Retraction Note: Nuclear factor of activated T cells 5 maintained by Hotair suppression of miR-568 upregulates S100 calcium binding protein A4 to promote breast cancer metastasis.

The authors are retracting this article [1] after an investigation by the Ethics Committee of the Fourth Military Medical University (Xi'an, Shaanxi, China) of the following concerns that had been raised with respect to two of the figures.

Nuclear factor of activated T cells 5 maintained by Hotair suppression of miR-568 upregulates S100 calcium binding protein A4 to promote breast cancer metastasis.

INTRODUCTION: The onset of distal metastasis, which underlies the high mortality of breast cancers, warrants substantial studies to depict its molecular basis. Nuclear factor of activated T cells 5 (NFAT5) is upregulated in various malignancies and is critically involved in migration and invasion of neoplastic cells. Nevertheless, the metastasis-related events potentiated by this transcriptional factor and the mechanism responsible for NFAT5 elevation in carcinoma cells remain to be fully elucidated. METHODS: The correlation of NFAT5 with breast cancer invasiveness was investigated in vitro and clinically. The genes transcriptionally activated by NFAT5 were probed and their roles in breast cancer progression were dissected. The upstream regulators of NFAT5 were studied with particular attempt to explore the involvement of non-coding RNAs, and the mechanism underlying the maintenance of NFAT5 expression was deciphered. RESULTS: In metastatic breast cancers, NFAT5 promotes epithelial-mesenchymal transition (EMT) and invasion of cells by switching on the expression of the calcium binding protein S100A4, and facilitates the angiogenesis of breast epithelial cells and thus the development of metastases by transcriptionally activating vascular endothelial growth factor C (VEGF-C). NFAT5 is directly targeted by miR-568, which is in turn suppressed by the long non-coding RNA, Hotair, via a documented in trans gene silencing pattern, that is recruitment of the polycomb complex (Polycomb Repressive Complex 2; PRC2) and LSD1, and consequently methylation of histone H3K27 and demethylation of H3K4 on the miR-568 loci. CONCLUSION: This study unravels a detailed role of NFAT5 in mediating metastatic signaling, and provides broad insights into the involvement of Hotair, in particular, by transcriptionally regulating the expression of microRNA(s), in the metastasis of breast cancers.

c-Myc-mediated epigenetic silencing of MicroRNA-101 contributes to dysregulation of multiple pathways in hepatocellular carcinoma.

UNLABELLED: The MYC oncogene is overexpressed in hepatocellular carcinoma (HCC) and has been associated with widespread microRNA (miRNA) repression; however, the underlying mechanisms are largely unknown. Here, we report that the c-Myc oncogenic transcription factor physically interacts with enhancer of zeste homolog 2 (EZH2), a core enzymatic unit of polycomb repressive complex 2 (PRC2). Furthermore, miR-101, an important tumor-suppressive miRNA in human hepatocarcinomas, is epigenetically repressed by PRC2 complex in a c-Myc-mediated manner. miR-101, in turn, inhibits the expression of two subunits of PRC2 (EZH2 and EED), thus creating a double-negative feedback loop that regulates the process of hepatocarcinogenesis. Restoration of miR-101 expression suppresses multiple malignant phenotypes of HCC cells by coordinate repression of a cohort of oncogenes, including STMN1, JUNB, and CXCR7, and further increases expression of endogenous miR-101 by inhibition of PRC2 activation. In addition, co-overexpression of c-Myc and EZH2 in HCC samples was closely associated with lower expression of miR-101 (P < 0.0001) and poorer prognosis of HCC patients (P < 0.01). CONCLUSIONS: c-Myc collaborates with EZH2-containing PRC2 complex in silencing tumor-suppressive miRNAs during hepatocarcinogenesis and provides promising therapeutic candidates for human HCC.

MiR-568 inhibits the activation and function of CD4⁺ T cells and Treg cells by targeting NFAT5.

