atherosclerotic mice. METHODS: ApoE knock-out mice were randomly assigned to 3 groups: control group, GFP transfection group and SHP-1 transfection group. All mice were placed with carotid collars on the right common carotid arteries near its bifurcation, following feeding with high-fat diet for 8 weeks and then transfected with GFP blank vector or SHP-1 lentivirus (SHP-1-LV). The fluorescence density of the plaques, body weight, the levels of plasma total cholesterol (TC) and triglyceride (TG) were determined at 1st, 2nd, and 6th week after lentivirus transfection. Furthermore, the mRNA and protein expression of SHP-1, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP)-2 and MMP-9 were analyzed by real-time PCR and Western blot. Additionally, pathological analysis of the plaques was also performed by HE and oil red O staining. RESULTS: The fluorescence of the plaques was observed at 1st, 2nd, and 6th week after lentivirus transfection, with a highest density at 2nd week. The body weight and the levels of TC and TG in the mice were not influenced by lentivirus transfection. Moreover, SHP-1-LV transfection significantly upregulated the expression of SHP-1 at mRNA and protein levels, but inhibited the expression of IL-6, TNF-α, MMP-2 and MMP-9. In addition, SHP-1-LV transfection also decreased the plaque size ratio and lipid content in right common carotid arteries. CONCLUSION: SHP-1 overexpression accelerates the regression of atherosclerotic plaque, thus emerging SHP-1 as a target for prevention and treatment of atherosclerosis.     

Neuroprotective effect of astrocyte protein phosphatase 2A up-regulation on APP/PS1 double transgenic mice

AIM: To investigate the protective effects of astrocyte protein phosphatase 2A(PP2A) up-regulation on APP/PS1 double transgenic mice.METHODS: An eGFP-wtPP2A lentivirus with glial fiber acidic protein promoter was constructed to specifically increase PP2A expression in the astrocytes. The mice were divided into wild -type mice+vector virus group(Con), APP/PS1 transgenic mice+vector virus group(APP/PS1) and APP/PS1 transgenic mice+eGFP-wtPP2A lentivirus group(PP2A) by lateral ventricular injection of the lentivirus. Four weeks after injection of the virus, the immunofluorescence of brain slices were used to detect the level of β-amyloid protein(Aβ). Golgi staining was used to detect the changes of dendritic spine density and morphology. Electron microscopy was applied to detect the thickness of postsynaptic density(PSD). The Morris water maze test was applied to examine the learning and memory abilities of the mice.RESULTS: Up-regulation of PP2A in the astrocytes attenuated Aβ level increasing in APP/PS1 group. Up-regulation of PP2A in the astrocytes significantly attenuated both decreases in the dendritic spine density and the percentage of mushroom-like dendritic spines in the hippocampal CA3 region of APP/PS1 mice. Up-regulation of PP2A in the astrocytes significantly attenuated the reduced thickness of PSD in APP/PS1 group. Up-regulation of PP2A in the astrocytes attenuated the escape latency extending in APP/PS1 group.CONCLUSION: Up-regulation of PP2A in the astrocytes reduces AD-like pathological changes, and attenuates synaptic impairment, synaptic plasticity deficits and cognitive impairment in the APP/PS1 double transgenic mice.     

Molecular mechanism of BMSC intracerebral transplantation in improving learning and memory abilities of AD mice

