Analysis of relationships between expression of RIP 2 and invasion in colorectal cancer / 国际检验医学杂志

Objective To explore the expression of RIP2 in colorectal cancer(CRC)tissues,and the inva-sion and migration was observed after RIP2 was increased in CRC cell line.Methods 48 cases of CRC paraf-fin-embedded specimens and 56 cases of tumor-adjacent tissues were collected,and expressions of RIP2 was tested by immunohistochemistry(IHC).pEGFP-RIP2 plasmid and JetPRIME were employed to increase the RIP2 in SW480 cells,and variety of invasion and migration was tested in SW480 cells.Results It was showed that expressions of RIP2 was lower in CRC tumors than in tumor-adjacent tissues(P<0.05).After RIP2 in-creased,migration ability of SW480 cells weakened.Conclusion The expression of RIP2 was decreased in CRC tissues,and it was closely related to tumor cell's invasion and metastasis.

Expression of VDR and GLi1 in prostate cancer cell line PC-3 and its correlation / 实用医学杂志

Objective To investigate the effect of lentivirus carrying shRNA-VDR vector on GLi1 in pros-tate cancer PC-3 cells. Methods The cells were cultured according to the culture conditions of PC-3 cells. Expression of VDR and GLi1 in PC-3 cells was detected by fluorescence quantitative PCR and immunocytochemistry SP method.The efficiency of PC-3 cell virus infection was evaluated.The effect of VDR gene interference and GLi1 transcription level on PC-3 cells was detected by RT-PCR.Results Cell culture,cell status was recorded and PC-3 cells were in good condition and the passages was 4 days. Fluorescence quantitative and immunocytochemi-cal SP showed that VDR and GLi1 were expressed in PC-3 cells.The virus infection efficiency showed that the in-fection efficiency was about 95% when adding LV3-NC lentivirus to PC-3 cells at 1:10 ratio. RT-PCR showed that VDR-shRNA lentivirus successfully disturbed VDR expression and decreased by 85%(P < 0.05)compared with the control group after 72 days of VDR-shRNA lentivirus transfection. Transcription level of GLi1 gene in the experimental group increased by 9% compared with the control group(P < 0.05). The transcription level of GLi1 gene in the experimental group increased by 248% compared with the control group(P < 0.05). Conclusion The cultured PC-3 cells were in good condition. VDR and GLi1 genes were expressed in PC-3 cells. Lentivirus showed highest efficiency in infecting PC-3 at 1:10 ratio. When VDR was disturbed,the expression of GLi1 in-creased.In prostate cancer cells,vitamin D can inhibit the Hh signaling pathway and cause GLi1 expression down expression.

Effect of silencing the VDR gene on the migration and invasion of prostate cancer cells / 中华男科学杂志

Objective@#To investigate the effect of small interfering RNA silencing the vitamin D receptor (VDR) on the biological behavior of prostate cancer PC-3 cells.@*METHODS@#We constructed the VDR-shRNA lentiviral vector and determined the mRNA and protein expressions of VDR by RT-PCR and Western blot. Using scratch wound healing and Transwell chamber assays, we detected the changes in the migration and invasiveness of the PC-3 cells after silencing VDR.@*RESULTS@#The VDR-shRNA plasmid significantly interfered the VDR expression and successfully screened the cell lines with stable VDR-shRNA interference. The rate of scratch wound healing was markedly lower in the VDR interference group than in the blank control and LV3 negative control groups (59% vs 73.6% and 77.8%, P 0.05), and so was the count of permeable cells (P 0.05). The migration ability and invasiveness of the VDR-treated cells were remarkably decreased as compared with those of the control cells.@*CONCLUSIONS@#Down-regulated expression of the VDR gene may reduce the migration and invasiveness of prostate cancer cells.

