Mechanism of transforming growth factor- β1 induce renal fibrosis based on transcriptome sequencing analysis / 浙江大学学报·医学版

OBJECTIVES@#To explore the mechanism of transforming growth factor-β1 (TGF-β1) induce renal fibrosis.@*METHODS@#Renal fibroblast NRK-49F cells treated with and without TGF-β1 were subjected to RNA-seq analysis. DESeq2 was used for analysis. Differentially expressed genes were screened with the criteria of false discovery rate<0.05 and l o g 2 F C >1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed for differentially expressed genes. Genes encoding transcription factors were further screened for differential expression genes. Then, the expression of these genes during renal fibrosis was verified using unilateral ureteral obstruction (UUO)-induced mouse renal fibrosis model and a public gene expression dataset (GSE104954).@*RESULTS@#After TGF-β1 treatment for 6, 12 and 24 h, 552, 1209 and 1028 differentially expressed genes were identified, respectively. GO analysis indicated that these genes were significantly enriched in development, cell death, and cell migration. KEGG pathway analysis showed that in the early stage of TGF-β1 induction (TGF-β1 treatment for 6 h), the changes in Hippo, TGF-β and Wnt signaling pathways were observed, while in the late stage of TGF-β1 induction (TGF-β1 treatment for 24 h), the changes of extracellular matrix-receptor interaction, focal adhesion and adherens junction were mainly enriched. Among the 291 up-regulated differentially expressed genes treated with TGF-β1 for 6 h, 13 genes (Snai1, Irf8, Bhlhe40, Junb, Arid5a, Vdr, Lef1, Ahr, Foxo1, Myc, Tcf7, Foxc2, Glis1) encoded transcription factors. Validation in a cell model showed that TGF-β1 induced expression of 9 transcription factors (encoded by Snai1, Irf8, Bhlhe40, Junb, Arid5a, Vdr, Lef1, Myc, Tcf7), while the expression levels of the other 4 genes did not significantly change after TGF-β1 treatment. Validation results in UUO-induced mouse renal fibrosis model showed that Snai1, Irf8, Bhlhe40, Junb, Arid5a, Myc and Tcf7 were up-regulated after UUO, Vdr was down-regulated and there was no significant change in Lef1. Validation based on the GSE104954 dataset showed that IRF8 was significantly overexpressed in the renal tubulointerstitium of patients with diabetic nephropathy or IgA nephropathy, MYC was highly expressed in diabetic nephropathy, and the expressions of the other 7 genes were not significantly different compared with the control group.@*CONCLUSIONS@#TGF-β1 induces differentially expressed genes in renal fibroblasts, among which Irf8 and Myc were identified as potential targets of chronic kidney disease and renal fibrosis.

Migration and invasion abilities of normal fibroblast-like synoviocytes versus fibroblast-like synoviocytes in rheumatoid arthritis / 中国组织工程研究

BACKGROUND:Fibroblast-like synoviocytes (FLS) in the synovial lining layer are related to the cell proliferation, invasion, migration and apoptosis as well as bone resorption in rheumatoid arthritis. OBJECTIVE:To compare the migration and invasion abilities of FLS (MH7A) in rheumatoid arthritis and normal FLS (HFLS). METHODS:The capacities of cell migration and invasion were evaluated by Transwell cell migration and invasion assays. The primers of the indicated microRNAs were designed and synthesized, and the expression levels of miRNAs were determined by real-time PCR according to the SYBR?PrimeScript?miRNA RT-PCR Kit instruction. RESULTS AND CONCLUSION:MH7A possessed stronger migration and invasion abilities than HFLS. Compared with HFLS, obviously upregulated miR-132, -155, -203, -223 and -124, and significantly downregulated miR-15a, -16, 18a, -19a, -26a and -146a were found in MH7A. These findings suggest that the differentially expressed 11 kinds of rheumatoid arthritis-associated miRNAs participate in the pathogenesis of rheumatoid arthritis probably by enhancing the migration and invasion capacities of MH7A.

Effect of genistein on the angiogenesis-related factors secreted by fibroblast-like synoviocytes from rats with collagen Ⅱ induced arthritis / 中华风湿病学杂志