CD4(+) T cells play critical roles in orchestrating adaptive immune responses. Their activation and proliferation are critical steps that occur before they execute their biological functions. Despite the important role of this process, the underlying molecular events are not fully understood. MicroRNAs (miRNAs) have been shown to play important roles in lymphocyte development and function. However, the miRNAs that regulate T-cell differentiation, activation and proliferation are still largely unknown. In our previous study, using a miRNA array, we found that several miRNAs (including miR-202, 33b, 181c, 568 and 576) are differentially expressed between resting and activated CD4(+) T cells. In this study, we focused on the function of miR-568 during CD4(+) T-cell activation. We showed that the expression level of miR-568 decreased during the activation of T cells, including Jurkat cells and human peripheral blood CD4(+) T cells. When Jurkat or human peripheral blood CD4(+) T cells were transfected with miR-568 mimics, cell activation was significantly inhibited, as shown by the inhibited expression of activation markers such as CD25, CD69 and CD154; decreased IL-2 production; and inhibited cell proliferation. Using software predictions and confirmatory experiments, we demonstrated that nuclear factor of activated T cells 5 (NFAT5) is a target of miR-568. Treg cells are an important CD4(+) T-cell subpopulation, so we also evaluated the function of miR-568 in Treg-cell activation and differentiation. We showed that the miR-568 level decreased, while the NFAT5 protein level increased during CD4(+)CD25(+) Treg-cell activation, and the transfection of miR-568 mimics inhibited the NFAT5 expression, inhibited the production of both TGF-ß and IL-10 and also inhibited the proliferation of Treg cells. Our further study showed that over-expression of miR-568 can inhibit Treg-cell differentiation and can inhibit the suppressive effect of these cells on effector cells. In addition, inhibition of NFAT5 by siRNA-mediated knockdown can inhibit the activation and differentiation of Treg cells. These findings reveal that miR-568 can inhibit the activation and function of both CD4(+) T cells and Treg cells by targeting NFAT5. Since miR-568 plays an important role in both CD4(+) T cells and Treg cells, these findings may provide leads for the development of novel treatments for human inflammatory and autoimmune diseases.

B7-H1 expression is associated with poor prognosis in colorectal carcinoma and regulates the proliferation and invasion of HCT116 colorectal cancer cells.

BACKGROUND AND OBJECTIVE: The investigation concerning the B7-H1 expression in colorectal cancer cells is at an early stage. It is unclear whether B7-H1 expression may have diagnostic or prognostic value in colorectal carcinoma. Additionally, how B7-H1 is associated with the clinical features of colorectal carcinoma is not known. In order to investigate the relationship between B7-H1 and colorectal cancer, we analyzed B7-H1 expression and its effect in clinical specimens and HCT116 cells. METHODS: Paraffin-embedded specimens from 143 eligible patients were used to investigate the expression of CD274 by immunohistochemistry. We also examined whether B7-H1 itself may be related to cell proliferation, apoptosis, migration and invasion in colon cancer HCT116 cells. RESULTS: Our results show that B7-H1 was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status and TNM (Tumor Node Metastasis) stage. Patients with positive B7-H1 expression showed a trend of shorter survival time. Using multivariate analysis, we demonstrate that positive B7-H1 expression is an independent predictor of colorectal carcinoma prognosis. Our results indicate that B7-H1 silencing with siRNA inhibits cell proliferation, migration and invasion. Furthermore, cell apoptosis was also increased by B7-H1 inhibition. CONCLUSIONS: Positive B7-H1 expression is an independent predictor for colorectal carcinoma prognosis. Moreover, knockdown of B7-H1 can inhibit cell proliferation, migration and invasion.

Tamoxifen represses miR-200 microRNAs and promotes epithelial-to-mesenchymal transition by up-regulating c-Myc in endometrial carcinoma cell lines.

