Transcriptome array screening and verification of oral leukoplakia carcinogenesis-related hypoxia-responsive gene and microRNA / 华西口腔医学杂志

OBJECTIVES@#To study the hypoxia response gene and microRNA (miRNA) expression profiles in the pathogenesis and progression of oral leukoplakia (OLK).@*METHODS@#Affymetrix GeneChip human transcriptome array 2.0 was used to detect the transcriptome of normal mucosa, low-risk OLK, high-risk OLK, and early squamous cell carcinoma (SCC). Gene ontology function analysis was used to screen genes and key miRNAs whose biological role is hypoxia response. Quantitative reverse transcription polymerase ch-ain reaction (qRT-PCR) was used to verify the expression of hypoxia response genes and miRNAs.@*RESULTS@#A total of 7 different genes of hypoxia response between normal mucosa and low-risk OLK, 10 genes between low-risk and high-risk OLK, and 21 genes between high-risk OLK and SCC were identified. The results of qRT-PCR showed that the expression of hypoxia-inducible factor 1α, chemokine cc-motif ligand 2, and matrix metalloproteinase 3 mRNA and miR-21 in normal mucosa, OLK, and SCC increased in a stepwise manner. The expression difference between OLK and SCC was statistically significant and consistent with the results of transcriptome array.@*CONCLUSIONS@#The hypoxia response gene and related miRNA play roles in the development and progression of OLK.

Single-stranded adeno-associated virus serotype 9 mediated gene expression in astrocyte via intravenous delivery / 上海交通大学学报（医学版）

Objective: To investigate the expression of LacZ gene mediated by intravenous injection of the single-stranded adeno-associated virus serotype 9 (ssAAV9) containing hypoxia-responsive element (HRE) promoter in the cerebral ischemic area, and further identify the types of brain cells that can be transfected by the vector. Methods: A mouse model of permanent left distal middle cerebral artery occlusion (dMCAO) was established. The expression of hypoxia-inducible factor-1 (HIF-1) in cerebral ischemic area was detected at 1 and 5 days after ischemia. The ssAAV vector containing HRE-regulated LacZ gene was packaged into the capsid of AAV9 virus (AAV9-H9LacZ), and AAV9-H9LacZ was injected into mice through jugular vein 1 h after the establishment of dMCAO model. Five days after injection of AAV9-H9LacZ, X-gal staining was used to detect the expression of β-galactosidase (β-gal) encoded by the LacZ gene in the ischemic area and liver tissue. Immunofluorescence double staining was used to detect the expression of β-gal in astrocyte, neurons and vascular endothelial cells. Results: The expression of HIF-1 was increased 1 and 5 days after ischemia. β-gal was mainly expressed in ischemic penumbra of mice injected with AAV9-H9LacZ. There was no positive expression of β-gal in the liver tissue of mice. β-gal was mainly co-expressed with glial fibrillary acidic protein as an astrocyte-specific marker, and a little of β-gal was co-expressed with neuron-specific nuclear protein. Conclusion: After cerebral ischemia, intravenous injection of AAV9-H9LacZ can effectively mediate gene expression in astrocyte in the cerebral ischemia area. HRE can effectively control the expression of the LacZ gene in cerebral ischemia.

Determination of the binding site of hypoxia inducible factor-2a to the MMP-2 promoter / 临床与实验病理学杂志

Purpose To identify the binding of hypoxia in-ducible factor-2a (HIF-2 a) to the hypoxia-response element(HRE) of the matrixmetallo proteinases-2 (MMP-2) gene promoter region and clear the binding site. Methods Electro-phoretic mobility shift assay (EMSA) was used to identify the binding of HIF-2 a to HRE of the MMP-2 gene promoter region in vitro. At the same time, the chromatin immunoprecipitation assay (CHIP) was used to further determine the binding site. Results Successful prediction of two potential HIF-2a binding sites of MMP-2 the promoter region, which were-217～-204 and-1 029 ～-1 007, respectively. Probe test shows that the marked efficiency of sense chain and antisense chain was above 50％, and they could be used for EMSA-electrophoretic mobility shift assay. The results of EMSA showed that there was a binding site of HIF-2 a sense chain and antisense chain moter region int-217～-204. The results of chromatin immuno-precipitation showed that in the experimental group and control group an about 250 bp fragment in MMP-2 promoter containing HRE region was amplified, suggesting that the protein of HIF-2a binded to the HRE in MMP-2 promoter region in vivo. Conclusion HIF-2 a in MMP-2 promoter regionne promoter region in vitro and in vivo.

Advances in the hypoxia-responsive antitumor drug nanocarriers and tumor hypoxia relieve / 中国药科大学学报

Hypoxia,a salient feature of solid tumors,is often associated with invasiveness,metastasis and resistance to anticancer drugs.The strategies including the use of oxygen-carriers based on hyperbaric oxygen and blood substitutes to transport oxygen into tumors or in situ generation of O2from the tumor microenvironment endogenous H2O2have been explored to relieve the tumor hypoxia and to improve therapeutic efficiency.In addi-tion,it is potential to design hypoxia-responsive nanocarriers based on tumor hypoxia microenvironment to deliver anticancer drugs to the targeted tumor site,thereby improve drug concentrations in targeted site,significantly increase the antitumor efficiency and reduce the side-effects of drugs.This review gives an overview of the advances in relieving tumor hypoxia and hypoxia-responsive nanocarriers for tumor to provide a reference for the research and development of new antitumor drugs.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20 percent O2) and hypoxia (less than 5 percent O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50 percent lower under hypoxia, while the HRE plasmid was about 50 percent higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.