Objective To investigate the effect of genistein (gen) on the secretion of vascular endo-thelial growth factor (VEGF) and matrix metalloproteinases (MMPs-1, 2, 3, 9) by fibroblast-like synoviocytes (FLS) from rats with collagen Ⅱ induced arthritis (CIA). Methods The CIA was induced by collagen Ⅱ and complete Freund's adjuvant (CFA) into rats. The rats were scored based on the arthritis index(AI) once a week. At the sixth week, the X-ray of joints was taken. The synovial tissues from knee joints were examined pathologically. The primary fibroblast-like synowocytes were separated from the synovial tissue by collagenase digestion and cultured. Then the expression of VCAM-1 was estimated by flow cytometry. After adding gen (100, 200, 400 μmol/L) at different concentrations into the FLS, VEGF and MMP-1, 2, 3, and 9 of the supernatants were tested by indirect ELISA. Results After 3 days of type Ⅱ collagen and CFA injec-tion, the rats started to catch arthrocele and their arthritis index increased gradually. The arthrocele was most remarkable at the 3rd week. The AI, X-ray and pathological examination indicated that the model group were significantly different from the control group. After the synoviocytes were cultured to the 4th generation, the expression of VCAM-1 was as high as about 85.5%. It showed that most synoviocytes were changed to fibro-blast-like synoviocytes. Different concentrations of gen (100, 200, 400 μmol/L) added to FLS were compared and revealed that the VEGF and MMP-1, 2, 3, and 9 in the supematants were suppressed evidently and in a dose-dependent manner. Conclusion The CIA model can be successfully constructed by collagen type Ⅱ and CFA. Tthe primary FLS of rats' joint can be separated and cultured well by collagenase digestion. Certain levels of gen can suppress the secretion of VEGF and MMP-1, 2, 3 and 9 hy FLS. The affect is dose-depen-dent.

TIMP-1 secreted by fibroblasts inhibits tumor cell invasion and metastasis in mouse melanoma / 生物医学工程学杂志

We constructed a recombinant adenoviral vector expressing human tissue inhibitors of metalloproteinase-1(TIMP-1), and evaluated the inhibition of TIMP-1 secreted by primary fibroblasts after infection with adenovirus-mediated TIMP-1 gene (Ad-TIMP-1) on tumor cell invasion and metastasis in mouse melanoma. It was found that TIMP-1 was detected in the supernatants of cultured mouse primary fibroblasts after infection with Ad-TIMP-1 by indirect enzyme-linked immunosorbent assay (ELISA). The TIMP-1 secreted by Ad-TIMP-1 infected primary fibroblast significantly inhibited B16BL6 cell invasion and metastasis both in vitro and in vivo. We also demonstrated that the primary fibroblasts transfected by Ad-TIMP-1, after being subcutaneously injected into mouse, can secreted TIMP-1 into the blood of mouse and maintained at the therapeutic in vivo levels of TIMP-1. These results suggest that the preparation of Ad-TIMP-1 infected primary fibroblast be an effective method to deliver TIMP-1 gene in vivo, which provides a new strategy of gene therapy and has the potential for clinical applications in the treatment of tumor cell metastasis.

Effects of arsenic trioxide on expressions of vascular endothelial growth factor and P-glycoprotein in multidrug resistant leukemia cell line K562/A02 / 中西医结合学报

OBJECTIVE: To investigate the effects of arsenic trioxide (ATO) on the expressions of vascular endothelial growth factor (VEGF) and P-glycoprotein (P-gp) in K562/A02 cells and to explore the correlation between VEGF and P-gp. METHODS: The inhibition rate of K562/A02 cell proliferation was detected by using methyl thiazolyl tetrazolium assay (MTT); the level of VEGF was detected by enzyme-linked immunosorbent assay (ELISA) and the expression rate of P-gp was determined by flow cytometry (FCM). RESULTS: 0.05 micromol/L ATO had no influences on the cell proliferation and the expression of VEGF in K562/A02 cells; 0.4 and 3.2 micromol/L ATO could significantly inhibit the K562/A02 cell proliferation and down-regulate the expression of VEGF in K562/A02 cells (P0.05). 3.2 micromol/L ATO could remarkably reduce the expression of P-gp in K562/A02 cells after 48- and 72-hour incubation with ATO (P<0.05). CONCLUSIONS: The down-regulation of P-gp and VEGF after being exposed to ATO probably contributes to the reversion of multidrug resistance in K562/A02 cells. The level of VEGF may be related to the expression rate of P-gp in K562/A02 cells.

AdH1-siRNA-Survivin inhibits tumor growth and metastasis of mouse melanoma / 第三军医大学学报

Objective To evaluate the effect of adenovirus-mediated siRNA targeting Survivin on tumor growth and metastasis of mouse melanoma.Methods A replication-defective recombinant adenovirus AdH1-siRNA-Survivin targeting mouse Survivin was constructed using the homologous recombination technique.The expression of Survivin protein in B16BL6 cells after infection was detected by Western blotting.Mouse melanoma models were established by subcutaneous injection of B16BL6 cells.AdH1-siRNA-Survivin of 2?109 pfu every week by multi-spot intratumoral or peritumoral injection was performed to every mouse(totally three times).The tumor volume was measured every five days after tumor cell implantation,and the microvessel density(MVD) of tumor was counted by immunohistochemistry under the microscope.For the experimental metastasis assay,B16BL6 cells infected with AdH1-siRNA-Survivin were injected into the tail veins of mice,then the visible black nodules on the lung surface were counted macroscopically at 21th day after tumor cell injection.Results AdH1-siRNA-Survivin could obviously decrease the expression of Survivin protein in B16BL6 cells.When compared with the blank control and AdH1-empty control,the tumor growth was inhibited and MVD was less in the mice injected with AdH1-siRNA-Survivin(P