Although tamoxifen (TAM), a selective estrogen receptor modulator, has been widely used in the treatment of hormone-responsive breast cancer, its estrogen-like effect increases the risk of endometrial cancer. However, the molecular mechanisms of TAM-induced endometrial carcinoma still remain unclear. In this report, we explored the role of microRNAs (miRNAs) in TAM-induced epithelial-mesenchymal transition (EMT) in ECC-1 and Ishikawa endometrial cancer cell lines and found miR-200 is involved in this process via the regulation of c-Myc. When treated with TAM, ECC-1 and Ishikawa cells were characterized by higher invasiveness and motility and underwent EMT. miR-200, a miRNA family with tumor suppressive functions in a wide range of cancers, was found reduced in response to TAM treatment. Consistent with zinc finger E-box binding homeobox 2, which was confirmed as a direct target of miR-200b in endometrial cancer cell lines, some other key factors of EMT such as Snail and N-cadherin increased, whereas E-cadherin decreased in the TAM-treated cells, contributing to TAM-induced EMT in these endometrial cancer cells. In addition, we showed that c-Myc directly binds to and represses the promoter of miR-200 miRNAs, and its up-regulation in TAM-treated endometrial cancer cells leads to the down-regulation of miR-200 and eventually to EMT. Collectively, our data suggest that TAM can repress the miR-200 family and induce EMT via the up-regulation of c-Myc in endometrial cancer cells. These findings describe a possible mechanism of TAM-induced EMT in endometrial cancer and provide a potential new therapeutic strategy for it.

TSA suppresses miR-106b-93-25 cluster expression through downregulation of MYC and inhibits proliferation and induces apoptosis in human EMC.

Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to decrease proliferation and increase apoptosis in different cancer cells. A significant number of genes have been identified as potential effectors responsible for the anti-tumor function of HDAC inhibitor. However, the molecular mechanisms of these HDAC inhibitors in this process remain largely undefined. In the current study, we searched for microRNAs (miRs) that were affected by HDAC inhibitor trichostatin (TSA) and investigated their effects in endometrial cancer (EMC) cells. Our data showed that TSA significantly inhibited the growth of EMC cells and induced their apoptosis. Among the miRNAs that altered in the presence of TSA, the miR-106b-93-25 cluster, together with its host gene MCM7, were obviously down-regulated in EMC cells. p21 and BIM, which were identified as target genes of miR-106b-93-25 cluster, increased in TSA treated tumor cells and were responsible for cell cycle arrest and apoptosis. We further identified MYC as a regulator of miR-106b-93-25 cluster and demonstrated its down-regulation in the presence of TSA resulted in the reduction of miR-106b-93-25 cluster and up-regulation of p21 and BIM. More important, we found miR-106b-93-25 cluster was up-regulated in clinical EMC samples in association with the overexpression of MCM7 and MYC and the down-regulation of p21 and BIM. Thus our studies strongly indicated TSA inhibited EMC cell growth and induced cell apoptosis and cell cycle arrest at least partially through the down-regulation of the miR-106b-93-25 cluster and up-regulation of it's target genes p21 and BIM via MYC.

Ectopically expressed perforin-1 is proapoptotic in tumor cell lines by increasing caspase-3 activity and the nuclear translocation of cytochrome C.

Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell's plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.) into the cytoplasm provides the proteases with access to numerous protein substrates that promote apoptosis after cleavage. These proteases are believed to be the main executioners of target cell apoptosis. Although the PRF and granzyme components are both critical to this process and in some way involved in inducing cell death in target cells, the inhibition of tumor growth could still be efficient in granzyme-deficient mice. It is unclear whether PRF alone can suppress tumors. In this study, we discovered that forced ectopic expression of PRF alone, in the absence of granzymes, could mediate cell death in cancer cells. Notably, transient expression of both full-length and truncated active-form PRF in human Hep G2, SK-BR-3, and HeLa cells was found to induce apparent cell growth inhibition and cell death, as evidenced by chromosome condensation and DNA fragmentation, increased caspase-3 activity, and the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. This PRF-induced cell death could be abrogated by pan-caspase inhibitor (Z-VAD) and mitochondria protector (TAT-BH4). The implication of these results is that ectopically expressed PRF has apoptosis-inducing abilities, and PRF alone is sufficient to induce apoptotic cell death in cells with ectopic expression. Taking this into consideration, our results suggest the possibility of using PRF as a pro-apoptotic gene for tumor therapeutics.

MicroRNA-320a suppresses human colon cancer cell proliferation by directly targeting ß-catenin.

Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-320a) in human colorectal carcinoma. However, its expression pattern and underlying mechanisms in the development and progression of colorectal carcinoma has not been elucidated clearly. Here, we performed real-time PCR to examine the expression levels of miR-320a in colon cancer cell lines and tumor tissues. And then, we investigated its biological functions in colon cancer cells by a gain of functional strategy. Further more, by the combinational approaches of bioinformatics and experimental validation, we confirmed target associations of miR-320a in colorectal carcinoma. Our results showed that miR-320a was frequently downregulated in cancer cell lines and colon cancer tissues. And we demonstrated that miR-320a restoration inhibited colon cancer cell proliferation and ß-catenin, a functionally oncogenic molecule was a direct target gene of miR-320a. Finally, the data of real-time PCR showed the reciprocal relationship between miR-320a and ß-catenin's downstream genes in colon cancer tissues. These findings indicate that miR-320a suppresses the growth of colon cancer cells by directly targeting ß-catenin, suggesting its application in prognosis prediction and cancer treatment.

MicroRNA-203 leads to G1 phase cell cycle arrest in laryngeal carcinoma cells by directly targeting survivin.

Previous studies have shown that miR-203 acts as a tumor-suppressive microRNA in various cancers, but its roles in laryngeal carcinoma are still contradicted. Here, we found that miR-203 inhibited the growth of laryngeal cancer cells and survivin was a direct target of miR-203. Moreover, silencing of survivin recapitulated the effect of miR-203 on cell cycle progression, whereas overexpression of survivin reversed this effect. Additionally, qRT-PCR showed the reciprocal relationship between miR-203 and survivin in laryngeal cancer tissues. These findings indicate that miR-203 inhibits the proliferation of laryngeal carcinoma cells by directly targeting survivin, suggesting its application in anti-cancer therapeutics.

HIV-TAT-fused FHIT protein functions as a potential pro-apoptotic molecule in hepatocellular carcinoma cells.

Accumulating evidence has demonstrated that FHIT (fragile histidine triad) is a bona fide tumour suppressor gene in a large fraction of human tumours, including hepatocellular cancer. A virus-based delivery system has been developed to transfer the FHIT gene into many types of cancer cells to inhibit growth or even induce apoptosis. However, a protein-based replacement strategy for FHIT has not been performed in cancer cells. Here, we used HIV-TAT (transactivator of transcription)-derived peptide to transfer the purified FHIT protein into HCC (hepatocellular carcinoma) cells and determine the biological effect of this fusion protein in inducing apoptosis. Affinity chromatography was used to purify TAT peptide-fused human FHIT (TAT-FHIT) protein from BL21 Escherichia coli. Immunofluorescence staining and Western blot analysis were performed to identify the expression and internalization of TAT-FHIT in HCC cells compared with the purified FHIT protein. Our study showed that TAT-FHIT protein can translocate into cancer cells in 1 h after incubation at 37°C. Furthermore, the results of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, Annexin-V staining and Western blotting demonstrated that TAT-FHIT can robustly inhibit growth and induce apoptosis of HCC cells in vitro. In addition, a mechanistic study showed that both exogenous and intrinsic apoptotic pathways were involved in TAT-FHIT-mediated apoptosis and this effect could be attenuated partially by a mitochondrial protector TAT-BH4, indicating that mitochondrion plays a critical role in TAT-FHIT-mediated pro-apoptotic effect in cancer cells. Taken together, our study suggests that TAT-FHIT is a potential pro-apoptotic molecule in HCC cells and strengthen the hypothesis of its therapeutic application against HCC.

[Construction and expression of human anti-HER2 scFv-9R fusion protein and identification of its activity].

AIM: To construct, express and purify a human fusion protein, which is composed of a single-chain antibody fragment scFv that recognizes HER2 protein and an oligo-9-arginine, and to analyze the binding activity of the expressed fusion protein. METHODS: Pairs of oligonucleotide primers were designed and used to amplify the scFv-9R. The fusion protein gene scFv-9R was then cloned into expression vector pQE30 and expressed in E.coli M15.Expressed protein was detected by SDS-PAGE and Western blot and purified by Ni-NTA chelating agarose. Then, the purified protein was refolded by dialysis and concentrated by ultrafiltration. The antigen-binding activity of the scFv-9R fusion protein was confirmed by ELISA, and the siRNA binding ability was confirmed by electrophoretic mobility shift assay (EMSA). RESULTS: HER2 scFv-9R encoding sequence was correctly cloned into the expression vector. The recombinant protein was insolubly expressed in E.coli M15 induced by IPTG. ELISA confirmed that it had specific antigen binding activity; EMSA assured that it had siRNA binding activity. CONCLUSION: The scFv-9R fusion protein can specially bind with both HER2 antigen and siRNA.