Effects of IRF3 gene interference on dynamic changes of cytokine secretions in early of LPS stimulation in primary Kupffer cells / 中国免疫学杂志

Objective:To investigate the effects of IRF 3 shRNA adenovirus on dynamic changes of early cytokines in LPS-stimulated primary Kupffer cells ( KCs ).Methods: Rat KCs were isolated and purified by means of in situ perfusion.After being infected with adenovirus carrying IRF 3 shRNA for 48 h, KCs were stimulated with LPS.Cell culture supernatants were collected respectively at 0,2,4 and 6 h after LPS stimulation as well as cells at 6 h.Supernatant cytokine secretion levels were detected by enzyme-linked immunosorbent assay ( ELISA).Intracellular gene expressions were tested by RT-PCR and Westeron blot.Results:IRF3 mRNA and protein were induced by LPS ,but suppressed by IRF 3 shRNA adenovirus in LPS-stimulated or non-stimulated KCs.IFN-βsecretions rose in the very early stage ( at 2 h) ,reached the peak at 4 h,and began to reduce but still remained high levels at 6 h after LPS stimulation in KCs.Interference adenovirus pretreatment suppressed IFN-βsecretions ( especially the secretion peak ) at each time point after LPS stimulation.IFN-βsecretions reached normal levels at 6 h after the stimulation in adenovirus-pretreated cells;TNF-αse-cretions rapidly increased in the very early stage and reached the peak at 2 h,then began to decrease gradually ,but remained high levels at 6 h after LPS stimulation in KCs.Interference adenovirus pretreatment inhibited LPS-induced TNF-αsecretions, especially the secretion peak;IL-1βsecretions did not increase untill 4 h, but reached a higher level at 6 h after LPS stimulation.Interference adenovirus suppressed IL-1βsecretions in the early stage of LPS stimulation;IL-10 secretions began to rise in the very early stage ,and gradually increased over time after LPS stimulation in KCs.Pretreatment of adenovirus with IRF 3 shRNA promoted upregulations of IL-10 secretions at each time point of the early of LPS stimulation.Conclusion:IRF3 gene expression can be silenced by IRF 3 shRNA ad-enovirus.IRF3 can promote its downstream signaling molecule IFN-βand pro-inflammatory cytokines including TNF-αand IL-1β,and block anti-inflammation cytokine IL-10 secretions in LPS-stimulated primary KCs.Therefore,IRF3 may play a central role in immune inflammatory injury of liver tissues.

Effect of inositol-requiring enzyme-1 signaling pathway on the differentiation of osteoblasts induced by fluoride / 中华地方病学杂志

Objective To observe the expression of the unfolded protein response especially the inositol-requiring enzyme-1 (IREl)-Xbp1 signaling pathway, and the change trend of osteogenic markers after inhibition of IREl expression through siRNA interference in osteoblasts exposed to fluoride. Methods Proliferation activity of MC3T3-E1 cells was detected by CCK-8 assay, and 0.0, 0.1, 1.0, 2.0, 8.0, 16.0, 20.0, 32.0, 64.0 mg/L groups were set up. Then representative doses of low, medium and high fluoride (2.0, 8.0, 20.0 mg/L) were selected to treat MC3T3-E1 cells and the expression of the unfolded protein response related genes and osteogenic markers [alkaline phosphatase (ALP), osteocalcin (OCN), Runx2, osterix, binding immunoglobulin protein (Bip), protein kinase-like endoplasmin reticulum kinase ( PERK ) , activated transcription factor 6 (ATF6), Xbp1] was detected by Real-time PCR. MC3T3-E1 cells were transfected with IRE1 siRNA and then exposed to fluoride, and the expression of IRE1 signaling pathway and osteogenic markers was detected by Western blotting and real-time PCR. Results CCK-8 results showed the bidirectional effect of fluoride on the activity of osteoblasts. Compared with the 0.0 mg/L group [1.00 ± 0.01 (d 1), 1.00 ± 0.02 (d 3), 1.00 ± 0.08 (d 7)], the osteoblast activity was significantly enhanced at 2.0 mg/L [1.11 ± 0.02 (d 1), 1.29 ± 0.02 (d 3)], 8.0 mg/L [1.16 ± 0.02 (d 1), 1.44 ± 0.03 (d 3), all P<0.05], while 20.0 mg/L inhibited cell activity [0.83 ± 0.01 (d 1), 0.81 ± 0.01 (d 3), 0.96 ± 0.04 (d 7), all P< 0.05]. Compared with the 0.0 mg/L group [6.86 ± 2.13 (ALP), 4.58 ± 1.52 (OCN), 2.65 ± 0.38 (Runx2), 12.48 ± 3.96 (osterix)], 2.0 mg/L significantly induced the expression of intracellular ALP (12.80 ± 3.62), Runx2 (6.61 ± 0.48) and osterix (21.42 ± 1.56), and the differences were statistically significant (all P< 0.05), while 20.0 mg/L inhibited the expression of ALP (0.88 ± 0.17), OCN (0.16 ± 0.05) and osterix (1.35 ± 0.51), and the differences were statistically significant (all P<0.05). Compared with the 0.0 mg/L group [1.36 ± 0.58 (IRE1), 0.96 ± 0.45 (Xbp1)], the expression of endoplasmic reticulum stress related genes IRE1 [14.84 ± 2.57 (2.0 mg/L), 4.10 ± 0.52 (8.0 mg/L), 5.30 ± 0.63 (20.0 mg/L)] and Xbp1 [2.62 ± 0.66 (2.0 mg/L), 1.97 ± 0.47 (20.0 mg/L)] were significantly increased in the corresponding fluoride groups (all P<0.05). After IRE1 gene knockout, compared with the control group [gene:3.25 ± 0.48 (OCN), 5.62 ± 1.86 (Runx2), 2.67 ± 0.35 (ALP); protein: 0.16 ± 0.03 (OCN), 0.34 ± 0.27 (ALP)], the gene expression of OCN [0.63 ± 0.46 (2.0 mg/L), 0.81 ± 0.36 (8.0 mg/L), 0.62 ± 0.31 (20.0 mg/L)], Runx2 [0.18 ± 0.03 (2.0 mg/L), 0.12 ± 0.01 (8.0 mg/L), 1.09 ± 0.33 (20.0 mg/L)] and ALP [1.01 ± 0.12 (8.0 mg/L), 0.38 ± 0.09 (20.0 mg/L)] in the corresponding fluoride groups were significantly decreased (all P < 0.05), protein expression of OCN [0.06 ± 0.02 (2.0 mg/L), 0.06 ± 0.02 (8.0 mg/L), 0.07 ± 0.03 (20.0 mg/L)], and ALP [0.02 ± 0.01 (8.0 mg/L), 0 (20.0 mg/L)] were significantly decreased (all P< 0.05). Conclusion Unfolded protein response is observed under different doses of fluoride in osteoblasts, and IRE1 gene knockout has inhibited the expression of ALP, OCN, osterix and Runx2 in osteoblasts induced by fluoride, which suggests that IRE1 signaling pathway may play a key role in the differentiation of osteoblasts exposed to fluoride.

