Eukaryotic Expression Vector pcDNA3 Research Articles

Development of a novel multi-epitope oral DNA vaccine for rabies based on a food-borne microbial vector

Rabies has been with humans for a long time, and its special transmission route and almost 100 % lethality rate made it once a nightmare for humans. In this study, by predicting the rabies virus glycoprotein outer membrane region and nucleoprotein B-cell antigenic epitopes, the coding sequence of the predicted highly antigenic polypeptide region obtained was assembled using the eukaryotic expression vector pcDNA3.1(-), and then E. coli was used as the delivery vector.The immunogenicity and protective properties of the vaccine were verified by in vivo and in vitro experiments, which demonstrated that the vaccine could produce antibodies in mice and prolong the survival time of mice exposed to the strong virus without any side effects. This study demonstrated that the preparation of an oral rabies DNA vaccine using food-borne microorganisms as a transport vehicle is feasible and could be a new strategy to eradicate rabies starting with wild animals.

A high efficacy DNA vaccine against Tilapia lake virus in Nile tilapia (Oreochromis niloticus)

Tilapia lake virus (TiLV) is the main tilapia-infecting virus worldwide, causing serious economic losses to the tilapia aquaculture. However, there is no vaccine for this viral disease. Here, TiLV ORF10 (TiLV-ORF10) encoding a protein with abundant epitopes was cloned into the eukaryotic expression vector pcDNA3.1 as a vaccine candidate, and then used to evaluate the protective effects against TiLV in Nile tilapia (Oreochromis niloticus). RT-PCR and Western blot analyses confirmed pcDNA3.1–ORF10 expression in tilapia. The transcription levels of immunity-related genes such as immunoglobulin M, Toll-like receptor 2, myeloid differentiation factor 88, interleukin 8, tumor necrosis factor alpha, gamma-IFN, and nuclear factor κB were significantly upregulated in the spleen, liver, and kidney of the vaccinated tilapias (P < 0.05). TiLV challenge experiments showed that relative percent survival (RPS) was significantly enhanced in fish vaccinated with the DNA vaccine. Moreover, the RPS was significantly higher in fish vaccinated with a high dose of the DNA vaccine (85.72% RPS at a dosage of 45 μg DNA vaccine plasmid). Meanwhile, vaccination with pcDNA3.1–ORF10 significantly reduced virus replication, as evidenced by the lower amount of virus in the spleen, liver, and kidney of vaccinated tilapia than tilapias vaccinated with the empty pcDNA3.1 plasmid. Thus, pcDNA3.1–ORF10 could induce protective immunity in tilapia and may be a potential vaccine candidate for controlling disease caused by TiLV.

Development of a Nanoparticle Multiepitope DNA Vaccine against Virulent Infectious Bronchitis Virus Challenge.

To develop a safe and effective nanoparticle (NP) multiepitope DNA vaccine for controlling infectious bronchitis virus (IBV) infection, we inserted the multiepitope gene expression box SBNT into a eukaryotic expression vector pcDNA3.1(+) to construct a recombinant plasmid pcDNA/SBNT. The NP multiepitope DNA vaccine pcDNA/SBNT-NPs were prepared using chitosan to encapsulate the recombinant plasmid pcDNA/SBNT, with a high encapsulation efficiency of 94.90 ± 1.35%. These spherical pcDNA/SBNT-NPs were 140.9 ± 73.2 nm in diameter, with a mean ζ potential of +16.8 ± 4.3 mV. Our results showed that the chitosan NPs not only protected the plasmid DNA from DNase degradation but also mediated gene transfection in a slow-release manner. Immunization with pcDNA/SBNT-NPs induced a significant IBV-specific immune response and partially protected chickens against homologous IBV challenge. Therefore, the chitosan NPs could be a useful gene delivery system, and NP multiepitope DNA vaccines may be a potential alternative for use in the development of a novel, safe, and effective IBV vaccine.

The Expression of MDM2 Gene Promoted Chondrocyte Proliferation in Rats with Osteoarthritis via the Wnt/β-Catenin Pathway.