AIM: To investigate the effect of bone marrow mesenchymal stem cell (BMSC) transplantation on learning and memory abilities and pathological changes of Alzheimer disease (AD) mice and the molecular mechanisms. METHODS: C57/BL6 wild-type (WT) and transgenic (Tg) mice were randomly divided into 4 groups:WT/PBS group, WT/BMSCs group, Tg/PBS group and Tg/BMSCs group. The mice were administered with PBS or BMSCs via intracerebroventricular injection. Spatial learning and memory abilities of the mice were evaluated by Morris water maze test on the 3rd day after surgery. Real-time PCR was applied to detect the mRNA expression of CX3C chemokine ligand 1 (CX3CL1), CX3C chemokine receptor 1 (CX3CR1), IL-1β, TNF-α, Nurr1, YM1, insulin-degrading enzyme (IDE) and matrix metalloproteinase 9 (MMP9). The protein levels of CX3CL1 and Aβ42 were measured by ELISA. Western blot was used to detect the protein expression of postsynaptic density protein 95 (PSD95) and synaptophysin (SYP). RESULTS: The transplanted BMSCs were observed near the hippocampus of APP/PS1 mice on the 10th postoperative day. The escape latency of the mice in Tg/PBS group was significantly longer than that in the WT/PBS mice (P<0.05). Compared with Tg/PBS group, the escape latency of Tg/BMSCs group was significantly shorter (P<0.05), and the mRNA and protein levels of CX3CL1 in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.01). The results of immunohistofluorescence staining showed that BMSC transplantation promoted the activation of microglia in the brain of WT and Tg mice. The mRNA expression of YM1 was up-regulated in WT/BMSCs group and Tg/BMSCs group (P<0.05). Compared with WT/PBS mice, the mRNA expression of TNF-α in the cortex and hippocampus of Tg/PBS group was significantly increased (P<0.05), and the mRNA expression of Nurr1 in the cortex was significantly decreased (P<0.01). Meanwhile, the mRNA expression of TNF-α in the cortex of Tg/BMSCs mice was decreased (P<0.01) and the mRNA expression of CX3CR1 and Nurr1 was up-regulated compared with Tg/PBS group (P<0.05). The results of Western blot showed that the protein levels of PSD95, p85, p110 and p-Akt in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.05). Finally, BMSC transplantation reduced the protein level of Aβ42 in APP/PS1 mice (P<0.05), and increased the mRNA expression of IDE and MMP9 in the hippocampus (P<0.05). CONCLUSION: BMSC transplantation modulates neuroinflammatory responses and promotes neuroprotective factor and synaptic protein expression, thus improving the learning and memory abilities in the APP/PS1 mice, which may be achieved by up-regulating the expression of CX3CL1.     

Hsa-miR-218 inhibits growth of cervical cancer HeLa cells by targeting to LASP1

AIM: To study the effect of hsa-miR-218 on cervical cancer HeLa cell growth and the underlying molecular mechanism.METHODS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed. pmiR-218 was transfected into HeLa cells. The number of viable HeLa cells was counted by the method of Trypan blue exclusion. The inhibitory rate of cell activity was detected by WST-8 assay. The expression of LIM and SH3 protein 1(LASP1) at mRNA and protein levels was determined by real-time PCR and Western blot. The interaction between miR-218 and LASP1 was examined using a luciferase reporter assay.RESULTS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed successfully and confirmed by DNA sequencing. Over-expression of miR-218 inhibited the activity of HeLa cells with the inhibitory rates of 15%, 26% and 65% at 24 h, 48 h and 72 h, respectively. The difference between transfection group and blank control/negative control group was statistically significant. The luciferase activity was reduced when co-transfection with miR-218 mimics and LASP1-3,UTR plasmid. The relative expression of miR-218 was increased after transfection with pmiR-218. Over-expression of miR-218 down-regulated the LASP1 expression at mRNA and protein levels by 25% and 75% respectively. Compared with blank control group and negative control group, the difference was statistically significant(P<0.05).CONCLUSION: pmiR-218 effectively inhibits the growth of HeLa cells in a time-dependent manner. miR-218 targets to the 3,UTR of LASP1, thus down-regulating the expression of LASP1 in HeLa cells.     

