Targeting lentiviral vectors to primordial germ cells(PGCs):An efficient strategy for generating transgenic chickens

Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.

Differentiation Character of Adult Mesenchymal Stem Cells andTransfection of MSCs with Lentiviral Vectors

This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lenti- viral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and trans- fection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Ag- gracan was positive in chondrogenic lineages and the expression of aggracan and type Ⅱ collagenwas significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.

Efficient production of transgenic chickens using self-inactive HIV-based lentiviral vectors

We demonstrated the simple and effective production of transgenic chickens, in which the enhanced green fluorescence protein （EGFP） was expressed by using third-generation self-inactive HIV-based lentiviral vectors. In our experiments, lentiviruses were injected into 204 fertilized eggs, from which 30 （15%） chickens were hatched. The exogenous gene was detected in the genomes of 16 out of 30 （53%） chickens. The green fluorescence signal was observed directly in various body parts, and was particularly significant in the testes. The transgenes were also found in the offspring of these chickens. The results indicate that HIV-based lentiviral vectors can be used to generate transgenic birds economically and effectively [ Current Zoology 55 （5）： 383 - 387,2009].

Construction of a lentiviral vector over - expressing BDNF and its transfection into adipose derived stem cell lines in vitro

Objective:To construct a recombinant lentiviral vector that co-express ZsGreen and BDNF and establish an Adipose derived stem cell (ADSC) line with stable BDNF expression down- regulation.Methods: BDNF were designed and inserted into the lentiviral vector p HBLV-CMVIE-Zs Green-Puro. After identification by DNA sequencing, the lentiviral vectors carrying BDNF were packaged in 293 cells. The transfection efficiency was observed under fluorescence microscope;the lentiviral particles were collected to infect mouse ADSCs, and BDNF level were assessed using real-time PCR and Western blotting.Results: DNA sequencing demonstrated successful construction of the BDNF lentivirus vectors. Real- time PCR and Western blotting showed a high level BDNF mRNA and protein.Conclusion: We have successfully established an ADSC model with stable BDNF expression which establish the basis for the potential application of ADSCs-BDNF in the treatment of AD.

Understanding the role of transmembrane 9 superfamily member 1 in bladder cancer pathogenesis

In this editorial we comment on the article by Wei et al,published in the recent issue of the World Journal of Clinical Oncology.The authors investigated the role of Transmembrane 9 superfamily member 1(TM9SF1)protein in bladder cancer(BC)carcinogenesis.Lentiviral vectors were used to achieve silencing or overexpression of TM9SF1 gene in three BC cell lines.These cell lines were then subject to cell counting kit 8,wound-healing assay,transwell assay,and flow cytometry.Proliferation,migration,and invasion of BC cells were increased in cell lines subjected to TM9SF1 overexpression.TM9SF1 silencing inhibited proliferation,migration and invasion of BC cells.The authors conclude that TM9SF1 may be an oncogene in bladder cancer pathogenesis.

STUB1基因RNA干扰慢病毒载体的构建与鉴定

目的:构建人STUB1基因RNA干扰(RNA interference,RNAi)慢病毒表达载体并进行鉴定。方法:针对筛选确定的人STUB1基因RNAi有效靶点序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经AgeI和EcoRI酶切后的pMagic 4.0载体连接,产生短发卡RNA慢病毒载体。PCR筛选阳性克隆,测序鉴定,并包装成慢病毒颗粒。结果:PCR鉴定与DNA测序证实,合成的含STUB1 shRNA慢病毒载体寡核苷酸链插入正确。STUB1 shRNA慢病毒载体在293T细胞中成功包装成慢病毒颗粒。结论:成功构建人STUB1基因RNAi慢病毒载体以及包装成功慢病毒颗粒,为研究STUB1在胶质瘤发生发展过程中相关信号通路的作用,提供了稳定感染细胞的载体。