This study aimed to investigate the regulatory mechanism of MDM2 gene expression on cartilage cell proliferation in Osteoarthritis (OA) rats. For this purpose, 22 SD rats were randomly divided into normal control (10 cases) and treated (12 cases) groups. Treated group was used for OA modelling with the modified Hulth method. After a week, RT-PCR was used to detect MDM2 in cartilage tissue of rats, Wnt 1, Wnt 3 a, Wnt 10 b and β-catenin genes mRNA expression. Rat chondrocytes were isolated and cultured, and the recombinant eukaryotic expression vector pcDNA3.1 myc-siRNA-MDM2-β-catenin and co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-β-catenin was used to transfect chondrocytes and the proliferation and related gene expression levels of the transfected chondrocytes were detected by MTT method and RT-PCR. The results showed that compared with the control group, MDM2, Wnt 1, Wnt 3 a, Wnt 10b and β-catenin genes in OA rat cartilage constructed by Hulth method were increased (p<0.05). The pcDNA3.1 myc-beta-catenin transfection slowed down the proliferation of OA chondrocytes, different from the non-transfected OA group (p<0.001), and increased Wnt 1, Wnt 3a, Wnt 10b and β-catenin genes expression compared with the Control group (p<0.05), but did not affect the expression of MDM2. The transfection of siRNA-MDM2 was opposite to pcDNA3.1 myc-β-catenin. The co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-beta-catenin transfection did not affect the proliferation of OA chondrocytes. In general, the high expression of MDM2 in OA rats restricts the proliferation of chondrocytes, which may be related to the main pathogenesis of the occurrence and development of OA in vivo, and the regulation of MDM2 on the proliferation of chondrocytes may be achieved through the Wnt/ β-catenin pathway.

Experimental Nanovaccine Offers Protection Against Repeat Exposures to Trypanosoma cruzi Through Activation of Polyfunctional T Cell Response.

A parasitic protozoan Trypanosoma cruzi (T. cruzi) is the etiologic agent of Chagas disease. Previously, we have identified T. cruzi antigens TcG2 and TcG4 as potential vaccine candidates, cloned in eukaryotic expression vector pCDNA3.1 (referred as p2/4) and tested their ability to elicit protection from T. cruzi infection. In the present study, we subcloned the two antigens in a nanoplasmid that is optimized for delivery, antigen expression, and regulatory compliance standards, and evaluated the nanovaccine (referred as nano2/4) for prophylactic protection against repeat T. cruzi infections. For this, C57BL/6 mice were immunized with two doses of p2/4 or nano2/4 at 21 days interval, challenged with T. cruzi 21 days after 2nd immunization, and euthanized at 10- and 21-days post-infection (pi) corresponding to parasite dissemination and replication phase, respectively. Some mice were re-challenged 21 days pi and monitored at 7 days after re-infection. Without the help of a vaccine, T. cruzi elicited delayed and sub-par T cell activation and low levels of effector molecules that failed to control tissue dissemination and replication of the parasite and provided no protection against repeat challenge infection. The nano2/4 was most effective in eliciting an early activation and production of IFN-γ by CD4+T effector/effector memory (TEM) cells and cytolytic perforin (PFN) and granzyme B (GZB) molecules by CD4+ and CD8+ TEM subsets at 10 days pi that was followed by robust expansion of CD4+ and CD8+ TEM and TCM cells with further increase in IFN-γ production at 21 days pi. Consequently, nano2/4-immunized mice exhibited potent control of parasite dissemination at 10 days pi, and tissue parasite burden and tissue inflammatory infiltrate and necrosis were barely detectable at 21 days pi. Furthermore, nano2/4-immunized mice responded to re-challenge infection with high levels of effector molecules production by CD4+ and CD8+ TEM subpopulations that offered even better control of tissue parasite burden than was observed after 1st infection. In comparison, non-vaccinated/infected mice exhibited clinical features of sickness and 59% mortality within 7 days after re-infection. In conclusion, we show that delivery of TcG2 and TcG4 in nanoplasmid offers excellent, protective T cell immunity against repeat T. cruzi infections.

Effect of microRNA-7 on proliferation, invasion, migration and EMT of hepatoma cell line SMMC-7721.

