Hypoxia enhances CXCR4 expression favoring microglia migration via HIF-1alpha activation

Migration toward pathological area is the first critical step in microglia engagement during the central nervous system (CNS) injury, although the molecular mechanisms underlying microglia mobilization have not been fully understood. Here, we report that hypoxia promotes stromal cell-derived factor-1α (SDF-1α) induced microglia migration by inducing the CXC chemokine receptor 4 (CXCR4) expression. Exposure to hypoxia significantly enhanced CXCR4 expression levels in N9 microglia cell. Then, cell migration induced by SDF-1, a CXCR4-specific ligand, was observed accelerated. Blockade of hypoxia inducible factor-1α (HIF-1α) activation by inhibitors of phosphoinositide-3-kinase (PI3K)/Akt signaling pathway abrogated both of hypoxia-induced CXCR4 up-regulation and cell-migration acceleration. These results point to a crucial role of Hypoxia-HIF-1α-CXCR4 pathway during microglia migration.     

Hypoxia-induced expression of HIF-1alpha and its target genes in umbilical venous endothelial cells of Tibetans and immigrant Han

The better adaptation of native Tibetans to hypoxia is thought to be partly due to improved umbilical circulation, which results in reduced pre- and postnatal fatalities. We hypothesized that the difference in umbilical circulation between native Tibetans and other high-altitude inhabitants was due to differences in the expression of hypoxia-induced factor (HIF-1) and its target genes vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). We tested this hypothesis by examining the effect of hypoxia on the expression of HIF-1alpha, VEGF, and iNOS in cultured umbilical venous endothelial cells (UVECs) from native Tibetans and immigrant Hans. UVECs were collected and cultured under hypoxic (0.5% oxygen) or normoxic conditions for 2, 4, 12 and 24 h. The mRNA levels of HIF-1alpha, VEGF, endothelial nitric oxide synthase (eNOS) and iNOS and the protein level of HIF-1alpha were determined with RT-PCR and Western blot analyses, respectively. In both immigrant Han and Tibetans, HIF-1alpha mRNA was constitutively expressed under normoxic condition, and remained constant after hypoxic exposure. In contrast, HIF-1alpha protein was undetectable under normoxic condition, but underwent dynamic changes in response to hypoxia. It was induced at 4 h, peaked at 12 h, and remained elevated at 24 h. Concurrent with the induction of HIF-1alpha protein, the mRNA levels of VEGF and iNOS were also up-regulated whereas that of eNOS was down-regulated. The lack of a hypoxia-related difference in the expression of HIF-1alpha and its target genes suggests that HIF-1alpha does not play a critical role in high altitude adaptation. Alternative mechanisms may be responsible for the better adaptation of native Tibetans.     

HIF-1alpha信号通路在肿瘤细胞调控中的研究进展

缺氧诱导因子（HIF-1alpha）是肿瘤细胞生长过程中重要的调控因子,研究其作用机制有利于实现对肿瘤细胞增殖的抑制作用。 HIF-1alpha可引起多种基因转录,使肿瘤细胞耐受低氧环境,进而使癌症患者在治疗过程中产生耐受反应,最终影响治疗效果,甚至 放弃治疗。因此,以HIF-1alpha为靶点是治疗肿瘤的重要手段和方法。本文对HIF-1alpha的基本概况及其主要信号通路（PI3K 通路、 HSP90 通路及MAPK通路）以及不同通路抑制剂（如LY294002、17AAG、PD98059、U0126、SB203580、SP600125 等）进行综述,并 对HIF-1alpha的应用前景进行展望。     

RNA interference as a tool for exploring HIV-1 robustness

Short interfering RNAs (siRNAs) that target viral genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, there is the potential for viral escape, particularly with a highly mutable target such as HIV-1. We present a novel strategy for anticipating and preventing viral escape using second-generation siRNAs. The evolutionary capacity of HIV-1 was tested by exerting strong selective pressure on a highly conserved sequence in the HIV-1 genome. We assayed the antiviral efficacy of five overlapping siRNAs directed against an essential region of the HIV-1 protease. Serial viral transfers in U87-CD4-CXCR4 cells were performed using four of the siRNAs. This procedure was repeated until virus breakthrough was detected. After several serial culture passages, resistant virus with a single point mutation in the targeted region was detected in the culture supernatants. The emergence of resistant virus was confirmed by molecular cloning and DNA sequencing of viral RNA. The most common escape route was the D30N mutation. Importantly, the addition of a second-generation siRNA that matched the D30N mutation restored viral inhibition and delayed development of escape variants. Passages performed with both siRNAs prevented the emergence of the D30N escape mutant and forced the virus to develop new escape routes. Thus, second-generation siRNAs can be used to block escape from RNA interference (RNAi) and to search for new RNAi escape routes. The protocol described here may be useful for exploring the sequence space available for HIV-1 evolution and for producing attenuated or deleterious viruses.     