E2F-1 overexpression inhibits human gastric cancer MGC-803 cell growth in vivo

AIM: To evaluate the influence of E2F-1 on the growth of human gastric cancer(GC) cells in vivo and the mechanism involved. METHODS: E2F-1 recombinant lentiviral vectors were injected into xenograft tumors of MGC-803 cells in nude mice, and then tumor growth was investigated. Overexpression of transcription factor E2F-1 was assessed by reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting analysis. Apoptosis rates were determined using a terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL) assay. Expression levels of certain cell cycle regulators and apoptosis-related proteins, such as Bax, survivin, Bcl-2, cyclin D1, S-phase kinaseassociated protein 2, and c-Myc were examined by Western blotting and RT-PCR. RESULTS: Xenograft tumors of MGC-803 cells in nude mice injected with E2F-1 recombinant lentiviral vectors stably overexpressed the E2F-1 gene as measured by semi-quantitative RT-PCR(relative m RNA expression: 0.10 ± 0.02 vs 0.05 ± 0.02 for control vector and 0.06 ± 0.03 for no infection; both P < 0.01) and Western blotting(relative protein expression: 1.90 ± 0.05 vs 1.10 ± 0.03 in control vector infected and 1.11 ± 0.02 for no infection; both P < 0.01). The growth-curve of tumor volumes revealed that infection with E2F-1 recombinant lentiviral vectors significantly inhibited the growth of human GC xenografts(2.81 ± 1.02 vs 6.18 ± 1.15 in control vector infected and 5.87 ± 1.23 with no infection; both P < 0.05) at 15 d after treatment. TUNEL analysis demonstrated that E2F-1 overexpression promoted tumor cell apoptosis(18.6% ± 2.3% vs 6.7% ± 1.2% in control vector infected 6.3% ± 1.2% for no infection; both P < 0.05). Furthermore, lentiviral vector-mediated E2F-1 overexpression increased theexpression of Bax and suppressed survivin, Bcl-2, cyclin D1, Skp2, and c-Myc expression in tumor tissue.CONCLUSION: E2F-1 inhibits growth of GC cells via regulating multiple signaling pathways, and may play an important role in targeted therapy for GC.

Peroxisome proliferator-activated receptor gamma inhibits hepatic fibrosis in rats

BACKGROUND:Hepatic fibrosis is a necessary step in the development of hepatic cirrhosis.In this study we used lentiviral vector-mediated transfection technology to evaluate the effect of peroxisome proliferator-activated receptor gamma(PPAR-γ) on rat hepatic fibrosis. METHODS:Hepatic fibrosis in rats was induced by CCl4 for 2 weeks(early fibrosis)and 8 weeks(sustained fibrosis).The rats were randomly divided into four groups:normal control, fibrosis,blank vector,and PPAR-γ.They were infected with the recombinant lentiviral expression vector carrying the rat PPAR-γgene by portal vein injection.The liver of the rats was examined histologically and hydroxyproline was assessed.In vitro primary hepatic stellate cells(HSCs)were infected with the recombinant lentiviral expression vector carrying the rat PPAR-γgene.The status of HSC proliferation was measured by the MTT assay.The protein levels of PPAR-γ,α-smooth muscle actin(α-SMA)and type I collagen expression were evaluated by the Western blotting method. RESULTS:In vitro studies revealed that expression of PPAR-γ inhibited expression ofα-SMA and type I collagen in activated HSCs(P<0.01)as well as HSC proliferation(P<0.01).In vivo experiments indicated that in the early hepatic fibrosis group,the hydroxyproline content and the level of collagen I protein in the liver in the PPAR-γtransfected group were not significantly different compared to the hepatic fibrosis group and the blank vector group;whereas the expressions of PPAR-γ andα-SMA were different compared to the hepatic fibrosis group(P<0.01).In the sustained hepatic fibrosis group,there were significant differences in the hydroxyproline content and the expression of PPAR-γ,α-SMA,and type I collagen between each group.CONCLUSION:PPAR-γcan inhibit HSC proliferation and hepatic fibrosis,and suppressα-SMA and type I collagen expression.

Adipose-derived stem cells modified by BDNF gene rescue erectile dysfunction after cavernous nerve injury

