Inhibitory Effects of Anti-sense PTTG on Malignant Phenotype of Human Ovarian Carcinoma Cell Line SK-OV-3

To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3.1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3.1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5 % and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3.1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.

Anti-sense oligonucleotide labeled with technetium-99m using hydrazinonictinamide derivative and N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline: A comparison of radiochemical behaviors and biological properties

AIM:To explore and compare the radiochemical behavior and biological property of anti-sense oligonuc-leotide (ASON) labeled with technetium-99m using N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycl ine (NHS-MAG3) and hydrazinonictinamide derivative (HYNIC). METHODS:After HYNIC and NHS-MAG3 were synthesized, ASON was labeled with technetium-99m using HYNIC and NHS-MAG3 as a bifunctional chelator. The in vivo and in vitro stability, binding rates of labeled compounds to serum albumen, biodistribution of 99mTc-MAG3-ASON and 99mTc-HYNIC-ASON in BALB/C mouse and its HT29 tumor cellular uptake were compared. RESULTS:The labeling efficiency and stability of 99mTc-MAG3-ASON were significantly higher than those of 99mTc-HYNIC-ASON (P = 0.02, and P = 0.03, respectively). 99mTc-MAG3-ASON had a significantly lower rate of binding to serum albumen than 99mTc-HYNIC-ASON (P < 0.05). In contrast to 99mTc-HYNIC-ASON, the biodistribution of 99mTc-MAG3-ASON was significantly lower in blood, heart, liver and stomach (P < 0.05), slightly lower in intestines and spleen (P > 0.05) and significantly higher in lung and kidney (P < 0.05). The HT29 tumor cellular uptake rate of 99mTc-MAG3-ASON was significantly higher than that of 99mTc-HYNIC-ASON (P < 0.05). CONCLUSION:99mTc-MAG3-ASON shows superior radiochemical behaviors and biological properties than 99mTc-HYNIC-ASON. 99mTc-MAG3-ASON is a potential radiopharmaceutical agent for in vivo application.

Expression of heparanase mRNA in anti-sense oligonucleotide-transfected human esophageal cancer EC9706 cells

AIM： To investigate the effed3 of anti-sense oligonucleotides （ASODNs） on mRNA expression of heparanase in human esophageal cancer EC9706 cells. METHODS： One non-sense oligonucleotide （N-ODN） and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied by in situ hybridization. RESULTS： The expression of heparanase mRNA could be inhibited by ASODNs.There was no significant difference among five ASODNs （P〉0.05）, but there was a significant difference between ASODNs and N-ODN or non-transfected group （ASODNI： 2.25±0.25, ASODN2： 2.21±0.23, ASODN3： 2.23±0.23, ASODN4：2.25±0.24 vs N-ODN： 3.47±2.80 or non- transfected group： 3.51±2.93 respectively, P〈0.05）. CONCLUSION： The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.

研究者们在探求“反义”(Anti-Sense)技术在医学和农业上的应用

对于正在探索所谓"反义因子"这项新技术的研究者来说,在培育各种美味水果、蔬菜,治疗各种遗传病,乃至艾滋病方面都可能取得成功。反义因子阻碍基因的正常功能。反义机制能够控制细菌的基因表达,对此人们已经知道10年了。不过,仅仅在几年前才发现,在真核细胞中也可以操纵这类因子米抑制一些"坏基因"的影响,譬如编码引起食品腐坏的一些酶的基因以及病毒复制所需的基因。

Effects of rAAV-CD151 and rAAV-antiCD151 on the Migration of Human Tongue Squamous Carcinoma Cell Line Tca8113

Summary: This study was designed to determine the effects of the recombinant adeno-associated virus vector containing sense CD151 gene (rAAV-CD151) and antisense CD151 gene (rAAV-antiCD151) on the migration of Tca8113 cell. Functional fragment of CD151 gene was amplified by RT-PCR, and inserted into the vector pAAV in the sense direction and antisense direction, respectively. The rAAV-CD151 and rAAV-antiCD151 were produced and the titers were determined by dot blot. The CD151, at protein level, was detected by Western blot. The Transwell chamber was used to detect the effects of the rAAV-CD151 and rAAV-antiCD151 on the tumor cell migration. The titers of the rAAV-CD151 and rAAV-antiCD151 were 2×10~11 pfu/ml and 1.0×10~11 pfu/ml, respectively. The expression of CD151 was increased by 108 % in the cells transfected with rAAV-CD151 and decreased by 79 % in the cells transfected with rAAV-antiCD151, as compared with non-transfected cells, respectively. The number of the migrating cells was significantly increased in the cells transfected with rAAV-CD151 (93.56±11.59) and decreased in the cells transfected with rAAV-antiCD151 (24.00±4.36) as compared with non-transfected and rAAV-GFP transfected cells (53.00±6.56 and 46.00±7.00, P<0.05). It is an important molecular mechanism of the tumor metastasis that the overexpression of CD151 promotes the migration of the tumor cells. The rAAV-antiCD151 is a novel tool, which can reduce the expression of CD151 and inhibit the migration of the tumor cells, and brings us a new approach of anti-sene gene therapy targeted at CD151 in human carcinoma.