HER2 interacts with CD44 to up-regulate CXCR4 via epigenetic silencing of microRNA-139 in gastric cancer cells.

BACKGROUND & AIMS: Human epidermal growth factor receptor 2 (HER2) (neu/ERBB2) is overexpressed on many types of cancer cells, including gastric cancer cells; HER2 overexpression has been associated with metastasis and poor prognosis. We investigated the mechanisms by which HER2 regulates cell migration and invasion. METHODS: HER2 expression or activity was reduced in gastric cancer cell lines using small interfering RNAs or the monoclonal antibody, trastuzumab. We identified proteins that interact with HER2 or microRNAs (miRNAs) involved in HER2 signaling. We used various software programs to identify miRNAs that regulate factors in the HER2 signaling pathway. We analyzed expression patterns of these miRNAs in gastric cancer cell lines and tumor samples from patients. RESULTS: We found that CD44 binds directly to HER2, which up-regulates the expression of metastasis-associated protein-1, induces deacetylation of histone H3 lysine 9, and suppresses transcription of microRNA139 (miR-139) to inhibit expression of its target gene, C-X-C chemokine receptor type 4 (CXCR4). Knockdown of HER2 and CD44 reduced invasive activity of cultured gastric cancer cells and suppressed tumor growth in nude mice. Lymph node metastasis was associated with high levels of HER2, CD44, and CXCR4, and reduced levels of miR-139 in human metastatic gastric tumors. Cultures of different types of metastatic cancer cells with histone deacetylase inhibitors and/or DNA methyltransferase resulted in up-regulation of miR-139. CONCLUSIONS: HER2 interaction with CD44 up-regulates CXCR4 by inhibiting expression of miR-139, at the epigenetic level, in gastric cancer cells. These findings indicate how HER2 signaling might promote gastric tumor progression and metastasis.

The effect of direct translocation across endosomes on the cytotoxicity of the recombinant protein e23sFv-Fdt-casp6 to HER2 positive gastric cancer cells.

HER2-positive cancers represent a class of malignancies with high metastasis and poor prognosis. We previously generated the e23sFv-PEA II-casp6 recombinant, which contains an anti-HER2 single-chain antibody (e23sFv), a Pseudomonas exotoxin A translocation domain (PEA II), and a constitutively active caspase-6 (casp6), and demonstrated its potent selective anti-tumor activities. In this study, we generated a smaller-sized recombinant e23sFv-Fdt-casp6, in which the PEA II domain was replaced with the furin cleavage sequence from diphtheria toxin (Fdt), and explored its translocation pathway and specific killing mechanism. We found that e23sFv-Fdt-casp6 proteins, following their receptor-mediated endocytosis in HER2-positive gastric cancer cells, underwent furin-mediated cleavage in endosome and engaged in direct translocation of the released C-terminal fragment (active caspase-6) instead of via the trans-Golgi and the endoplasmic reticulum (ER) pathway. The active caspase-6 cleaved its well-documented substrate, Lamin A, and subsequently triggered the apoptosis of cancer cells. The e23sFv-Fdt-casp6 proteins produced from genetically modified cells showed a selective cytotoxicity to cultured HER2-positive gastric cancer cells. Similar to the results of our previous research on e23sFv-PEA II-casp6, the delivery of liposome-encapsulated e23sFv-Fdt-casp6 constructs in tumor-adjacent muscles also inhibited tumor growth and prolonged animal survival in a nude mouse xenograft tumor model. Moreover, e23sFv-Fdt-casp6 proteins were also cytotoxic to trastuzumab-resistant gastric cancer cells characterized by downregulated HER2 expression. Accordingly, e23sFv-Fdt-casp6 recombinant provides a promising therapeutic alternative for HER2-positive and trastuzumab-resistant gastric cancers.