Cavernous nerve injury is the main cause of erectile dysfunction following radical prostatectomy.The recovery of erectile function following radical prostatectomy remains challenging.Our previous studies found that injecting adipose-derived stem cells(ADSCs)into the cavernosa could repair the damaged cavernous nerves,but the erectile function of the treated rats could not be restored to a normal level.We evaluated the efficacy of ADSCs infected with a lentiviral vector encoding rat brain-derived neurotrophic factor(lenti-rBDNF)in a rat model of cavernous nerve injury.The rats were equally and randomly divided into four groups.In the control group,bilateral cavernous nerves were isolated but not injured.In the bilateral cavernous nerve injury group,bilateral cavernous nerves were isolated and injured with a hemostat clamp for 2 minutes.In the ADSCGFP and ADSCrBDNF groups,after injury with a hemostat clamp for 2 minutes,rats were injected with ADSCs infected with lenti-GFP(1×106 in 20μL)and lenti-rBDNF(1×106 in 20μL),respectively.Erectile function was assessed 4 weeks after injury by measuring intracavernosal pressures.Then,penile tissues were collected for histological detection and western blot assay.Results demonstrated that compared with the bilateral cavernous nerve injury group,erectile function was significantly recovered in the ADSCGFP and ADSCrBDNF groups,and to a greater degree in the ADSCrBDNF group.Neuronal nitric oxide synthase content in the dorsal nerves and the ratio of smooth muscle/collagen were significantly higher in the ADSCrBDNF and ADSCGFP groups than in the bilateral cavernous nerve injury group.Neuronal nitric oxide synthase expression was obviously higher in the ADSCrBDNF group than in the ADSCGFP group.These findings confirm that intracavernous injection with ADSCs infected with lenti-rBDNF can effectively improve erectile dysfunction caused by cavernous nerve injury.This study was approved by the Medical Animal Care and Welfare Committee of Wuhan University,China(approval No.2017-1638)on June 20,2017.

miRNA-155 Modulates the Malignant Biological Characteristics of NK/T-Cell Lymphoma Cells by Targeting FOXO3a Gene

This study investigated the effects of miRNA-155 on malignant biological characteristics of NK/T-cell lymphoma cell lines and the possible mechanism. The expression of miRNA-155 was detected in lymphoma cell lines from different sources （SNK-6, YTS, Jurkat and DOHH2） by real-time PCR. Lentiviral vectors （pLL3.7） that could overexpress or downexpress miRNA-155 were constructed. Recombinant lentiviral particles were prepared and purified, and their titers determined. The expression of miRNA-155 in the infected SNK-6 cells and the cell proliferation were detected by PCR and CCK-8, respectively. Flow cytometry was used to determine the apoptosis of infected SNK-6 cells. The target of miRNA155 was predicted from Targetscan website. The effect of miRNA155 on FOXO3a expression was examined by Western blotting. The results showed that among the human NK/T-cell lymphoma cell lines SNK-6, YTS, Jurkat and DOHH2, the expression of miRNA-155 was highest in SNK-6. The infection efficiency of the recombinant lentivirns in SNK-6 was more than 70% at multiplicity of infection （MOI） of 100. The expression of miRNA-155 was significantly increased in SNK-6 cells infected by lentivirus vectors with high expression of miRNA-155 （4 times higher than the control group）, and profoundly decreased in those infected with lentivirnses with low expression of miRNA-155. The proliferation of letivirns-infected SNK-6 cells was decreased as the expression of miRNA-155 reduced. The apoptosis rate was increased with the reduction in the expression of miRNA-155. FOXO3a was found to be a possible target of miRNA155, as suggested by Targetscan website. Western blotting showed that the expression of FOXO3a was significantly elevated in SNK-6 cells with miRNA-155 inhibition. It was concluded that reduction in miRNA-155 expression can inhibit the proliferation of SNK-6 lymphoma cells and promote their apoptosis, which may be associated with regulation of FOXO3a gene.

Exosomes derived from bone marrow mesenchymal stem cells inhibit neuroinflammation after traumatic brain injury

Exosomes derived from bone marrow mesenchymal stem cells can inhibit neuroinflammation through regulating microglial phenotypes and promoting nerve injury repair.However,the underlying molecular mechanism remains unclear.In this study,we investigated the mechanism by which exosomes derived from bone marrow mesenchymal stem cells inhibit neuroinflammation.Our in vitro co-culture experiments showed that bone marrow mesenchymal stem cells and their exosomes promoted the polarization of activated BV2 microglia to their anti-inflammatory phenotype,inhibited the expression of proinflammatory cytokines,and increased the expression of anti-inflammatory cytokines.Our in vivo experiments showed that tail vein injection of exosomes reduced cell apoptosis in cortical tissue of mouse models of traumatic brain injury,inhibited neuroinflammation,and promoted the transformation of microglia to the anti-inflammatory phenotype.We screened some microRNAs related to neuroinflammation using microRNA sequencing and found that microRNA-181b seemed to be actively involved in the process.Finally,we regulated the expression of miR181b in the brain tissue of mouse models of traumatic brain injury using lentiviral transfection.We found that miR181b overexpression effectively reduced apoptosis and neuroinflamatory response after traumatic brain injury and promoted the transformation of microglia to the anti-inflammatory phenotype.The interleukin 10/STAT3 pathway was activated during this process.These findings suggest that the inhibitory effects of exosomes derived from bone marrow mesenchymal stem cells on neuroinflamation after traumatic brain injury may be realized by the action of miR181b on the interleukin 10/STAT3 pathway.