This study aimed to observe the effect of microRNA-7 on the proliferation and migration of human hepatocellular carcinoma cell line SMMC-7721 and its effect on epithelial-mesenchymal transition (EMT). Eukaryotic expression vector pcDNA3.1(-)-pri-miR-7(p-miR-7) was used to instantaneously transfect human liver cancer cells of SMMC-7721 cells in vitro. The expression of microRNA-7 was detected by RT-qPCR. Western blot was used to detect the expression of EMT marker proteins E-cadherin, β-catenin, N-cadherin and Vimentin. The proliferation of SMMC-7721 cells was detected by CCK-8 assay, and the invasion and migration ability of cells was detected by transwell assay. Compared with the normal group, the expressions of E-cadherin and β-catenin in SMMC-7721 cells transfected with miR-7 were significantly increased (p<0.05), while the expressions of N-cadherin and Vimentin were significantly decreased (p<0.05). Meanwhile, the proliferation, invasion and migration ability of the cells were significantly weakened (p<0.05). The miR-7 can inhibit the proliferation and invasion of human hepatocellular carcinoma cell line SMMC-7721, and its mechanism may be related to upregulation of E-cadherin, β-catenin protein, and downregulation of N-cadherin and Vimentin proteins.

The immune protection induced by a serine protease from the Trichinella spiralis adult administered as DNA and protein vaccine

Trichinellosis is caused by Trichinella spiralis (T. spiralis), which is an important public health problem. In this study, a gene encoding a serine protease from adult worms of T. spiralis (Ts-Adsp) was screened from a cDNA library of adult worms and was cloned and expressed in a prokaryotic expression system. The gene Ts-Adsp was subcloned into the eukaryotic expression vector pcDNA3.1(+), which was named pcDNA3.1(+)-Adsp. Previous studies have found that recombinant Ts-Adsp protein (rTs-Adsp) can elicit partial protection against T. spiralis infection in mice. Our aim was to explore the protective effect of combining a DNA vaccine with the rTs-Adsp protein against T. spiralis. One week after the last vaccination, the serum and spleen were obtained. The levels of IgG, IgG1 and IgG2a and cytokine production in serum and spleen cells were analyzed. The results showed that the levels of humoral and cell-mediated immune responses increased in the pcDNA3.1(+)-Adsp/rTs-Adsp group mice and demonstrated that a Th1/Th2 mixed immune response was induced by pcDNA3.1(+)-Adsp/rTs-Adsp after vaccination. Moreover, mice vaccinated with pcDNA3.1(+)-Adsp/rTs-Adsp displayed a 69.50% reduction in muscle larvae burden. This study suggested that mixed immunity could improve the muscle larvae reduction rate.

Construction of eukaryotic expression vector for human platelet CD36 gene 220C>T and 429+4insg variants and analysis of their expressions in HEK293T cells

To construct eukaryotic expression vectors for human platelet CD36 gene 220 C>T and 429+4insg variants and analyze their expressions in HEK293T cells. RNA was isolated from human platelets and reversely transcribed into cDNA. Sequences of 220C>T and 429+4insg variants were derived by PCR amplification. The target sequence was ligated into a pcDNA3.1/V5-His-TOPO vector by TA cloning, which was transformed into TOP10 E. coli. Positive plasmids were screened by blue-white selection. After sequencing, plasmid DNA carrying 220C>T or 429+4insg variant was used to transfect HEK293T cells with the help of effectene. Expression of CD36 protein was then analyzed by flow cytometry and Western blotting. An eukaryotic expression vector pcDNA3.1/V5-His-CD36 (220C>T/429+4insg) was constructed by TA cloning. After transfected into HEK293T cells, the 220C>T and 429+4insg variants resulted in CD36 deficiency in HEK cells, which was confirmed by flow cytometry and Western blotting. The 220C>T and 429+4insg variants can cause CD36 deficiency in human platelets. This system may be used for assessing the effect of 220C>T, 429+4insg, and other variants on the expression of CD36.

Immunogenicity evaluation of plasmids encoding Brucella melitensis Omp25 and Omp31 antigens in BALB/c mice.