RNA干扰引起的非特异性免疫反应及其机制研究探讨

基因的表达失控是疾病发生的主要原因之一,干扰靶基因的表达可能成为有效的治疗手段。RNA干扰技术是近年兴起的基因调控干预方法,其基础,特别是应用研究极受关注,人们期待RNA干扰能成为肿瘤、病毒感染等难治疾病的临床治疗手段。然而,这一新兴技术在应用研究过程中显现出诸多问题,如细胞毒性、引起机体非特异性反应等等。就RNA干扰引起的非特异性免疫反应展开综述,探讨其机制,期望为RNA干扰的应用研究提供一些思考。     

Silencing stromal interaction molecule 1 by RNA interference inhibits the proliferation and migration of endothelial progenitor cells

Knockdown of stromal interaction molecule 1 (STIM1) significantly suppresses neointima hyperplasia after vascular injury. Endothelial progenitor cells (EPCs) are the major source of cells that respond to endothelium repair and contribute to re-endothelialization by reducing neointima formation after vascular injury. We hypothesized that the effect of STIM1 on neointima hyperplasia inhibition is mediated through its effect on the biological properties of EPCs. In this study, we investigated the effects of STIM1 on the proliferation and migration of EPCs and examined the effect of STIM1 knockdown using cultured rat bone marrow-derived EPCs. STIM1 was expressed in EPCs, and knockdown of STIM1 by adenoviral delivery of small interfering RNA (siRNA) significantly suppressed the proliferation and migration of EPCs. Furthermore, STIM1 knockdown decreased store-operated channel entry 48 h after transfection. Replenishment with recombinant human STIM1 reversed the effects of STIM1 knockdown. Our data suggest that the store-operated transient receptor potential canonical 1 channel is involved in regulating the biological properties of EPCs through STIM1. STIM1 is a potent regulator of cell proliferation and migration in rat EPCs and may play an important role in the biological properties of EPCs.     

RNA干扰沉默缺氧诱导因子1α逆转肺癌细胞耐药性

目的：观察RNA干扰沉默缺氧诱导因子1α（HIF-1α）对肺癌细胞耐药性的影响。方法：构建靶向HIF-1α小干扰RNA基因,并转染到人肺腺癌耐顺铂细胞株A549／DDP细胞中。逆转录聚合酶链反应RT—PCR）检测细胞的HIF-1α、多药耐药基因-（MDR-1）以多药耐药相关蛋白基因（MRP）mRNA变化,免疫细胞化学法观察干扰后HIF-1α、P-糖蛋白以及MRP蛋白的变化。MTT法检测不同浓度的顺铂作用下细胞死亡率。结果：HIF-1αsiRNA组中H1F-1α、MDR—1、MRPmRNA水平显著降低（P〈0．05）。且蛋白水平也显著下降（P〈0．05）。HIF-1αsiRNA组细胞死亡率较未转染组均明显增高（P〈0．05）,转染siRNA阴性组不影响肿瘤细胞的耐药性。结论：HIF-1αsiRNA可显著降低A549／DDP细胞中H1F-1α、MDR-1、MRP表达,从而起到逆转肺腺癌A549／DDP细胞的耐药作用。     

Autologous blood transfusion promotes autophagy and inhibits hepatocellular carcinoma progression through HIF-1α signalling pathway

To explore the molecular mechanism of autologous blood transfusion promoting autophagy of hepatocellular carcinoma (HCC) cells and inhibiting the HCC progression through HIF-1α signalling pathway. This is a research paper. Rat hepatocellular carcinoma model and HepG2 cell model were built. The rats with HCC were conducted a surgery, and their blood was collected for detection to detect the recurrence and metastasis of the rats. Western blot was used to analysed the expression of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein. The apoptosis rate of HepG2 cells was detected by flow cytometry, and autophagosomes were observed by transmission electron microscopy. HIF-1α expression was measured by immunofluorescence assay. The expressions of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein were highest in model + autoblood group compared with the model group. HIF-1α content of model group was higher, but content of TP53, MDM2, ATG5 and ATG14 in the model group is the second. The highest apoptosis rate was found in HepG2 + autoblood group. The number of autophagosomes in HepG2 + autoblood was obviously larger than that of HepG2 + autoblood + inhibitor. HIF-1α expression of immunofluorescence assay showed that high expression of HIF-1α was clearly observed in HepG2 and HepG2 + autoblood group from confocal observation. However, there was no HIF-1α protein expression in HepG2 + autoblood + inhibitor group. The migration rate in HepG2 group, HepG2 + autoblood group and HepG2 + autoblood + inhibitor group was 85.71 ± 7.38%, 14.36 ± 6.54% and 61.25 ± 5.39%, respectively. Autologous blood transfusion promotes autophagy of HCC cells through HIF-1α signalling pathway, which further inhibits HCC migration and erosion.     

A fast, simple method for screening radiation susceptibility genes by RNA interference

Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes.     

Applications of RNA interference: current state and prospects for siRNA-based strategies in vivo

Within the recent years, RNA interference (RNAi) has become an almost-standard method for in vitro knockdown of any target gene of interest. Now, one major focus is to further explore its potential in vivo, including the development of novel therapeutic strategies. From the mechanism, it becomes clear that small interfering RNAs (siRNAs) play a pivotal role in triggering RNAi. Thus, the efficient delivery of target gene-specific siRNAs is one major challenge in the establishment of therapeutic RNAi. Numerous studies, based on different modes of administration and various siRNA formulations and/or modifications, have already accumulated promising results. This applies to various animal models covering viral infections, cancer and multiple other diseases. Continuing efforts will lead to the development of efficient and “double-specific” drugs, comprising of siRNAs with high target gene specificity and of nanoparticles enhancing siRNA delivery and target organ specificity.