17 research outputs found

    VCP positively regulated the progress of HCC.

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    <p>A: Analysis of the level of VCP in HCC tissues by qRT-PCR. The result showed that the level of VCP is frequently increased in human HCC tissues. *:P<0.05.**:P<0.01. B,C: Effect of si-VCP and miR-129-5p on tumor growth in nude mice model. si-VCP, miR-129-5p and NC-transfected HepG2 cells were suspended and then injected subcutaneously into either side of the posterior flank of the male BALB/c athymic nude mouse. Tumor growth was examined every four days. Tumor volume (V) was monitored by measuring the length (L) and width (W) of the tumor with calipers and was calculated with the formula V = (L×W<sup>2</sup>)×0.5. Photographs of dissected tumors from nude mice (B) and the curve of tumor growth (C) are shown. *:P<0.05. Results are representative of three animals in every time point per group. D:The level of VCP and Bcl-2 in the transplanted tumors was detected by IHC.</p

    miR-129-5p could directly regulate the expression of VCP.

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    <p>A: miR-129-5p could significantly suppress the luciferase activities of pGL3-VCP-3′UTR in HepG2 cells. Five predicted miRNAs (miR-103, miR-107, miR-129-5p, miR-136, miR-339-5p) were transfected in HepG2 cells, respectively, with VCP 3′UTR report. 72 h after the transfection, the cells were harvested for luciferase activity. Shown data are representative from three independent experiments. **:P<0.01. B: Bioinformatic analysis of miR-129-5p predicted binding sites in the VCP 3′UTR. There were two putative miR-129-5p target sites located in the VCP 3′UTR (162–168 and 505–511). Three mutant reporter vectors were constructed with the deletion of two target sites individually (pGL3-VCP-3′UTRm1, pGL3-VCP-3′UTRm2) or both (pGL3-VCP-3′UTRm3). C: The luciferase activity of the mutant VCP 3′UTR report genes were not regulated by miR-129-5p in HepG2 cells. Shown data are representative from three independent experiments.**:P<0.01. D: miR-129-5p could significantly suppress the luciferase activities of pGL3-VCP-3′UTR in MHCC-LM3 cells, while has no effect on three mutant reporter vectors. Shown data are representative from three independent experiments.**:P<0.01. E: The inhibiter of miR-129-5p could increase the luciferase activities of pGL3-VCP-3′UTR in SK-HEP1 cells, while has no effect on three mutant reporter vectors. Shown data are representative from three independent experiments. **:P<0.01. F: Analysis of the level of miR-129-5p in HCC tissues by qRT-PCR. The result showed that the level of miR-129-5p is frequently reduced in human HCC tissues.*:P<0.05.**:P<0.01. G: The level of miR-129-5p was negatively correlated with the expression of VCP in HCC. The 39 tissue samples of HCC were divided into two groups according to the level of VCP. The level of miR-129-5p in each sample from the two groups (level 1,n = 17;level 2,n = 22) was measured by qRT-PCR and compared. U6 snRNA was used as internal control gene. Dot plots showed an inverse relationship between U6 snRNA and miR-129-5p expression in HCC. Significant differences were determined using Student's t tests. **: P<0.01; Bars: mean±SD.</p

    miR-129-5p could regulate the cell growth, apopotosis and migration of HCC cells dependent on the regulation of VCP expression.

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    <p>A: HepG2 or SK-HEP1 cells were transfected and in the indicated time periods posttransfection, cell growth rate was evaluated using CCK8 assay. The result showed miR-129-5p and si-VCP could repress cell growth. The cell growth rate was restored after VCP expression vector were transfected into HepG2 or SK-HEP1 cells. Shown data are representative from three independent experiments. **:P<0.01. B: HepG2 cells were transfected and the apoptosis of cell was detected with FACS. Left panel showed miR-129-5p and si-VCP could increase the apoptosis of HepG2 cells at 48 h and 72 h after the transfection. Right panel showed the apoptosis of cell after miR-129-5p with/without VCP expression vector were transfected into HepG2 cells for 72 h. The experiment was repeated three times independently. NC: negative control RNA duplex; miR-129-5p: miR-129-5p mimic; miR-129-5p/pcDNA3.1: cells were co-transfected with miR-129-5p mimic and blank pcDNA3.1; miR-129-5p/VCP: cells were co-transfected with miR-129-5p mimic and VCP expression vector. The experiments were repeated three times independently.*:P<0.05. **:P<0.01. C: SK-HEP1 cells were transfected and the apoptosis of cells was detected with FACS. miR-129-5p and si-VCP could increase the apoptosis of SK-HEP1 cells at 72 h after the transfection. After the level of VCP was enhanced by transfecting VCP expression vector, the apoptosis rate of cells were returned. D:Detection the level of Bcl-2 and XIAP by RT-PCR and western blot. GAPDH served as the internal control. E:Transwell assay was performed to assess cell migration. Upper panel represented the photographs of treated and untreated cells at 24 h (Ă—40 magnification). Lower panel showed the number of cells invaded at 24 h. Shown data are representative from four replicates per group. *P<0.05.</p

    The <i>Δrnc</i> was less pathogenic compared with its parent strain.

