[Effect of Bmi-1 Expression on Chemotherapy Sensitivity in THP-1 Cells].

OBJECTIVE: To investigate the effect of Bmi-1 expression on the chemosensitivity of THP-1 cells and its relative mechanism. METHODS: The pGenesil-2-Bmi-1 1 siRNA, p-MSCV-Bmi-1 plasmid was transfected into THP-1 cells to reduce or increase the expression of Bmi-1. The expression of Bmi-1 mRNA and protein was verified by PCR and Western blot. The effect of camptothecin (CPT) on the proliferation and chemosensitivity of THP-1 cells affected by Bmi-1 gene were detected by MTT assay. The expression of DNA double-strand breaks marker-γ-H2AX was detected by immunofluorescence assay. Mitochondrial membrane potential and apoptosis were observed by flow cytometry. The expression of Cytochrome C, Caspase 3, Bax and BCL-2 was detected by Western blot. RESULTS: Silencing Bmi-1 could inhibit proliferation and enhance the sensitivity of THP-1 cells to CPT, while overexpressed Bmi-1 could promote the cell proliferation and attenucate sensitivity of THP-1 cells to CPT. Silencing Bmi-1 could enhance CPT-induced DNA double-strand breaks, decrease mitochondrial membrane potential and promote CPT-induced apoptosis. While increasing Bmi-1 gene expression could attenuate CPT-induced DNA double-strand breaks, enhamce mitochondrial membrane potential and significantly reduce CPT-induced apoptosis of cells. CONCLUSION: Bmi-1 expression could influence the sensitivity of THP-1 cells to CPT, and its relative mechanism may relate to DNA double-strand breaks and endogenous apoptotic pathways.

[Effect of Bmi-1 on Multidrug Resistance in K562/ADR Cells and Its Mechanisms].

OBJECTIVE: To investigate the effect of Bmi-1 gene silencing on drug resistance of leukemia cell K562/ADR and to explore its possible mechanism. METHODS: After two sequences of Bmi-1-siRNA were transfected into drug-resistant K562/ADR cells, the mRNA and protein expressions of Bmi-1 gene were detected. After Bmi-1 gene silencing the expression of P-gp and MDR1 were detected and the accumulation of doxorubicin in K562/ADR cells were detected by flow cytometry to determine the effect of Bmi-1 gene silencing on drug resistance of K562/ADR cells. The protein expression of NF-κB was analyzed after Bmi-1 gene silencing. Then after K562/ADR cells were treated with NF-κB inhibitor PDTC, the protein expression of P-gp and its functional changes were analyzed to determine the effect of NF-κB on drug resistance of leukemia cells. The protein expressions of PTEN, AKT and p-AKT after Bmi-1 gene silencing were detected and the effect of Bmi-1 gene silencing on PTEN/PI3K/AKT signaling pathway in drug-resistant cells was determined. After K562/ADR cells were treated with PI3K/AKT pathway inhibitor LY294002, the protein expressions of NF-κB and P-gp were analyzed to determine the regulation of AKT on the expression of NF-κB and P-gp. The protein expressions of AKT, p-AKT, NF-κB and P-gp were detected after the Bmi-1-siRNA transfected cells were treated by PTEN inhibitor BPV. Above-mentioned expression of mRNA was detected by RT-PCR, and the protein expression was detected by Western blot. RESULTS: The expression of Bmi-1 gene in K562/ADR cells decreased at both mRNA and protein levels and the doxorubicin accumulation increased after Bmi-1 gene silencing. The expression of MDR1/P-gp in Bmi-1-siRNA transfected cells was lower than that in K562/ADR cells (P＜0.05). After Bmi-1 gene silencing, the activity of NF-κB decreased. The activity of NF-κB and P-gp expression was inhibited and the function of P-gp in K562/ADR cells was reduced by using NF-κB inhibitor (PDTC). The protein expression of PTEN increased while the protein expression of p-AKT decreased after Bmi-1 gene silencing (P＜0.05). The protein expressions of p-AKT, P-gp and the activity of NF-κB were inhibited significantly by using PI3K/AKT inhibitor LY294002 (P＜0.05). After the Bmi-1-siRNA transfected cells were treated by PTEN inhibitor BPV, the activity of NF-κB and the protein expressions of P-gp were restored. CONCLUSION: Bmi-1 plays a key role in MDR-mediated multidrug resistance in K562/ADR cells, which may be mediated by activating PTEN/AKT pathway to regulate NF-κB.

[Effect of Bmi-1 Silencing on the Proliferation of K562/ADM Cells in vitro and in vivo].