Human activated CD4(+) T lymphocytes increase IL-2 expression by downregulating microRNA-181c.

MicroRNAs, a large family of small regulatory RNAs, are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner, thereby controlling diverse aspects of cell function, including immune reaction. In this study, we screened and identified a group of differentially expressed miRNAs in naive and activated CD4(+) T cells. Among the miRNAs studied, miR-181c was proven to have the potential to regulate CD4(+) T cell activation. miR-181c was downregulated in the process of CD4(+) T cell activation, and transfection of miR-181c mimics partially repressed the activation of both Jurkat cells and human peripheral blood mononuclear cells (PBMC) CD4(+) T cells. We further showed that miR-181c can bind to the IL-2 3' UTR and repress its expression by inhibiting translation. Moreover, miR-181c mimics reduced activated CD4(+) T cell proliferation. Taken together, our results show that miR-181c serves as a negative regulator that modulates the activation of CD4(+) T cells.

[Construction and expression of recombinant adenovirus encoding MDA-7/IL-24 and its inhibitory effect on proliferation in human hepatocellular carcinoma cells].

AIM: To construct recombinant plasmid and adenovirus harboring MDA-7 gene, and to investigate its biological function on the proliferation of human hepatocellular carcinoma cells. METHODS: The MDA-7 fragments from the T vectors were inserted into pCDNA-3 vector to construct expression plasmids named pCDNA3-MDA-7. To determine its effects on the proliferation of HCC cells, transfected the expression vector into cells and tested the ability of colony formation in cancer cells. Simultaneously, constructed recombinant adenovirus expressing MDA-7, and detected its effect on the proliferation of HCC cells by using MTT assay. RESULTS: Successfully constructed plasmid- and adenovirus-based system to express MDA-7. The data of colony formation assay and M'T test showed that MDA-7 can obviously suppress cell growth in HCC cells. CONCLUSION: MDA-7 may function as tumor suppressor in HCC cells, and the adenovirus-mediated MDA-7 can be a novel strategy for the anti-HCC therapy. Our study established the foundation for future research on the effect of MDA-7 in HCC.

Selective cytotoxicity to HER2-positive tumor cells by a recombinant e23sFv-TD-tBID protein containing a furin cleavage sequence.

PURPOSE: The HER2 antigen is a recognized target on breast cancer cells for immunotherapy. To overcome the immunogenicity and systemic toxicity of traditional immunotoxins, a novel human immunoproapoptotic molecule was developed and its antitumor activity was investigated. EXPERIMENTAL DESIGN: Recombinant e23sFv-TD-tBID, consisting of a single-chain anti-HER2 antibody fragment linked to a human active truncated Bid by a 10-amino acid residue furin cleavage sequence, was bacterially expressed. Purified e23sFv-TD-tBID was tested for binding, internalization, and cytotoxic activity in cell and for tumor localization and antitumor activity in athymic nude mice bearing established human tumor xenografts. RESULTS: e23sFv-TD-tBID selectively binds to HER2-positive cells and induces apoptotic cell death in vitro and in vivo. An investigation of its mechanism of action has revealed that e23sFv-TD-tBID was internalized on binding to the surface of HER2-positive tumor cells, proteolytically cleaved and transported directly to cytosol. The antitumor activity of e23sFv-TD-tBID was shown in a dose-dependent manner when injected i.p. into immunodeficient mice bearing human breast carcinomas. Moreover, this immunoproapoptotic protein, either given as a single dose or in combination with chemotherapy agents, significantly inhibited tumor growth without any observed toxic side effects on mice. Magnetic resonance imaging further showed the specific targeting and good penetration of tumors by e23sFv-TD-tBID in vivo. The therapeutic value of e23sFv-TD-tBID to human was shown by its cytotoxic effects on primary patient-derived breast tumor cells but not on endothelial cells. CONCLUSION: These data suggest that recombinant e23sFv-TD-tBID has therapeutic potential for HER2-positive tumors and warrant further testing for clinical applications.

[Expression and functional analysis of transcriptional factor JunB in hepatic cancer cells].