Expression of α-synuclein with aging in APP transgenic model of Alzheimers disease

AIM: To investigate the expression of α-synuclein in the brain of AD-like animal model at different age and to explore the pathology mechanism of α-synuclein in neural degeneration.METHODS: APP V717I transgenic (Tg) mouse model of Alzheimers disease was observed at age of 4,10 and 16 months.The Tg mice were randomly divided into 3 model groups (4,10 and 16 months of age).Control adopted the same age and background C57BL/6J mice.The mRNA expression of α-synuclein was measured by genechips and RT-PCR method.The protein of α-synuclein was detected by immuno-histochemistry and Western blotting.RESULTS: The expression of α-synuclein mRNA increased significantly in hippocampus and cortex in Tg mice at age of 4 months,10 months and 16 months compared with age matched controls.The production of α-synuclein protein also increased significantly in hippocampus and cortex in Tg mice at 3 groups.With aging,α-synuclein expression was increased and aggregated in Tg mice.CONCLUSION: Overexpression and aggregation of α-synuclein in different age of APP transgenic mice may play a key role in learning-memory deficit and the pathology of Alzheimers disease,aging,and neural degeneration.     

Effect of BDNF expression in cerebral cortex and hippocampus on ability of learning and memory in APP/PS1 transgenic mice

AIM: To investigate the expression changes of brain-derived neurotrophic factor (BDNF) in the cerebral cortex and hippocampus and their effects on the ability of learning and memory in the wild-type (WT) mice and APP/PS1 transgenic mice. METHODS: WT mice and APP/PS1 transgenic mice were selected as study subjects. Aβ plaques, apoptosis rate and BDNF expression in the cerebral cortex and hippocampus of WT mice and APP/PS1 transgenic mice were detected by the methods of Congo red staining, TUNEL, immunofluorescence and Western blot. The abilities of learning and memory were determined by Morris water maze test. RESULTS: The Aβ plaques appeared in the cerebral cortex and hippocampus of APP/PS1 transgenic mice, and the number of Aβ plaques in 12-month-old mice was larger than that in 6-month-old mice (P<0.05). The number of apoptotic neurons in the cerebral cortex and hippocampus of 12-month-old APP/PS1 transgenic mice was larger than that of WT mice (P<0.01). The expression level of BDNF in the cerebral cortex and hippocampus of WT mice was higher than that of APP/PS1 transgenic mice (P<0.01). The Morris water maze test showed that the escape latency in APP/PS1 transgenic mice was longer than that in WT mice, and the times across the platform quadrant in 60 s was less than that in WT mice (P<0.01). The swim-tracking path of APP/PS1 transgenic mice was disordered and irregular. CONCLUSION: The expression of BDNF in the cerebral cortex and hippocampus of APP/PS1 transgenic mice was lower than that of WT mice, accompanied by increased neuronal apoptosis and decreased spatial learning and memory ability. The decrease in learning and memory ability may be related to decreased BDNF expression in the cerebral cortex and hippocampus of APP/PS1 transgenic mice, leading to increased neuronal apoptosis, which may be one of the pathological mechanisms of Alzheimer disease.     

Effect of microRNA-132 transfection on lipopolysaccharide-induced inflammation in rat alveolar macrophages

AIM: To investigate the effect of microRNA-132 (miR-132) transfection on the lipopolysaccharide (LPS)-induced inflammation in rat alveolar macrophages. METHODS: The rat alveolar macrophage NR8383 cultured without pyrogen in vitrowere divided into blank control group, negative control group and transfected group. The cells in the 3 groups were transfected with phosphate buffer solution (PBS), Lipofectamine 2000 and synthesized miR-132 mimic respectively. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Real-time PCR was used to detect the expression of miR-132 in the cells. After NR8383 cells were stimulated with LPS for 6 h, the NF-κB DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA). The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in NR8383 cells was assayed by Western blotting.RESULTS: After transfection, the expression of miR-132 was significantly higher than that in blank control group and negative control group. The growth of NR8383 cells in transfected group was significantly inhibited compared with blank control group and negative control group (P<0.05). After the cells were stimulated with LPS, the productions of NF-κB, TNF-α and IL-6 in transfected NR8383 cells were decreased compared with blank control group and negative control group (P<0.05).CONCLUSION: Transfection of alveolar macrophages with miR-132 significantly suppresses the cell growth, and inhibits inflammatory responses induced by LPS.     