Objective(s):Vaccination is one of the most effective means to protect humans and animals against brucellosis. Live attenuated Brucella vaccines are considered effective in animals but they may be potentially infectious to humans, so it is vital to improve the immunoprotective effects and safety of vaccines against Brucella. This study was designed to evaluate the immunogenicity of DNA vaccines encoding B. melitensis outer membrane proteins (Omp25 and Omp31) against B. melitensis Rev1 in a mouse model. Materials and Methods: For this propose, Omp25 and Omp31 genes were cloned (individually and together) into the eukaryotic expression vector pcDNA3.1/Hygro (+). Expressions of recombinant plasmids were confirmed by SDS-PAGE and Western blot analysis. Six groups of BALB/c mice (seven mice per group) were intramuscularly injected with three recombinant constructs, native pcDNA3.1/Hygro (+) and phosphate-buffered saline (PBS) as controls and subcutaneous injection of attenuated live vaccine Rev1.Results:Results indicated that DNA vaccine immunized BALB/c mice had a dominant immunoglobulin G response and elicited a T-cell-proliferative response and induced significant levels of interferon gamma (INF-γ) compared to the control groups.Conclusion:Collectively, these finding suggested that the pcDNA3.1/Hygro DNA vaccines encoding Omp25 and Omp31 genes and divalent plasmid were able to induce both humoral and cellular immunity, and had the potential to be a vaccine candidate for prevention of B. melitensis infections.

Plasmid pcDNA3.1-s11 constructed based on the S11 segment of grass carp reovirus as DNA vaccine provides immune protection

Although some commercial vaccines against grass carp reovirus (GCRV) are available, given the many varieties of GCRV and limited types of vaccines, the disease caused by GCRV remains a major problem, which leads to economic losses in grass carp aquaculture. A reovirus strain (GCRV-HN14) was recently isolated from local diseased fish in our laboratory. The S11 segment of GCRV-HN14 was speculated to encode the virus capsid protein VP35. In our study, the S11 segment was cloned into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1-s11, which was then transfected into CIK cells, and the VP35 protein was successfully expressed. Grass carp was immunized with pcDNA3.1-s11, and the in vivo distribution and expression of the pcDNA3.1-s11 plasmids were analyzed by PCR and Western blot. Recombinant plasmids were detected in the blood, liver, spleen, kidney, and muscle. However, protein expression could only be detected in the muscle. The immune protection of the pcDNA3.1-s11 plasmid in grass carp was evaluated using a series of experiments. Results showed that the population of white blood cells significantly increased at 1, 7, and 14 days post-immunization (dpi) and reached a peak with (9.58 ± 0.72) × 107/ml at 7 dpi (P < 0.01 or P < 0.05). The percentage of neutrophils reached a peak with (24.13 ± 2.38)% at 7 dpi (P < 0.01), whereas the lymphocytes peaked with (93.30 ± 4.71)% at 14 dpi (P < 0.05). Serum antibody levels were significantly enhanced in immunized fish at 14, 21, and 28 dpi (P < 0.01). The mRNA expression levels of type I interferon, immunoglobulin M, Toll-like receptor 22, and major histocompatibility complex class I were significantly up-regulated in the head kidney and spleen of immunized fish (P < 0.05). Grass carp immunized with pcDNA3.1-s11 exhibited a higher survival percentage (70.4%–73.3%) than the controls (5%–13%). Overall, as a DNA vaccine, the pcDNA3.1-s11 plasmid could induce immune protection against GCRV.

Effect of spleen tyrosine kinase on nonsmall cell lung cancer.

To investigate the anti.tumor effect of spleen tyrosine kinase. (Syk) on the human nonsmall cell lung cancer cells in vitro and its mechanism. In this study, we constructed a eukaryotic expression vector pcDNA3.1D/V5-His-TOPO/Syk and transfected it into human nonsmall cell lung cancer cells A549. Then, we not only analyzed the expression of Syk in transfected cells and its invasion but also the expression of matrix metalloproteinase-9 (MMP-9). The results showed that overexpressed Syk significantly inhibited A549 cell invasive ability by decreasing the expression of MMP-9. The overexpressed Syk plays an important role in tumor invasion and metastasis, and a negative role in human nonsmall cell lung cancer cells.

Gastrin induces multidrug resistance via the degradation of p27Kip1 in the gastric carcinoma cell line SGC7901.