AIM: To construct the eukaryotic expression vector harboring transcriptional factor JunB and to observe the expression, localization and biological function of JunB in hepatic cancer cells. METHODS: The JunB gene was amplified by PCR from the human liver tissue cDNA library. After confirmed by DNA sequencing in the T-vector, the JunB gene was subcloned into pcDNA3.1(-) and pEGFP-C3 vectors, respectively. The recombinant vectors pcDNA-JunB and pEGFP-JunB were confirmed by Kpn I and BamH I restriction enzyme digestion. The recombinant vectors were transiently transfected into the hepatic cancer cells HepG2 using Lipofectamin2000. Western blotting was used to detect the expression of exogenous JunB and fluorescence microscopy was applied to observe the localization of JunB protein coupled with enhanced green fluorescent protein (EGFP) in hepatic cancer cells. Double luciferase reporter assay was performed to determine the effect of JunB on the transcriptional regulation of target genes. RESULTS: The recombinant eukaryotic expression vector carrying JunB or JunB-EGFP fusion gene was constructed and transiently transfected into HepG2 cells. Western blot analysis confirmed the exogenous expression of JunB. JunB-EGFP was observed uniquely in the nuclei. Double luciferase assay showed the transcriptional up-regulation of the VEGF in the presence of JunB. CONCLUSION: We constructed the recombinant eukaryotic expression vectors harboring JunB and JunB-EGFP successfully. The exogenous JunB is localized in the nuclei of transiently transfected HepG2 cells, suggesting JunB may function as a transcriptional factor. The result of reporter assay showed that JunB up-regulates the expression of VEGF, a tumor-angiogenesis associated molecule, at a transcriptional level. These demonstrate that JunB might be a novel target for an anti-angiogenesis treatment for hepatic cancers.

[Effects of exogenous nitric oxide on highbush blueberry PSII photochemical activity and antioxidant system under high temperature stress].

Taking the test tube 'Duke' highbush blueberry (Vaccinium corymbosum) seedlings having been transplanted to the field for 6 months as test materials, this paper studied the effects of exogenous nitric oxide (NO) on their growth, PS II photochemical activity, and antioxidant system under high temperature stress. Applying 0.2, 0.5, and 1.0 mmol x L(-1) of exogenous sodium nitroprusside (SNP) could alleviate the decrease of maximum photochemical efficiency (Fv/Fm), actual photochemical efficiency under light (phi PS II), photochemical quench (q(P)), and nonphotochemical quench (NPQ) caused by high temperature, and prevented the damage of high temperature on photosynthetic apparatus. Comparing with the control, treatments NO decreased the leaf membrane permeability and MDA content, increased the SOD and CAT activities significantly, and promoted proline accumulation. Appropriate concentration SNP could significantly alleviate the damage of high temperature stress on highbush blueberry seedlings, and 0.5 mmol x L(-1) of SNP had the most satisfactory effect.

[Expression and localization of adenovirus-mediated transcriptional factor Runx3 in malignant glioblastoma cells].

AIM: To construct the replicative deficient adenovirus Ad-Runx3 expressing Runx3, and to express it in U251 malignant glioblastoma cells. METHODS: The runx3 gene with a flag tag was amplified by PCR using pCMV5-AML2 as a template, and was confirmed by DNA sequencing. The adenovirus shuttle vector pShuttle-CMV-Runx3 was constructed by introducing runx3 DNA fragment into the sites of Kpn I and Xho I of pShuttle-CMV vector. This recombinant plasmid was linearized by PmeI and electronically transfected into BJ5183 cells to get the recombinant adenovirus vector Ad-Runx3. The recombinant adenovirus expressing Runx3 was infected into U251 malignant glioblastoma cells. The expression of exogenous Runx3 was observed by immonoblotting and its localization was detected by immunostaining using anti-Flag tag antibody. RESULTS: The recombinant adenovirus expressing Runx3 with a Flag tag was constructed and infected into U251 glioblastoma cells. The exogenous Runx3 protein was detected only in the nuclei. CONCLUSION: The recombinant adenovirus expressing Runx3 with a Flag tag is constructed successfully, and the Runx3 protein expressed in the nuclei of infected cells. The study laid a foundation for further research of the function of Runx3 in gliocarcinogenesis.