OBJECTIVE: To investigate the effect of Bmi-1 gene silencing on the proliferation of K562/ADM cells in vitro and in vivo and to explore its possible molecular mechanism. METHODS: The small interference RNA (siRNA) sequences of Bmi-1 were transfected into K562/ADM cells for decreasing the expression of Bmi-1. The effect of Bmi-1 silencing on the proliferation of K562/ADM cells in vitro and in vivo was detected by using MTT method, cell colony-forming test and tumoriganicity of nude mice; the expression of Bmi-1, PTEN and PAKT proteins was detected by Wertern blot. The immunohistochemistry assay was used to analyze the expression of Bmi-1 and Ki-67. RESULTS: The Bmi-1 silencing could significantly inhibit the proliferation activity of K562/ADM cells, the cell colony-forming ability and tumorigenicity were reduced. LY294002 decreased p-AKT expression, cell colony-forming ability and tumorigenicity. Bpv reduced the PTEN expression but increased p-AKT expression and restored the colony-forming ability and tumorigenesis of K562/ADM-S1-Bpv cells. Bmi-1-siRNA dramatically suppressed the Bmi-1 and Ki-67 protein levels in xenograft tumor tissue. CONCLUSION: Bmi-1 can mediate the proliferation of K562/ADM cell, the PTEN/p-AKT might be involved in this pathway.

[Effects of antisense Bmi-1 RNA on the proliferation of lung cancer cell line A549].

OBJECTIVE: To study the effects of antisense Bmi-1 (B cell-specific moloney murine leukemia virus insertion site 1) RNA on the growth, cell cycle and apoptosis of lung cancer cell line A549. METHODS: Recombinant plasmids carrying antisense Bmi-1 RNA were transfected into A549 cells, which expressed a high level of endogenous Bmi-1. The mRNA level of A549 cell was analyzed by real time quantitative RT-PCR and the protein level was determined using Western blot. MTT growth curve and plate colony forming assay were used to measure the effect of antisense Bmi-1 RNA expression on the growth of A549. Flow cytometry was used to analyze cell cycle and apoptosis. RESULTS: Antisense Bmi-1 RNA reduced the Bmi-1 expression at the protein level, but did not alter the mRNA level in A549 cells. Compared with the control cells, A549 cells transfected with antisense Bmi-1 RNA showed a strong inhibition of the cell growth. The number of plate colony formation of the antisense Bmi-1 transfected cells (0.67 +/- 0.50) was less than those of the control (73.0 +/- 4.1) and cells transfected with empty vector (67.0 +/- 4.0, P < 0.01). Transfection of antisense Bmi-1 RNA arrested the A549 cells at G0/G1 phase of the cell cycle and did not increase the apoptosis. CONCLUSION: Antisense Bmi-1 RNA expression inhibits A549 cells proliferation, likely through the interference of Bmi-1 leading to an arrest of the proliferating cells at the G0/G1 phase.

[Boldness of the alpine musk deer in captivity during non-mating season and its relationships with the musk secretion and reproduction succes]. / åœˆå »é©¬éºéžç¹æ®–å­£èŠ‚èƒ†é‡æ°´å¹³åŠä¸Žéºé¦™åˆ†æ³Œå’Œç¹æ®–æˆæ•ˆçš„å ³ç³».

This study was conducted in 2017 from July 1st to August 31st at Xinglongshan musk deer farm in the Xinglongshan National Nature Reserve of Gansu Province, where we recorded behaviors and locations of 29 captive musk deer using the integrated methods of focal sampling and all occurrence recording. Based on the location selection patterns under artificial stress, we defined the animal's stress level and quantified individuals' boldness by combination with the occupation time ratio at each level. Then, the effects of gender, age-class, and other factors on boldness were analyzed along with musk secretion and reproduction success. The results showed that musk deer in better health (1.731±0.347) were significantly bolder than those in ill health (0.915±0.789). Female musk deer (1.901±0.391) were significantly bolder than the males (1.035±0.120). The boldness of sub-adults (1.450±0.463) was higher than those of the adults (1.093±0.321) and the old (1.430±0.189). No significant difference in boldness was observed among three age-classes under the same gender. Deer living in groups (1.397±0.179) were not significantly bolder than those living alone (0.853±0.326). More individuals in groups, less boldness. Furthermore, there was negative correlation between male's boldness and musk secretion. The litter size had positive relationship with boldness. The non-pregnant percentage was strongly correlated to the boldness, namely the bolder females had lower non-pregnant ratio. Our results provide reference for forecasting the musk secretion and reproduction success of captive musk deer, and provide new ideas for the study of boldness in captive animals.

[Bmi-1-siRNA Regulates the Proliferation of K562 Leukemia Cells in vitro and in vivo by PTEN/pAKT Pathway].