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    <p><b>A: Analysis of apoptosis and necrosis of MDBK cell after treatment with the supernatant from different strains.</b> Flow cytometric analysis was prepared to observe the apoptosis and necrosis of MDBK cell after treated with the supernatant of <i>S. aureus</i>. Comparing with its parent stain, the percentage of apoptosis and necrosis induced by the supernatant of <i>Δrnc</i> was significantly decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). <b>B: Analysis of the production of the heat-stable toxins from different stains.</b> The heat-stable toxins were obtained as the described method and incubated with MDBK cell. The survival cell number was determined by MTT method. Comparing with its parent strain, the heat-stable toxins of <i>Δrnc</i> supernatant was decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). <b>C: The detection of the pathogenicity of the different strains with the acute peritonitis animal model.</b> Groups of 10 Balb/c mice were injected intra-abdominally with 500 µl of <i>Δrnc</i> and its parent strain (1×10<sup>8</sup> CFU). The number of the survival mice was recorded at different time points. The survival rate was calculated. The result showed that the pathogenicity of <i>Δrnc</i> was decreased.</p

    Detection of the protein profile from different phases of WT and <i>Δrnc</i>.

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    <p>Equal number of <i>S. aureus</i> cells was harvested at the indicated time points. The total proteins of whole-cell and supernatant proteins were extracted. The results showed that the supernatant proteins of the <i>Δrnc</i> were decreased significantly compared with WT, while the total proteins of whole-cell did not show the same change as the supernatant proteins. The experiment has been repeated for three times. 1,4,7: WT, wild type, <i>S. aureus</i> 8325-4; 2,5,8: <i>Δrnc</i>; 3,6,9: rncR.</p

    Stability of RNAs.

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    <p>Half-lives of <i>secY2</i> mRNA and RNAIII were determined in the presence of rifampicin (500 µg ml<sup>-1</sup>) in the WT and <i>Δrnc</i> strains. Percentage of RNA was calculated normalizing with 5s rRNA.</p

    The decrease of <i>secY2</i> resulted in the inhibition of extracellular protein secretion in <i>Δrnc</i> at 1.5 h.

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    <p><b>A: Detection of the mRNA level of </b><b><i>secA1, secY1</i></b><b>,</b><b><i>secA2</i></b><b> and </b><b><i>secY2.</i></b> The mRNA levels of <i>secA1, secY1</i>,<i>secA2</i> and <i>secY2</i> were detected at 1.5 h by qRT-PCR. The results showed that the level of <i>secY2</i> was decreased in <i>Δrnc</i>. (**: P<0.01). Then the decrease of <i>secY2</i> mRNA was confirmed by Northern blot. 16s rRNA was used as the internal control. WT: wild type, <i>S. aureus</i> 8325-4; <i>Δrnc</i>: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in <i>Δrnc</i>. <b>B: Detection of the profile of extracellular proteins and the expression of Efb in the different strains.</b> The pOS1-secY2 plasmid was constructed and transferred to <i>Δrnc</i> to recover the level of SecY2. At the same time, the double mutant <i>Δrnc/secY2</i> was constructed. And then the extracellular proteins were extracted. The results showed that the production of the extracellular proteins was significantly increased at 1.5 h after the recovery of the level of <i>secY2</i>. The mRNA level of <i>secY2</i> was measured by RT-PCR. 16s rRNA was used as the internal control. At the same time, the expression of Efb was determined by Western blot. The result showed that Efb was restored after the level of <i>secY2</i> was recovered in <i>Δrnc</i>. 1: wild type; 2: <i>Δrnc</i>; 3: secY2r(the <i>Δrnc</i> strain transferred with the plasmid pOS1-secY2); 4, <i>Δrnc/secY2;</i> 5. <i>ΔsecY2</i>.</p

    RNAIII regulates the levels of extracellular proteins at 6 h and 12 h.

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    <p><b>A: The expression level of RNAIII was analyzed by Northern blot.</b> The level of RNAIII in different strains at 6 h and 12 h was detected by Northern blot. 16s rRNA was used as the internal control. WT: wild type, <i>S. aureus</i> 8325-4; <i>Δrnc</i>: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in <i>Δrnc</i>. <b>B: Detection of the extracellular proteins of WT and </b><b><i>ΔRNAIII</i></b><b> at the different time points.</b> The extracellular proteins from the equal number of cells were extracted at the indicated time points. The results of SDS-PAGE showed that the extracellular proteins of <i>ΔRNAIII</i> were decreased in comparing with WT at 6 h and 12 h. 1,4,7: wild type; 2,5,8: <i>ΔRNAIII</i> (RNAIII deletion mutant); 3,6,9: ΔRNAIIIR (the restoration of RNAIII in <i>ΔRNAIII</i>). <b>C: Detection of the extracellular proteins from different strains.</b> The pOS1-RNAIII plasmid was constructed to recover the level of RNAIII in <i>Δrnc</i>. At the same time, the double mutant <i>Δrnc/RNAIII</i> was constructed. Then the extracellular proteins were extracted. The results showed that the extracellular proteins were increased at 6 h and 12 h after the level of RNAIII was recovered in <i>Δrnc</i>. The level of RNAIII was measured by RT-PCR. 16s rRNA was used as the internal control. 1,5: WT, wild type; 2,6: <i>Δrnc</i>; 3,7: RNAIIIr(the <i>Δrnc</i> strain transferred with the plasmid pOS1-RNAIII); 4,8, <i>Δrnc/RNAIII</i>. The experiment has been repeated for three times.</p
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