Multidrug resistance (MDR) is one of the major reasons for the failure of chemotherapy-based gastric carcinoma (GC) treatments, hence, biologically based therapies are urgently needed. Gastrin (GAS), a key gastrointestinal(GI) hormone, was found to be involved in tumor formation, progression, and metastasis. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining analysis revealed a high level of expression of GAS in drug-insensitive GC tissues (P<0.01) and similar results were revealed in GC cell lines SGC7901 and its multidrug-resistant variants SGC7901/VCR and SGC7901/ADR. We constructed a eukaryotic expression vector pCDNA3.1(+)/GAS for GAS overexpression and recombinant lentiviral vectors for specific siRNA (siGAS). Transfection of pCDNA3.1(+)/GAS increased (P<0.05) while transfection of siGAS (P<0.05) and co-treated with paclitaxel(TAX) and vincristine (VCR) combination (TAX-VCR) decreased (P<0.01) the cell viability of SGC7901, SGC7901/VCR and SGC7901/ADR. Apoptosis rates of SGC7901/VCR and SGC7901/ADR were reduced by pCDNA3.1(+)/GAS and increased by siGAS (P<0.05). The apoptosis rates of SGC7901/VCR, SGC7901/ADR and SGC7901 were all upregulated (P<0.01) when cells were co-treated with a combination of siGAS and TAX-VCR. Additionally, siGAS significantly downregulated the expression of Bcl-2 and multidrug-resistant associate protein (MRP1) and P-glycoprotein (Pgp) (P<0.05) in SGC7901/VCR and SGC7901/ADR cells. Moreover, GAS overexpression in SGC7901 cells significantly inhibited p27Kip1 expression but increased phosphorylation levels of p27Kip1 on Thr(187) and Ser(10) sites (P<0.05), as well as increasing nuclear accumulation of S-phase kinase-associated protein 2 (Skp2) and cytoplasmic accumulation of the Kip1 ubiquitination-promoting complex (KPC) (P<0.05). Silencing of Skp2 blocked the promoting effects of pCDNA3.1(+)/GAS on viability, the expression of MRP1 and Pgp and the inhibitory effects of pCDNA3.1(+)/GAS on apoptosis. In conclusion, we suggest that GAS contributes to the emergence of MDR of SGC7901 cells via the degradation of p27Kip1.

Cell injury induced by 3D protein of different enterovirus 71 strains

Objective To compare the degree of cell injury induced by 3D protein (SDLY11 and SDLY107) of enterovirus 71 (EV71) strains. Methods EV71 strains SDLY11 and SDLY107 were respectively isolated from children with mild and severe hand foot mouth disease. The target genes 11-3D-Flag and 107-3D-Flag were amplified by reverse transcription-polymerase chain reation (RT-PCR) and inserted into the eukaryotic expression vector pcDNA3.1. The recombinant plasmids 11-3D-Flag-pcDNA3.1 and 107-3D-Flag-pcDNA3.1 were transformed into Escherichia. coli DH5α, respectively, and were identified by enzyme digestion and sequencing. The recombinant plasmids were transfected into rhabdomyosarcoma (RD) cells, respectively. Expression of 3D protein was detected by indirect immunofluorescence assay and western blot. Cell injury induced by 3D protein was detected with lactate dehydrogenase (LDH) test, cell proliferation was detected by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenylthiazolium bromide (MTT) test, and cell apoptosis was detected with Annexin-V and PI. Multiple comparisons among groups were analyzed using LSD-t test if multiple sets of variables were consistent with homogeneity of variance. If not, Dunnett T3 test was used. Results The 1 400 bp fragments were amplified by reverse tramscription (RT)-polymerase chain reaction (PCR), and the recombinant plasmids were digested by enzyme and the 1 400 bp and 5 400 bp fragments were obtained and identified. Gene sequencing showed that the sequences were consistent with the target genes. The specific fluorescence was observed by indirect immunofluorescence assay, and the western blot showed that the molecular weight of the target protein was 55×103. The LDH test showed that the A490 of SDLY11 3D protein transfection group (0.790±0.048) was higher than that of SDLY107 3D protein transfection group (0.641±0.018). The difference was statistically significant (t=5.14, P<0.05). The cell membrane damage caused by SDLY11 3D protein was more severe than SDLY107 3D protein. The MTT test showed that the A570 of SDLY11 3D protein transfection group (1.028±0.020) was lower than that of SDLY107 3D protein transfection group (1.081±0.002), and the difference was statistically significant (t=3.31, P<0.05). The effect on cell proliferation activity of SDLY11 3D protein was greater than SDLY107 3D protein. The results of Annexin-V/PI showed that the percentage of apoptotic cells of SDLY11 3D protein transfection group and SDLY107 3D protein transfection group were (1.471±0.246)% and (1.465±0.237)%, respectively, and the difference was not statistically significant (t=0.04, P=0.973). Conclusions Compared with the SDLY11 3D protein, SDLY107 3D protein induces slighter cell injury, has weaker effect on cell proliferation activity, and is more favorable for virus replication in cells. Key words: Enterovirus A, human; 3D protein; Cell injury