Preparation of polyethylene glycol-modified polyarginine for gene delivery and in vitro evaluation / 第二军医大学学报

Objective To modify polyargine (PLR) with polyethylene glycol (PEG) and to observe the effect of PEG modilication on PLR cytotoxicity and efticiency of PLR-mediated RNA interference. Methods1 HNMR was used to characterize PLR-PEG and gel electrophoresis was adopted to determine the siRNA-packing capacity of PLR-PEG. The cytotoxicity of PLR-PEG, cellular uptake and RNA interference efficiency of PLR-PEG/siRNA complexes were investigated using prostate cancer stem-like cells(RC-92a/hTERT). Results1 HNMR results showed the successful synthesis of PLR-PEG. It was found that PEG modiiication decreased cytotoxicity of PLR and reduced cellular uptake of PLR/siRNA complexes, but the reduction of cellular uptake was limited when N/P was high. The modiiication also inhibited the efticiency of PLR-mediated RNA interference, but the influence of PEG moditication was not notable within a certain range. Conclusion PEG-moditied PLR may be a promising vector for gene therapy targeting prostate cancer stem cells.

Research of interference Livin gene inhibits growth of QBC939 cell / 中华肝胆外科杂志

Objective To study the effect of transfection of livin antisense oligodeoxynucleotide (Livin ASODN) on Livin mRNA and Livin protein expression and proliferation of QBC939 cells.Methods Livin ASODN was transfected into cell line QBC939 by LipofectamineTM 2000. Fluorescence microscopy was used to observe the ASODN transfected cells and to calculate the rate of transfection.to measure Livin mRNA and Livin protein expression by RT-PCR and immunohistochemistry and con-focal laser scanning microscopy after the transfection. Changes in cell proliferation were detected by MTT. Results The highest efficiency was at 24 hours after 500 nmol/L Livin ASODN transfection.The results of MTT showed that the inhibition of cell proliferation of QBC939 cells was most obvious at 60 hours after Livin ASODN transfection (P<0. 05). The level of Livin mRNA and Livin protein expression in the ASODN group was obviously lower than that in the control group (P<0. 05).Conclusion The transfection of Livin ASODN inhibited Livin gene and Livin protein expression, and obviously inhibited the proliferation and depressed the vitality of QBC939 cells.