Effect of lentivirus-mediated DKK3 overexpression on apoptosis of hypertrophic scar fibroblasts

AIM:To investigate the effect of lentivirus-mediated DKK3 overexpression on the apoptosis of hypertrophic scar fibroblasts. METHODS:Human hypertrophic scar fibroblasts were isolated and cultured in vitro. The cells were divided into control group, vector (negative control lentivirus infection) group and DKK3 (pcDNA3.1-DKK3 lentivirus infection) group. The overexpression effect was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein levels of cleaved caspase-9, collagen type Ⅱ (COL Ⅱ), COL I and cleaved caspase-3 in the cells, and cytochrome C in the cytoplasm and mitochondrion were detected by Western blot. RESULTS:After transfection with pcDNA3.1-DKK3, the expression of DKK3 at mRNA and protein levels was increased in the hypertrophic scar fibroblasts (P<0.05). The viability of hypertrophic scar fibroblasts in DKK3 group was decreased, and the apoptotic rate was increased. The protein levels of cleaved caspase-9 and cleaved caspase-3 were increased in the cells, and the protein levels of COL Ⅱ and COL I were decreased. The protein level of cytochrome C was increased in the cytoplasm, while the protein level of cytochrome C in the mitochondrion decreased. Compared with vector group, these differences were statistically significant (P<0.05). CONCLUSION:Lentivirus-mediated DKK3 overexpression induces apoptosis and reduces collagen synthesis in the fibroblasts from hypertrophic scars.     

miR-21 enhances resistance of glioma cells to carmustine by down-re-gulating PTEN

AIM:To explore the function of miR-21 in human glioma cells resistant to carmustine and to elucidate its related mechanism. METHODS:SWOZ2 cells were transfected with miR-21 mimics(SWOZ2-miR-21mimics) or miRNA mimics negative control(control group) by the method of jetPRIME. The real-time fluorescence quantitative PCR was used to detect and compare the levels of miR-21 expression between BCNU-resistant cell line SWOZ2-BCNU and BCNU-sensitive cell line SWOZ2, or between SWOZ2-miR-21 mimic group and control group. The drug sensitivity of these cells to BCNU was determined by CCK-8 assay. The protein expression of phosphatase and tensin homology deleted on chromosome 10(PTEN), phosphorylated protein kinase B(p-Akt) and P-glycoprotein(P-gp) in these cells were also detected by Western blotting. RESULTS:The expression level of miR-21 was remarkably higher in SWOZ2-BCNU cells than that in SWOZ2 cells. The expression level of miR-21 was significantly higher in SWOZ2-miR-21 mimics group than that in control group. The half-maximal inhibitory concentration(IC₅₀) of BCNU was obviously higher for SWOZ2-BCNU cells than that for SWOZ2 cells. The IC₅₀ of BCNU was markedly higher in SWOZ2-miR-21 mimics group than that in control group. PTEN protein expression was remarkably lower, but p-Akt and P-gp protein expression levels were markedly higher in SWOZ2-BCNU cells than those in SWOZ2 cells. The protein level of PTEN was significantly lower, but the protein levels of p-Akt or P-gp were distinctly higher in SWOZ2-miR-21 mimics group than those in control group. CONCLUSION:miR-21 enhances the resistance of human glioma cells to BCNU by down-regulating the expression of PTEN protein.     

