Résumé
Background:Fusobacterium nucleatum (Fn)is a common oral pathogen. Studies have shown that Fn is closely related to the occurrence and development of colorectal cancer,especially the inflammation-related colorectal cancer. Aims:To investigate the mechanism of Fn in forming an inflammatory microenvironment in colon cancer cells. Methods:An inflammation model of Caco-2 cells infected by Fn was constructed,and miRNA sequencing was performed. miR-181b mimics or inhibitor was transfected into Fn infected Caco-2 cells. mRNA and protein expressions of TNF-α were determined by qRT-PCR and Western blotting,respectively,and concentration of TNF-α in supernatant was measured by ELISA, number of lymphocyte penetrating the membrane was measured by Transwell chamber. Results:Compared with control group,mRNA and protein expressions of TNF-α were significantly increased (P < 0. 05),concentration of TNF-α in supernatant was significantly increased (P < 0. 05),and number of lymphocyte penetrating the membrane was significantly increased in Fn group (P < 0. 05). miRNA sequencing and qRT-PCR results showed that expression of miR-181b was significantly decreased in Fn group than in control group (P < 0. 05). Compared with control group,mRNA and protein expressions of TNF-α were significantly decreased in miR-181b mimics + Fn group (P < 0. 05),however,mRNA and protein expressions of TNF-α were significantly increased in miR-181b inhibitor group (P < 0. 05). Bioinformatics tools and Luciferase assay confirmed that TNF-α might be the target gene of miR-181b in Caco-2 cells. Conclusions:Fn can up-regulate the expression of TNF-α by inhibiting miR-181b in Caco-2 cells and recruiting lymphocytes to form an inflammatory microenvironment.
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Background:Fusobacterium nucleatum (Fn)is a common oral pathogen. Studies have shown that Fn is closely related to the occurrence and development of colorectal cancer,especially the inflammation-related colorectal cancer. Aims:To investigate the mechanism of Fn in forming an inflammatory microenvironment in colon cancer cells. Methods:An inflammation model of Caco-2 cells infected by Fn was constructed,and miRNA sequencing was performed. miR-181b mimics or inhibitor was transfected into Fn infected Caco-2 cells. mRNA and protein expressions of TNF-α were determined by qRT-PCR and Western blotting,respectively,and concentration of TNF-α in supernatant was measured by ELISA, number of lymphocyte penetrating the membrane was measured by Transwell chamber. Results:Compared with control group,mRNA and protein expressions of TNF-α were significantly increased (P < 0. 05),concentration of TNF-α in supernatant was significantly increased (P < 0. 05),and number of lymphocyte penetrating the membrane was significantly increased in Fn group (P < 0. 05). miRNA sequencing and qRT-PCR results showed that expression of miR-181b was significantly decreased in Fn group than in control group (P < 0. 05). Compared with control group,mRNA and protein expressions of TNF-α were significantly decreased in miR-181b mimics + Fn group (P < 0. 05),however,mRNA and protein expressions of TNF-α were significantly increased in miR-181b inhibitor group (P < 0. 05). Bioinformatics tools and Luciferase assay confirmed that TNF-α might be the target gene of miR-181b in Caco-2 cells. Conclusions:Fn can up-regulate the expression of TNF-α by inhibiting miR-181b in Caco-2 cells and recruiting lymphocytes to form an inflammatory microenvironment.
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Objective To investigate the alternation of insulin -like growth factor -Ⅱ( IGF-Ⅱ) gene promoter P4 methylation status in hepatocellular carcinoma ( HCC) and explore its relationship with expression of P4 mRNA levels.Methods Liver specimens of 43 patients with HCC and normal liver specimens of 9 control patients were collected in operation .Tissue DNA and total RNA were extracted from these specimens .IGF-Ⅱ P4 methylation status and P4 mRNA expression levels were detected .Results (1)The incidence of IGF -ⅡP4 methyl-ation in HCC group was significantly lower than that in normal liver specimens (16.28%vs 88.89%,χ2 =19.12,P<0.01).(2)The expression level of IGF -ⅡP4 mRNA in HCC group was significantly higher than that that in normal liver specimens[(0.96 ±0.74) vs (0.25 ±0.19),t=5.48,P<0.01].(3)In HCC group,the IGF-Ⅱ P4 mRNA expression level with hypomethylation gene was significantly higher than that without hypomethylation gene [(1.18 ± 0.76) vs (0.32 ±0.27),t=5.28,P<0.01].Conclusion The hypomethylation alternation of IGF -Ⅱ P4 gene promoter which is accomplished by up -regulate P4 mRNA expression has a close relationship with HCC .
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Objective To investigate the inhibitory effect of small interference RNA(siRNA)on the exDression of tumor necrosis factor(TNF)-oligo nucleotide fragments were designed and synthesized according to the sequence of TNF-insert downstream to H1 promoter to construct plasmids pHS-A and pHS-B for expression of short hairpin RNA(shRNA).Then the recombinant pSilence3.1-TNF-phages(RAW264.7)by lipofectamin 2000.The inhibition of TNF-examined bv real-time PCR and ELISA,respectively.Results After LPS stimulating 6 hours,TNF-expression was increased in a specific time-dependent manner,and reached high peak at 9-12 hours.The macrophages treated with plasmids pHS-A showed a significant decrease in TNF- mRNA(0.021 34±0.009 60)and protein(149.93,P＜0.01)compared with untransfected group[TNF-mRNA:0.021 34±0.009 60,protein:1 922.30±149.05]pg/ml].The relative rate of inhibition was 83.3％.No inhibitory effects was found in plasmids pHS-B and control group.Conclusions LPS stimulation results in a increasing expression of TNF-of TNF-
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Objective To investigate the effect of transplantation of allogeneic bone marrow hematopoietic cells(HCs)and mesenchymal stem cells(MSCs)on experimental colitis(EC)in rats.Methods The HCs and MSCs obtained from SD male rats were cultured and expanded in vitro.In experiment 1 and 2 groups,HCs were labeled with bromodeoxyuridine(BrdU)and MSCs were obtained using the tube wall attach technique,respectively.Seventy-two female rats were infused with trinitrobenzene sulfonic acid(TNBS)to induce EC models.After 24 hours,HC or MSC suspensions were injected into the rats in experimental 1(n=18)and 2(n=18)groups via caudal veins,respectively.Control animals were injected with isotonic saline.The whole colon was removed on day 7,14 and 21 after transplantation and examined histopathologically.BrdU labeled HCs were tested with immunohistochemical staining and MSCs were detected for sex-determining gene(sry)by PCR.Results EC models were successfully established.The HCs or MSCs grew rapidly in the culture suspension.On day 7,14 and 21 after transplantation,the BrdU immunoreactive cells were detected in the colon(6/6),and the positive expression of the sry gene was found in 1/6,2/6 and 3/6,respectively.No positive labeled cell was found in controls.There was no significant improvement in histopathological scores on the colon in two experimental groups compared with the controls.Conclusions Allogeneic HCs and MSCs may localize in the colon of EC models.The ability of localization is higher in HCs than MSCs.The transplantation of HCs and MSCs can not obviously improve histopathologically.