Inhibition of SW620 human colon cancer cells by upregulating mi RNA-145

AIM: To investigate the targeted inhibition of proliferation and migration of SW620 human colon cancer cells by upregulating mi RNA-145(mi R-145).METHODS: Forty-five samples of colon cancer tissues and 45 normal control samples were obtained from the biological database of the First Affiliated Hospital of Liaoning Medical University. We performed quantitative analysis of mi R-145 and N-ras expression in tissues; reverse transcriptase polymerase chain reaction analysis of mi R-145 expression in SW620 colon cancer cells and normal colonic epithelial cells; construction of mi R-145 lentiviral vector and determination of mi R-145 expression in SW620 cells transduced with mi R-145 vector; analysis of the effect of mi R-145 overexpression on SW620 cell proliferation; analysis of the effect of mi R-145 overexpression on SW620 cell migration using a wound healing assay; and analysis of the effect ofmi R-145 on N-ras expression using Western blotting. RESULTS: mi R-145 expression was significantly downregulated in colon cancer tissues, with its expression in normal colonic tissues being 4-5-fold higher(two sample t test, P < 0.05), whereas N-ras expression showed the opposite trend. mi R-145 expression in SW620 cells was downregulated, which was significantly lower compared to that in colonic epithelial cells(two sample t test, P < 0.05). mi R-145 vector and control were successfully packaged; expression of mi R-145 in SW620 cells transduced with mi R-145 was 8.2-fold of that in control cells(two sample t test, P < 0.05). The proliferation of mi R-145-transduced SW620 cells was significantly decreased compared to control cells(two sample t test, P < 0.05). At 48 h in the wound healing experiment, the migration indexes and controls were(97.27% ± 9.25%) and(70.22% ± 6.53%), respectively(two sample t test, P < 0.05). N-ras expression in mi R-145-tranduced SW620 cells was significantly lower than others(one-way analysis of variance, P < 0.05). CONCLUSION: mi R-145 is important in inhibiting colon cancer cell proliferation and migration. This is a good foundation for development of colon cancer therapy by targeting tumor suppressor mi R-145.

TrkA regulates the regenerative capacity of bone marrow stromal stem cells in nerve grafts

We previously demonstrated that overexpression of tropomyosin receptor kinase A(TrkA)promotes the survival and Schwann celllike differentiation of bone marrow stromal stem cells in nerve grafts,thereby enhancing the regeneration and functional recovery of the peripheral nerve.In the present study,we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts.Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA,TrkA-shRNA or the respective control.The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect.Then,8 weeks after surgery,hematoxylin and eosin staining showed that compared with the control groups,the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged,whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group.Western blot assay showed that compared with the control groups,the TrkA overexpressing group had higher expression of the myelin marker,myelin basic protein and the axonal marker neurofilament 200.The TrkA overexpressing group also had higher levels of various signaling molecules,including TrkA,pTrkA(Tyr490),extracellular signal-regulated kinases 1/2(Erkl/2),pErk1/2(Thr202/Tyr204),and the anti-apoptotic proteins Bcl-2 and Bcl-xL.In contrast,these proteins were downregulated,while the pro-apoptotic factors Bax and Bad were upregulated,in the TrkA-shRNA group.The levels of the TrkA effectors Akt and pAkt(Ser473)were not different among the groups.These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway.All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University,China in December 2014(approval No.AEWC-2014-001219).