miR-496 over-expression inhibits growth and metastasis in colon cancer cells

AIM: To investigate the effect of miR-496 over-expression on the growth and metastasis of colon cancer cells and its molecular mechanism.METHODS: The proteins interacting with miR-496 were screened by bioinformatic method. The levels of miR-496, CTNNB1 mRNA and β-catenin protein in colon cancer cell lines, HT29, HCT116 and SW480, and normal colonic epithelial cell line NCM460 were detected by real-time PCR and Western blot. HT29, HCT116 and SW480 cells were transfected with miR-496 mimics using Lipofectamine 2000 and named as HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimics cells, respectively, and the cells transfected with the scramble served as negative control. The cell viability, lactate dehydrogenase (LDH) leakage, and colony formation and metastatic abilities were determined by MTT assay, LDH assay, colony formation assay and Transwell method, respectively. The promoter activity of miR-496 was measured using luciferase reporter gene assay. The protein levels of β-catenin, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p-4E-BP1, low-density lipoprotein receptor-related protein 6(LRP6), p-LRP6, MMP-7, MMP-9, MMP-13 and TIMP-2 were monitored by Western blot.RESULTS: Endogenous miR-406 interacted with β-catenin was found in the colon cancer cells. Low miR-496 expression in the HT29, HCT116 and SW480 cells and high miR-496 expression in NCM460 cells were detected. In contrast, high β-catenin expression was found in the HT29, HCT116 and SW480 cells and low β-catenin expression was observed in the NCM460 cells. Compared with control group, the cell viability, colony formation rate and the number of metastatic cells remarkably decreased in the HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimic cells (P<0.05). The promoter activity of miR-496 was significantly increased in colon cancer cells transfected with miR-496 mimics, and was 1.75, 2.04 and 1.61 times as high as control group. miR-496 over-expression inhibited β-catenin levels, and p-4E-BP1 and p-LRP6 protein levels were also reduced. siRNA- or over-expressed miR-496-mediated β-catenin down-regulation inhibited MMP-7 and MMP-9 expression, but promoted TIMP-2 expression.CONCLUSION: The expression level of miR-496 in the colon cancer cells is low, but in the normal colonic epithelial cells is high. miR-496 over-expression inhibits the protein levels of MMP-7 and MMP-9, and promotes the protein expression of TIMP-2 via inhibiting Wnt/β-catenin pathway, thus suppressing malignant phenotype in the colon cancer cells.     

miR-193b enhances cytotoxicity of doxorubicin by targeting Mcl-1 in breast cancer

AIM: To investigate the effect of microRNA(miR)-193b on doxorubicin therapy in breast cancer in vitro.METHODS: miR-193b level in plasma was detected by real-time PCR in the patients with breast cancer or the healthy controls. MTT assay was performed to measure the inhibitory effect of miR-193b plus doxorubicin on the growth of MDA-MB-231 cells. Bioinformatics, real-time PCR and Western blot were performed to determine whether the expression of Mcl-1 was regulated by miR-193b. Mcl-1 expression vector was constructed, and the role of Mcl-1 vector toward miR-193b plus doxorubicin-induced cytotoxicity in MDA-MB-231 cells was observed by MTT assay.RESULTS: Down-regulation of miR-193b was found in breast cancer patients. The miR-193b plus doxorubicin group showed a higher growth inhibition than cisplation group in MDA-MB-231 cells. The expression of Mcl-1 at both mRNA and protein levels was down-regulated after miR-193b transfection. The growth inhibition of MDA-MB-231 cells treated with miR-193b plus doxorubicin was significantly decreased after the transfection of Mcl-1 expression vector.CONCLUSION: miR-193b sensitizes doxorubicin-induced cytotoxicity by targeting Mcl-1 in breast cancer.     

Effect of Kangshuaiyizhi formula I on learning and memory behavior and expression of ChAT in hippocampus of mice

AIM: To evaluate the role of kangshuaiyizhi formula I (KYF I, a Chinese medicine) on improving the ability of learning and memory in mice and to explore the mechanism related to choline acetyltransferase (ChAT) in hippocampus. METHODS: The mice were divided into 4 groups by gastragavege of distilled water, low-, middle-, and high-doses of KYF I, respectively for 40 successive days. Morris water maze test was used to assess the behavior performance of the mice with different treatment. The activities of AChE and ChAT in hippocampus were measured and the expression of ChAT in hippocampus was observed by immunohistochemistry and Western blotting after treatment. RESULTS: Compared to control group, the mice in high-dose KYF I group showed better performance in Morris water maze test (P<0.05). The activity of ChAT increased (P<0.01) and the activity of AChE decreased in high-dose KYF I treated mice. Moreover, the number of ChAT positive neurons and the expression of ChAT increased significantly in the hippocampus of high-dose KYF I treated animals (P<0.05). CONCLUSION: High-dose KYF I improves the ability of learning and memory in mice. The results suggest that KYF I promotes the level of ACh in brain by inhibiting the activity of AChE, enhancing the activity of ChAT and upregulating the expression of ChAT in hippocampus.     