Long noncoding RNA HAND2-AS1 re duce d the viability of hepatocellular carcinoma via targeting microRNA-300/SOCS5 axis

Background:Hepatocellular carcinoma(HCC)is one of the most prevalent human cancers with high mortality.Long non-coding RNA heart and neural crest derivatives expressed 2 anti-sense 1(HAND2-AS1)is down-regulated in several cancers including HCC,yet the precise mechanisms how HAND2-AS1 regulates cell survival in HCC remains poorly understood.Methods:The expression levels of HAND2-AS1 and miR-300 were measured using quantitative real-time PCR.The protein levels of suppressor of cytokine signaling 5(SOCS5),Bcl-2,Bax and cleaved caspase-3 were determined by Western blot.Cell viability and cell proliferation were assessed using cell counting kit-8 and clone formation assay,respectively.Cell apoptosis was detected using flow cytometry.The interactions between HAND2-AS1 and miR-300,miR-300 and SOCS5 were validated using luciferase reporter assay.Results:HAND2-AS1 was down-regulated in HCC tissues and cell lines,and the expression level of HAND2-AS1 was positively correlated to patient survival.HAND2-AS1 over-expression reduced viability and proliferation in HCC cells.Elevated HAND2-AS1 level induced apoptosis in HCC cells,accompanied with increased Bax and cleaved caspase-3 levels and decreased Bcl-2 level.We also validated that HAND2-AS1 acted as a sponge of miR-300,and there was a negative correlation between expression levels of HAND2-AS1 and miR-300 in HCC tissues.Furthermore,we found that SOCS5 was a downstream target of miR-300.In addition,miR-300 mimics abolished HAND2-AS1-mediated inhibition of cell viability and proliferation.miR-300 mimics also reversed the HAND2-AS1-induced apoptosis in HCC cells.Conclusion:lncRNA HAND2-AS1 inhibits proliferation in HCC through regulating miR-300/SOCS5 axis.

Transformation of Chinese Cabbage(Brassica rapa L. ssp. pekinensis)by Agrobacterium Micro-Injection into Flower Bud

We obtained two lines of Chinese head cabbage(Brassica rapa L. ssp. pekinensis)selfed progenies containing both an anti-sense gene of BcpLH and a gene for resistance to kanamycin by micro-injecting buds of their primary transformants(T0)with Agrobacterium tumefaciens strain LBA4404. 31 positive plants resistant to kanamycien were recovered. Southern blot analysis confirmed the presence of T-DNA in two transgenic plants. One(DHZ-13-1)exhibits the characteristics of out-toward rosette and cauline leaves, and nested flower model in which secondary complete flower developed from the base of the primary ovary and the third flower from the ovary in the secondary flower, and so on, while another(DHZ-6-1)has no phenotype change. ABA and IAA affected the root growth of progeny of DHZ-13-1, but 6-BA was insensitive to hypocotyl growth during its seedling development.

Role of Smad7 in inflammatory bowel diseases

Crohn's disease and ulcerative colitis,the major forms of inflammatory bowel diseases(IBD) in man,are complex diseases in which genetic and environmental factors interact to promote an excessive mucosal immune response directed against normal components of the bacterial microflora.There is also evidence that the pathologic process is due to defects in counterregulatory mechanisms,such as those involving the immunosuppressive cytokine transforming growth factor(TGF)-1.Indeed,studies in human IBD tissues and murine models of colitis have documented a disruption of TGF-1 signalling marked by a block in the phosphorylation of Smad3,a signalling molecule associated with the activated TGF-receptor,due to up-regulation of Smad7,an intracellular inhibitor of Smad3 phosphorylation.Knock-down of Smad7 with a specific antisense oligonucleotide restores TGF-1/Smad3 signalling,thus resulting in a marked suppression of inflammatory cytokine production and attenuation of murine colitis.These findings together with the demonstration that Smad7 antisense oligonucleotide is not toxic when administered in mice have paved the way for the development of a Smad7 antisense oligonucleotidebased pharmaceutical compound that is now ready to enter the clinics.In this article we review the available data supporting the pathogenic role of Smad7 in IBD and discuss whether and how Smad7 antisense therapy could help dampen the ongoing inflammation in IBD.

