1.Construction of X IAP-3′UTR-luciferase reporter vector and its activity analysis
Ning DONG ; Gaoxia GE ; Weiming ZHANG ; Wei ZHU ; Huaguo XU
International Journal of Laboratory Medicine 2014;(9):1098-1099,1102
Objective To construct the recombinant X-linked inhibitor of apoptosis protein(XIAP) gene 3′untranslational region (3′UTR)-luciferase reporter vector ,and analyze the microRNA(miRNA) which possibly regulate the expression of XIAP gene . Methods Polymerase chain reaction (PCR) was employed to amplify X IA P-3′UTR sequences from human cDNA ,in which luciferase reporter vector pGL3-Ctrl was inserted ,and the recombinant vector pGL3-Ctrl/XIAP was gained .Target Scan 6 .2 soft-ware was adopted to predict the miRNA which possibly combined with the X IA P-3′UTR .pGL3-Ctrl/XIAP recombinant plasmids and the miRNA were co-transfected into A549 cells ,and the X IA P-3′UTR-luciferase activity was measured .Results Confirmed by digestion and DNA sequencing ,the X IA P-3′UTR-luciferase reporter recombinant was successfully constructed .Prediction of miRNA target sites indicated that X IA P gene may be the target of miR-200b ,miR-200c and miR-429 .Compared with miRNA mim-ic ctrl group ,miR-200b ,miR-200c and miR-429 significantly reduced the luciferase activity of pGL 3-Ctrl/XIAP with statistically significant difference(P<0 .05) .Conclusion X IA P-3′UTR-luciferase reporter vector is successfully constructed .miR-200b ,miR-200c and miR-429 can obviously decrease the luciferase activity .
2.HIV-1 Nef promotes KSHV vIL-6-induced angiogenesis by regulating PTEN/PI3K signaling pathway
Shuihong YAO ; Xiaofei ZHU ; Chun LU ; Gaoxia GE
Chinese Journal of Microbiology and Immunology 2013;(8):627-633
Objective To investigate whether HIV-1 Nef could promote the angiogenesis and tu-morigenesis induced by KSHV vIL-6 through regulating PTEN/PI3K signaling pathway .Methods Lipo-some transfection was used to transfect cDNA of pPTEN , dominant-negative ( DN) construct of PI3K and control vector into endothelial cells , which stably express KSHV vIL-6 and HIV-1 Nef.Microtubule forma-tion assay and chicken chorioallantoic membrane ( CAM) assay were used to evaluate microtubule formation and angiogenesis , respectively .Expressions of PTEN and PI 3 K were measured by Western blot .Results Both overexpression of PTEN and inhibited expression of PI 3K suppressed the vIL-6-induced microtubule for-mation and angiogenesis in CAM mediated by Nef .Conclusion HIV-1 Nef enhances vIL-6-induced angio-genesis and tumorigenesis through regulating PTEN/PI3K signaling pathway .
3.MiR-503 regulates cisplatin resistance of human gastric cancer cell lines by targeting IGF1R and BCL2.
Tongshan WANG ; Gaoxia GE ; Yin DING ; Xin ZHOU ; Zebo HUANG ; Wei ZHU ; Yongqian SHU ; Ping LIU
Chinese Medical Journal 2014;127(12):2357-2362
BACKGROUNDStudies have shown that the drug resistance of gastric cancer cells can be modulated by abnormal expression of microRNAs (miRNAs). We investigated the role of miR-503 in the development of cisplatin resistance in human gastric cancer cell lines.
METHODSMiR-503 expression was measured by quantitative real-time PCR. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) and clonogenic assays were used to examine changes in cell viability and the drug resistance phenotype of cancer cells associated with upregulation or downregulation of the miRNA. A dual-luciferase activity assay was used to verify target genes of miR-503. Immunohistochemistry, Western blotting analysis, and a flow cytometric apoptosis assay were used to elucidate the mechanism by which miR-503 modulates drug resistance in cancer cells.
RESULTSMiR-503 was significantly downregulated in gastric cancer tissues and several gastric cancer cell lines. Additionally, downregulation of miR-503 in the cisplatin (DDP)-resistant gastric cancer cell line SGC7901/DDP was concurrent with the upregulation of insulin-like growth factor-1 receptor (IGF1R) and B-cell lymphoma 2 (BCL2) expression compared with the parental SGC7901 cell line. An in vitro drug sensitivity assay showed that overexpression of miR-503 sensitized SGC7901/DDP cells to cisplatin. The luciferase activity of reporters driven by IGF1R and BCL2 3'-untranslated regions in SGC7901/DDP cells suggested that IGF1R and BCL2 were both direct target genes of miR-503. Enforced miR-503 expression in SGC7901/DDP cells reduced expression of the target proteins, inhibited proliferation, and sensitized the cells to DDP-induced apoptosis.
CONCLUSIONOur findings suggest that hsa-miR-503 modulates cisplatin resistance of human gastric cancer cells at least in part by targeting IGF1R and BCL2.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Humans ; Immunohistochemistry ; MicroRNAs ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Real-Time Polymerase Chain Reaction ; Stomach Neoplasms ; genetics