Non-viral vector-mediated high efficiency expression of manganese superoxide dismutase gene in mouse liver

AIM: To investigate the transfection efficiency of mouse liver with non-viral vector containing manganese superoxide dismutase (Mn-SOD) gene. METHODS: The eukaryotic expression vector, gWiz/Mn-SOD, encoding human manganese superoxide dismutase was constructed. The plasmids of gWiz/Mn-SOD were mixed with cationic lipids, followed by injection into mice via branch of superior mesenteric vein, to induce Mn-SOD over-expression in murine liver detected by RT-PCR, Western blotting, SOD activity and immunohistochemical staining. RESULTS: gWiz/Mn-SOD transfection resulted in the obvious expression of exogenous Mn-SOD mRNA and protein in hepatic tissues at 8 hours after injection, and elevated mitochondria SOD activity 8.4 times in transfected hepatocytes than that in non-transfected cells at 72 hours after injection. It was showed that nearly 70% of mouse hepatocytes was obviously Mn-SOD positive after transfection. CONCLUSION: High expression efficiency of Mn-SOD gene in mouse liver is achieved safely, by injection of gWiz/Mn-SOD and cationic lipid mixture into branch of superior mesenteric vein.     

Influences of Vit-E on the mtDNA damage and Ca2+ homostasis of hippocampus and antioxidative ability in brain of aging mice induced by D-galactose

AIM: To study the influences of vitamin E (Vit-E) on the mtDNA damage and Ca²⁺ homeostasis in hippocampus and antioxidative ability in aging brain induced by D-galactose.METHODS: D-galactose (1 000 mg·k^-1·d^-1 ) was injected into mice hypodermically for 8 weeks to induce aging animal model, and Vit-E (100 mg·kg^-1; 250 mg·kg^-1) was administered for 6 weeks by ig at the 3rd week of making model. After Vit-E treatment for 8 weeks, water maze test was used to determine the ability of mice’s learning and memory. The activities of glutathione peroxidase (GSH-Px) and succinate dehydrogenase (SDH), the content of nitric oxide (NO) and activity of nitric oxide synthase (NOS) in the brain tissue were detected separately. Fura-2/AM, double-wave-length fluorospectrophotometer and PCR method were used to measure the concentration of calcium ion and mtDNA mutation in the hippocampus cells.RESULTS: Administration of Vit-E improved significantly the ability of learning and memory in model mice, inhibited the activity of NOS and decreased the amount of NO, and increased the activities of GSH-Px and SDH respectively in brain tissues, decreased the concentration of calcium ion (P<0.01, P<0.05), and prevented the damage of mtDNA in hippocampus.CONCLUSION: Vit-E can enhance the antioxidative ability, regulate the homeostasis of Ca²⁺ and inhibit the damage of mtDNA caused by oxidative stress in aging brain, and improve the ability of learning and memory in aging mice.     

miR-105 inhibits cell proliferation,migration and invasion abilities in non-small-cell lung cancer cells by targeting and negative regulation of KIFC1