OBJECTIVE: To investigate the effect of Bmi-1 gene silence on the proliferation ability of K562 cells in vitro and in vivo, and to explore the relation of molecular mechanism between proliferation ability of K562 cells in vitro and in vivo with PTEN/pAKT signaling pathway. METHODS: The Bmi-1 small interference RNA (siRNA) sequences were transfected into K562 cells for decreasing Bmi-1 expression. The effect of Bmi-1 siRNA on the proliferation of K562 cells in vitro and in vivo was detected by MTT method and colony-forming test, the effect of Bmi-1 siRNA on the tumorogenicity of K562 cells was observed by subcutaneous inoculation of K562 cells, LY294002 and Bpv treated K562 cells in nude mice, the expression of Bmi-1, PTEN and pAKT proteins were detected by Western blot. RESULTS: The Bmi-1 siRNA could inhibit the proliferation activity, colony-forming and tumor-forming abilities of K562 cells. After the silence of Bmi-1 gene, the PTEN expression in Bmi-1 gene-silenced group was significantly enhanced. While the pAKT expression in Bmi-1 gene-silenced group was significantly reduced; after the K562 cells were treated with LY294002 (an inhibitor of pAKT), the pAKT expression colony-forming and tumor forming abilities were reduced in comparison with untreated K562 cells; after the K562-S1 cells were treated with Bpv (an inhibitor of PTEN), the PTEN expression decreased, while the pAKT expression, colony forming and tumor-forming abilities were restored. CONCLUSION: The Bmi-1 gene possibly involves in regulation of K562 proliferation in vivo and in vitro, the effect of PTEN/pAKT signaling pathway maybe one of molecular mechanisms mediating this regulation.

Construction of antisense Bmi-1 expression plasmid and its inhibitory effect on K562 cells proliferation.

BACKGROUND: Bmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16) expression. METHODS: A transcriptional repressor, Bmi-1 cDNA was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) of its mRNA from K562 cells. A plasmid expressing antisense Bmi-1 mRNA was then constructed by reverse design of PCR primers and cloned to the plasmid pLNCX2; G418 was added to the medium after the plasmid was successfully introduced in K562 cells by lipofectin-mediated DNA transfection. The effects of the antisense expression on the proliferation of K562 cells were analyzed by using microculture tetrazolium and colony forming. Cell cycle was analyzed by using flow cytometry. The p16 expression of K562 cells was observed by immunofluorescence histochemical stain. RESULTS: K562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls (the parental K562 and cells transfected with empty plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected cells decreased significantly (P < 0.01) compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls. CONCLUSION: The antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.

Fitness traits and underlying genetic variation related to host plant specialization in the aphid Sitobion avenae.

Sitobion avenae (F.) is an important cereal pest worldwide that can survive on various plants in the Poaceae, but divergent selection on different host plants should promote the evolution of specialized genotypes or host races. In order to evaluate their resource use strategies, clones of S. avenae were collected from oat and barley. Host-transfer experiments for these clones were conducted in the laboratory to compare their fitness traits. Our results demonstrated that barley clones had significantly lower fecundity and tended to have longer developmental times when transferred from barley to oat. However, oat clones developed faster after they were transferred to barley. Clones from oat and barley had diverged to a certain extent in terms of fecundity and developmental time of the nymphs. The separation of barley clones and oat clones of S. avenae was also evident in a principal component analysis. Barley clones tended to have higher broad-sense heritabilities for fitness traits than oat clones, indicating the genetic basis of differentiation between them. Barley clones showed significantly higher extent of specialization compared to oat clones from two measures of specialization (i.e., Xsp and Ysp). Therefore, barley clones were specialized to a certain extent, but oat clones appeared to be generalized. The fitness of S. avenae clones tended to increase with higher extent of specialization. The evolution toward ecological specialization in S. avenae clones, as well as the underlying genetic basis, was discussed.

[Ecological characteristics of preferred habitat of reindeer of Daxing'an Mountain forest area Northeast China in summer].

In July and August of 2012 and 2013, habitat selection and use patterns of reindeer were studied using both line and strip-transect surveys. Twenty-three habitat factors were measured and compared in known reindeer range areas in northwestern China. A total of 72 sampling sites were designated as being used by reindeer, and 162 sites were designated as unused control plots. The results indicated that, compared to the non-used habitat plots, reindeer selected summer habitats with higher values in altitude (26.9 ± 0.8 m), arbor canopy (17.9% ± 2.4%), arbor DBH (35.5 ± 2.1 cm), arbor height (8.2 ± 0.5 m), arbor density (6.9 ± 0.5 ind · 400 m(-2)) and stump quan- tity (1.3 ± 0.2 ind · 400 m(-2)), and with a lower shrub height (54.2 ± 2.0 cm). Moreover, reindeer also selected habitats at intermediate positions of intermediate slope gradient, which provided good water accessibility, more distance from human disturbance and herder influence, but bad concealment and lee condition. Results of the principal component analysis showed that the disturbance intensity (i. e. residential dispersion, anthropogenic-disturbance dispersion), arbor characteristics (arbor height and arbor density, arbor DBH and arbor canopy), geography characteristics (i. e. slope position, slope aspect and soil moisture), food abundance (ground-plant cover and shrub cover), openness (concealment and lee condition) and slope gradient were the most important factors influencing the habitat selection of reindeer in summer. In summary, the summer habitat selection of reindeer is a multidimensional process, through which reindeer adapt according to their ecological needs of food resources, safety and anti-predation. Furthermore, the pattern of habitat selection of reindeer showed that reindeer in China has not yet been domesticated, and reindeer populations and their core habitats should be conserved from intensive disturbance.