Protective effects of ciliary neurotrophic factor on the retinal ganglion cells by injure of hydrogen peroxide

AIM： To explore the effect of ciliary neurotrophic factor （CNTF） on retinal ganglion cell （RGC）-5 induced by hydrogen peroxide （H2O2）. METHODS： After cell adherence, RGC-5 culture medium was changed to contain different concentrations of H2O2 from 50 to 150 μmol/L at four time points （0.5, 1, 1.5 and 2h） to select the concentration and time point for H2O2 induced model. Two different ways of interventions for injured RGC-5 cells respectively were CNTF as an addition in the culture medium or recombinant lentiviral plasmid carrying CNTF gene transfecting bone mesenchymal stem cells （BMSCs） for co-culture with RGC-5. RESULTS： Compared to the control group, H2O2 led to RGC-5 death closely associated with concentrations and action time of H2O2 and we chose 125 μmol/L and 2h to establish the H2O2-induced model. While CNTF inhibited the loss of RGC-5 cells obviously with a dose-dependent survival rate. Nevertheless two administration routes had different survival rate yet higher rate in recombinant lentiviral plasmid group but there were no statistically significant differences. CONCLUSION： Both the two administration routes of CNTF have effects on RGC-5 cells induced by H2O2. If their own advantages were combined, there may be a better administration route.

On the development of optical peripheral nerve interfaces

Limb loss and spinal cord injury are two debilitating conditions that continue to grow in prevalence. Prosthetic limbs and limb reanimation present two ways of providing affected individuals with means to interact in the world. These techniques are both dependent on a robust interface with the peripheral nerve. Current methods for interfacing with the peripheral nerve tend to suffer from low specificity, high latency and insufficient robustness for a chronic implant. An optical peripheral nerve interface may solve some of these problems by decreasing invasiveness and providing single axon specificity. In order to implement such an interface three elements are required:(1) a transducer capable of translating light into a neural stimulus or translating neural activity into changes in fluorescence,(2) a means for delivering said transducer and(3) a microscope for providing the stimulus light and detecting the fluorescence change. There are continued improvements in both genetically encoded calcium and voltage indicators as well as new optogenetic actuators for stimulation. Similarly, improvements in specificity of viral vectors continue to improve expression in the axons of the peripheral nerve. Our work has recently shown that it is possible to virally transduce axons of the peripheral nerve for recording from small fibers. The improvements of these components make an optical peripheral nerve interface a rapidly approaching alternative to current methods.

Tetracysteine as a Reporter for Gene Therapy

Objective To study the feasibility of using tetracysteine （TC） reporter in gene therapy. Methods Effects of TC reporter and conventional reporter genes encoding green fluorescence protein （GFP） and luciferase （Luc） on expression and function of the therapeutic gene MGMT^P140K were compared. Cytotoxicity and drug resistance were studied by Western blot. TC reporter used in therapy was analyzed by flow cytometry （FCM）. Results The TC reporter had no toxicity to cells and neither affected the expression or activity of therapeutic gene as compared to GFP and Luc. TC could be used in blood sample detection. Conclusion TC is a new kind of reporter gene for lentiviral vector in future gene therapy.

Effects of Over-expression of ANXA10 Gene on Proliferation and Apoptosis of Hepatocellular Carcinoma Cell Line HepG2

The effects of over-expression of ANXA10 gene on proliferation and apoptosis of hepatocellular carcinoma cell line HepG2 were elucidated.The human ANXA10 gene was subcloned into the lentiviral vector,PGC-FU,to generate the lentiviral expression vector,PGC-FU-ANXA10.The corrected ANXA10 was confirmed by endoenzyme digestion,and sequencing.Recombinant lentiviruses were produced by 293T cells following the co-transfection of PGC-FU-ANXA10 with the packaging plasmids pHelper1.0 and pHelper2.0.The resulting recombinant lentiviruses carrying ANXA10 were then used to infect human embryonic kidney epithelial cells,and lentiviral particles were produced.The ANXA10 expression in 293T cells was detected by using fluorescent microscope and Western blotting.HepG2 cells were infected,and divided into PGC-Fu-ANXA10 group,PGC-Fu group and HepG2 cell group.The changes of ANXA10 mRNA and protein expression were detected by using RT-PCR and Western blotting respectively.Flow cytometry and MTT assay were performed to examine the changes in cell apoptosis and proliferation respectively.The recombinant PGC-FU-ANXA10 vector was successfully constructed,the ANXA10 protein was detected by using Western blotting,and virus titer was 2×108 TU/mL.The recombinant lentiviruses were effectively infected into HepG2 cells in vitro and the infection efficiency was 70%.At 72 h after infection,the ANXA10 mRNA and protein expression levels in PGC-Fu-ANXA10 group were significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05);the in vitro growth inhibition rate of HepG2 cells in PGC-Fu-ANXA10 group was 24.65%,significantly higher than that in PGC-Fu group and HepG2 cell group (P<0.05),but there was no significant difference between PGC-Fu group and HepG2 cell group;the apoptosis rate in PGC-Fu-ANXA10 group,PGC-Fu group and HepG2 cell group was (51.92±1.41)%,(19.00±1.12)% and (3.59±0.89)% respectively.The apoptosis rate in PGC-Fu-ANXA10 group was significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05).The recombinant lentiviruses PGC-FU-ANXA10 were constructed successfully and infected into HepG2 cells.The overexpression of ANXA10 gene can significantly inhibit proliferation and promote apoptosis of HepG2 cells in vitro.