AIM:To study the effects of microRNA-105(miR-105) on the cell proliferation, migration and invasion abilities of non-small-cell lung cancer (NSCLC) H460 cells, and further to explore its mechanism. METHODS:The expression of miR-105 and kinesin family member C1 (KIFC1) mRNA in the NSCLC tissues and adjacent tissues and cells was detected by RT-qPCR. The protein expression of KIFC1 in the NSCLC tissues, adjacent normal tissues and cells was determined by Western blot. The H460 cells were divided into miR-105 group (transfection with miR-105 mimics), miR-negative control (NC) group (transfection with miR-NC), inhibitor-NC group (transfection with NC of inhibitor), inhibitor-miR-105 group (transfection with miR-105 inhibitor), si-NC group (transfection with NC siRNA), si-KIFC1 group (transfection with KIFC1 siRNA), miR-105+vector group (miR-105 mimics and pcDNA 3.1 co-transfection) and miR-105+KIFC1 group (miR-105 mimics and pcDNA 3.1-KIFC1 co-transfection). The cell proliferation was measured by MTT assay and colony formation assay. The migration and invasion abilities were detected by Transwell methods. The relative luciferase acitivity was evaluated by double luciferase reporter assay. RESULTS:Compared with the adjacent tissues, the expression of miR-105 was significantly decreased and the expression of KIFC1 was significantly increased in NSCLC tissues (P<0.05). Compared with human normal embryonic lung fibroblasts MRC-5, the expression of miR-105 in the H460 cells was significantly decreased, and the expression of KIFC1 was significantly increased (P<0.05). miR-105 inhibited the relative luciferase activity of H460 cells with wild-type KIFC1 and negatively regulated the protein expression of KIFC1. Over-expression of miR-105 and knockdown of KIFC1 expression significantly inhibited the proliferation, migration and invasion abilities of H460 cells. Over-expression of KIFC1 reversed the inhibitory effect of miR-105 on the cell proliferation, migration and invasion abilities of H460 cells. CONCLUSION:miR-105 inhibits the proliferation, migration and invasion abilities of NSCLC cells. The mechanism may be related to targeting and negatively regulating expression of KIFC1.     

Effect of curcumin on scopolamine-induced memory impairment in mice

AIM:Scopolamine blocks cholinergic transmission and impairs learning and memory in mice. The purpose of this study was to evaluate the memory-improving properties of curcumin on scopolamine-induced memory impairment in mice. METHODS:The mice of memory impairment were induced by scopolamine. Step down test and Morris water maze test were used to observe the learning and memory ability in curcumin-treated mice. Biochemical assessments of AChE, MDA, and GSH-Px levels in brains were performed. RESULTS:Oral administration of curcumin significantly reduced the numbers of step-down errors (P<0.05) and prolonged the step-down latency induced by scopolamine (P<0.05). The escape latency time in mice treated with curcumin was remarkably reduced compared to that in scopolamine group by Morris water maze test (P<0.05). After the platform was removed, the total time that the mice swam in the target quadrant was also longer in curcumin group than that in model group (P<0.05). The data also indicated that curcumin significantly inhibited AChE activity (P<0.01) and prevented oxidative stress characterized by the significant reduction in MDA content and the significant increase in GSH-Px activities in the brain (P<0.01). CONCLUSION:Curcumin induces cognitive improvement by enhancing the function of cholinergic system and its antioxidant activity.     

Construction of a lentiviral vector for RNA interference of human EGFL7 gene and its stable expression in human laryngeal carcinoma cells

AIM：To investigate the role of epidermal growth factor-like domain 7 (EGFL7) in the pathogenesis and progress of laryngeal carcinoma via constructing a lentiviral expression vector for RNA interference (RNAi) of human EGFL7 gene and assessing the gene-silencing effect of the vector in human laryngeal epidermoid carcinoma (HEp-2) cells. METHODS：Specific RNAi target sequences were designed focused on human EGFL7 gene sequence. The double-stranded oligonucleotides were cloned into the pcDNA6.2-GW/EmGFP-miR plasmid after synthesis and annealing. A positive clone was subcloned into the pLenti6.3-MCS/V5-DEST vector after sequence analysis. The recombinant lentivirus was harvested from 293T cells co-transfected with the positive recombinant plasmid and lentiviral packing materials. HEp-2 cells were infected with the recombinant lentivirus and the cells with stable EGFL7 knockdown were screened by blasticidin selection. EGFL7 mRNA expression in the cells was determined by real-time fluorescence quantitative PCR. RESULTS：A recombinant lentiviral vector expressing short hairpin RNAs (shRNAs) against EGFL7 gene was obtained and confirmed by DNA sequencing. The virus titer was 5×1011 TU/L, and the silencing efficiency was 97%. CONCLUSION：A lentiviral vector targeting human EGFL7 gene, capable of stable EGFL7 gene knockdown in HEp-2 cells, has been successfully constructed, which provides a basis for further study of the relationship between human laryngeal carcinoma and EGFL7 protein.