[Summer habitat selection of alpine musk deer in Xinglongshan National Nature Reserve, Northwestern China].

During July and August from 2006 to 2008, summer habitat selection was studied in Alpine musk deer (Moschus sifanicus) in Xinglongshan National Natural Reserve in northwestern China. In total, seventy one musk deer utilized habitat plots and 246 random habitat plots were surveyed. Seventeen habitat characteristics were recorded and compared between the two habitat types, using Mann-Whitney U test and chi-square to compare the differences between utilized and random habitat plots, and principal component analysis (PCA) to determine the main factors influencing the habitat selection of musk deer. Mann-Whitney U test and chi-square was conducted to test whether there was significant difference between utilized and random plots. The results showed that musk deer prefers habitat with taller arbor height (7.57 +/- 0.83 ) m, higher food-plants abundance (12.97 +/- 1.80), increasing foliage cover for concealment, lower water dispersion, and higher anthropogenic disturbance. Furthermore, PCA results suggested that the arbor characteristics (arbor canopy and arbor DBH), altitude characteristic, food characteristics (ground-plant cover and food-plant abundance) and shrub characteristics (shrub canopy, shrub height and related arbor density) influence summer habitat selection patterns of alpine musk deer in Xinglongshan National Nature Reserve. The general pattern of summer habitat utilization and selection of alpine musk deer is an adaptive strategy to the changing food, concealment, water source and the physical condition of summer habitat.

[Inhibition effect of antisense Bmi-1 on Jurkat cells].

OBJECTIVES: To investigate whether antisense Bmi-1 plasmid could inhibit the proliferation of Jurkat cells. METHODS: The antisense plasmid was constructed by PCR amplification of a 171 bp segment spanning Bmi-1 start codon and zinc finger structure and the PCR product was subsequently inserted reversely to plasmid pLNCX2. The final construct was confirmed through restriction enzyme digestion. G418 was added into the medium after the plasmid was successfully introduced into Jurkat cells by using lipofectin-mediated DNA transfection. The proliferation of Jurkat cells were determined by MTT and colony formation assays. Cell cycle was determined by flow cytometry. The p16 expression of Jurkat cells was studied by immunofluorescent histochemistry. RESULTS: The growth rate of antisense Bmi-1 transfected Jurkat cells was significantly lower than that of the controls, and the colony forming capacity of the transfected cells decreased significantly (P < 0.01), the colony numbers being (90.7 +/- 9.07)/10(3) cells, (83.3 +/- 6.11)/10(3) cells and (56.0 +/- 5.56)/10(3) cells for control cells, empty plasmid transfected Jurkat cells and antisense Bmi-1 transfected Jurkat cells, respectively. The percentage of G, phase cells was increased and the p16 expression of antisense Bmi-1 transfected cells was significantly upregulated than that of control cells. CONCLUSION: Antisense Bmi-1 can inhibit the growth and upregulate the expression of p16 of Jurkat cells in vitro.

[Construction of antisense telomerase hTERT and its effect on K562 cells].

OBJECTIVES: To investigate whether antisense human telomerase reverse transcriptase (hTERT) could inhibit the activity of telomerase and the proliferation of K562 cells. METHODS: The antisense plasmid was constructed by reverse insertion of hTERT PCR product into plasmid pLNCX-neo. Then the constructed plasmid was introduced into K562 cells by liposomes-mediated DNA transfection. The inhibition effects of telomerase on the proliferation of K562 cells were analyzed by MTT and colony formation assay, the telomerase activity of K562 cells by TRAP-PCR ELISA methods. RESULTS: The growth rate of antisense hTERT transfected K562 cells was significantly lower than those of the controls, and the colony formation capacity of the transfected cells decreased significantly (P < 0.01), the colony number is (100.33 +/- 7.57)/10(3) cells, (92.67 +/- 5.86)/10(3) cells and (50.33 +/- 6.11)/10(3) cells for control K562 cells, K562 neo cells and antisense hTERT transfected HL60 cells, respectively. The telomerase activity of antisense hTERT transfected K562 cells was significantly inhibited. CONCLUSION: The expression of an antisense sequence to the mRNA sequence of telomerase protein subunit can inhibit the activity of telomerase, slow the cell growth and inhibit the capacity of colony formation of K562 cells.