Identification of host miRNAs that may limit human rhinovirus replication

AIM: To test whether the replication of human rhinovirus(HRV) is regulated by micro RNAs in human bronchial epithelial cells.METHODS: For the present study, the human cell line BEAS-2B(derived from normal human bronchial epithelial cells) was adopted. DICER knock-down, by si RNA transfection in BEAS-2B cells, was performed in order to inhibit micro RNA maturation globally. Alternatively, antisense oligonucleotides(anti-mi Rs) were transfectedto inhibit the activity of specific micro RNAs. Cells were infected with HRV-1B. Viral replication was assessed by measuring the genomic viral RNA by reverse transcription quantitative polymerase chain reaction(RT-q PCR). Association between micro RNA-induced-silencing-complex and viral RNA was detected by Ago2 co-immunoprecipitation followed by RT-q PCR. Targetscan v.6 was used to predict micro RNA target sites on several HRV strains.RESULTS: Here, we show that micro RNAs affect replication of HRV-1B. DICER knock-down significantly reduced the expression of mature micro RNAs in a bronchial epithelial cell line(BEAS-2B) and in turn, increased the synthesis of HRV-1B RNA. Additionally, HRV-1B RNA co-immunoprecipitated with argonaute 2 protein, an important effector for micro RNA activity suggesting that micro RNAs bind to viral RNA during infection. In order to identify specific micro RNAs involved in this interaction, we employed bioinformatics analysis, and selected a group of micro RNAs that have been reported to be under-expressed in asthmatic bronchial epithelial cells and were predicted to target different strains of rhinoviruses(HRV-1B,-16,-14,-27). Our results suggest that, out of this group of micro RNAs, mi R-128 and mi R-155 contribute to the innate defense against HRV-1B: transfection of specific anti-mi Rs increased viral replication, as anticipated in-silico.CONCLUSION: Taken together, our results suggest that pathological changes in micro RNA expression, as already reported for asthma or chronic obstructive pulmonary disease have the potential to affect Rhinovirus replication and therefore may play a role in virusinduced exacerbations.

Expression of human adrenomedullin gene in gastric adenocarcinoma and construction and identification of adrenomedullin overexpression vector and adrenomedullin-shRNA vector

Background:To explore the expression of adrenomedullin in gastric adenocarcinoma tissues,and to construct a eukaryotic expression vector that effectively overexpresses adrenomedullin gene and a short hairpin RNA eukaryotic expression vector that effectively inhibits adrenomedullin gene.This study lays the foundation for exploring the impact of adrenomedullin on solid tumors.Methods:A total of 60 samples of gastric adenocarcinoma tissues and adjacent tissues were collected.Immunohistochemical staining was used to verify the expression of adrenomedullin in gastric adenocarcinoma and the adjacent tissues.According to the adrenomedullin gene sequence in the National Center for Biotechnology Information and the design principle of the small interfering RNA target sequence,design and construct three specific short hairpin RNA expression vectors targeting the adrenomedullin gene mRNA and one homology using the lentiviral vector KLPO.1.The negative control vector,RT-PCR and Western blotting methods were used to detect the expression of the adrenomedullin gene,and the expression vector with the best inhibitory effect was selected.The eukaryotic expression vector pcDNA3 was used to construct an overexpression vector containing the full length of the adrenomedullin cDNA.RT-PCR and Western blotting methods were used to detect the expression of the adrenomedullin gene.Results:The PLKO.1-adrenomedullin with the best inhibitory effect and the human adrenomedullin gene overexpression vector pcDNA-adrenomedullin were successfully constructed and screened.Conclusion:Adrenomedullin is highly expressed in gastric cancer,and effectively inhibiting the expression of adrenomedullin in gastric cancer may have certain value in the treatment of gastric cancer.

CO-TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS WITH HUMAN BMP2 AND VEGF165 GENES

Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.