Fusions of Dendritic Cells and C6 Cells Transfected with TGF-β1 Antisense in Treatment of Intracranial Gliomas

Objective： To investigate the immunotherapy efficacy of fusion cells （dendritic-C6anti-TGF-β1 cells） in the treatment of intraeranial gliomas. Methods： Dendritic cells were isolated from rat bone-marrow precursors stimulated in vitro with granuloeyte-macrophage colony-stimulating factor （GM-CSF） and Interleukin-4 （IL-4）. C6anti-TGF-β1 cells originally from C6 cell line of a rat glioblastoma were transfected with plasmid of TGF-β1 anti-sense gene. Fusions of dendritic cells and C6anti-TGF-β1 cells were prepared by polyethylene glycol （PEG）. The DC/C6anti-TGF-β1 fusion cells were observed and confirmed by fight microscopy and scanning electron microscopy. Experimental rats were divided into three groups at random： C6 cells （Ⅰ）, dendritic-C6anti-TGF-β1 fusion cells and C6 cells （Ⅱ） and IMDM medium only （Ⅲ）. The cells were injected into right parietal lobe region of the rat with stereotaxic technique. Histology, tumor necrosis and survival time were evaluated. Results： Compared with the rats that received C6 cells （survival median time was less than 20 days, tumor region was seen in all fields of observed）, the rats injected with dendritic-C6anti-TGF-β1 fusion cells and C6 cells got a more prolonged life span （more than 59 days）, as well as less tumor region （5.01%-6.2%）. There was no tumor necrosis, but some glias were seen in surroundings. All rats were survived and no necrosis was observed in negative control group. Statistical analysis showed that group Ⅱ had significant difference compared with group Ⅰ. Conclusions： Dendritic-C6anti-TGF-β1 fusion cells could prolong the life span of rats, providing a strategy to achieve an antitumor response against tumors in the central nervous system.

Partial inhibition of CDK4 gene expression leads to the extension of G1 phase of V79-8 cells

V79-8 is an abnormal cell line which does not have detectable G1 and G2 phases in its cell cycle.This cell line is derived from V79 cell line which has G1 phase but lacks G2 phase. By using an anti-sense approach, CDK4 gene expression was partially inhibited to find whether CDK4 might contribute to the lack of G1 phase in V79-8 cells. Anti-CDK4 anti-sense plasmid was constructed and used to transfect V79-8 cells. Clones of transfected cells (V79-8-asCDK4) were examined, in comparison with V79-8 cells, to determine its growth curve, cell doubling-time (GT), the level of CDK4 gene expression and the levels of expression of some other growth related genes. V79-8-asCDK4 cells showed a slower growth rate with a doubling time 2.5-h longer than that of V79-8 cells. A flow cytometry (FCM) analysis demonstrated that the 2.5 h increase of the doubling time of V79-8-asCDK4 cells was mainly due to the appearance of G1 phase because its G2+M phase was not significantly different from that of V79-8 cells. The

Identification and characterization of a novel group of natural antisense transcripts from RNA1.2 gene locus of human cytomegalovirus

Background:Natural anti-sense transcripts(NATs),which are transcribed from the complementary DNA strand of annotated genes,exert regulatory function of gene expression.Increasing studies recognized anti-sense transcription widespread throughout human cytomegalovirus(HCMV)genome,whereas the anti-sense transcription of RNA1.2 gene locus has never been investigated.In this study,the transcription of the RNA1.2 anti-sense strand was investigated in clinically isolated HCMV strain.Methods:Strand-specific high-through RNA-sequencing(RNA-seq)was performed to find possible anti-sense transcripts(ASTs).For analyzing and visualization of RNA-seq data sets,Integrative Genomics Viewer software was applied.To confirm these possibilities,Northern blotting and rapid amplification of cDNA ends(RACE)were used.Results:Transcription of the opposite strand of RNA1.2 gene locus was detected by RNA-sequencing using RNAs extracted from human embryonic lung fibroblasts infected with HCMV clinical isolate HAN.At least three HCMV NATs,named RNA1.2 AST 1,RNA1.2 AST2,and RNA 1.2 AST3,were characterized by Northern blotting and RACE analyses.These RNA1.2 ASTs orientated from the complementary strand of RNA1.2 locus during the late phase of HCMV infection.The 5'-and 3'-termini of these transcripts were located within the opposite sequence of the predicted RNA1.2 gene.Conclusion:A cluster of novel NATs was transcribed from the opposite sequence of the HCMV RNA 1.2 gene region.