Molecular cloning, expression and characterization of rat tumor necrosis factor-α as potent anti-tumor candidate

Tumor Necrosis factor (TNF), a pleiotropic cytokine, has been shown to have direct anti-tumor activity killing tumor cells while sparing normal cells. In the present study, we amplified and cloned the rat TNF-α into pcDNA3.1 (+) eukaryotic expression vector for its future use as genetic adjuvant in the treatment of chemically induced skin tumor model (Wistar rats). The recombinant pcDNA.rat.tnf was confirmed by restriction enzyme digestion and nucleotide sequencing. The in-vitro expression of recombinant pcDNA.rat.tnf was checked both at transcriptional and translational levels in transfected HeLa cells by RT-PCR, flow cytometry and immunofluorescence. Further, the characterization of in-vivo expression of the protein was carried out by sandwich ELISA using serum from pcDNA.rat.tnf treated Wistar rats. We report successful cloning and expression of rat TNF-α in a eukaryotic expression vector pcDNA3.1 (+). The construct pcDNA.rat.tnf can now be used as an anti-tumor agent alone or along with other anti-tumor therapies as an adjuvant.

The construction of human prostate specific homeobox gene NKX3.1 eukaryotic expression vector and preliminary study on the biological function of NKX3.1

Objective To investigate the basic biological functions of NKX3.1 with construction of a series of eukaryotic expression vector of NKX3.1 to study its effects on cell cycle and apoptosis. Methods The full-length reading frames (ORF) of NKX3.1 was amplified from normal human prostate tissue with polymerase chain reaction (PCR), the ORF was cloned into T vector, and then NKX3.1 was cloned into eukaryotic expression vector pcDNA3.1(+ ) and pcDNA3.1(-) with this T vector as template. NKX3.1 in pEGFP C1 was transfected into PC3 cell line, and C1 pEFP was taken as control to observe the localization of NKX3.1 in cells. At the same time, NKX3.1 in pcDNA3.1 and pcDNA3.1 were transfected into PC3 cells and the cells were collected after 24~48 hours to detect cell cycle and apoptosis by flow cytometry. Results The eukaryotic expression vector of NKX3.1 was constructed successfully. Fluorescent protein was ocated in the nucleus after transfection of NKX3.1 in pEGFP C1, but pEGP C1 was both expressed in nucleus and cytoplasm. There was significant effect on the early apoptosis (P 0.05) after transfection of NKX3.1 compared to PC3 cell-transfected with empty vector of pcDNA3.1. Conclusions The result data suggest that NKX3.1 be nuclear protein and have effect on the early apoptosis of PC3 cell line based on the construction of eukaryotic expression vector. Key words: Genes, tumor suppressor; Genes, homeobox; Genetic vectors; Transfection; Prostatic neoplasms; Cell cycle; Apoptosis

Over-expression of PTEN on proliferation and apoptosis in canine mammary tumors cells

ABSTRACTPhosphatase and tensin homolog (PTEN) is an important tumor-suppressor gene which constitutes an important PI3K/Akt pathway by regulating the signaling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth has been gaining increasing attention. However, the role of PTEN in regulating apoptosis of canine mammary tumors cells still needs further investigation. In this experiment, the effect of PTEN on proliferation and apoptosis in canine mammary tumors (CMT) cells was analyzed. As a result, gene and protein expression levels of apoptosis-related genes were detected. Eukaryotic expression vector pcDNA3.1+-PTEN were successfully constructed and stably transferred into canine CMT cells after geneticin (G418) selection. After pcDNA3.1+-PTEN transfection, compared with control group, the cells proliferation was inhibited and the cell apoptosis was increased in CMT cells. The expression of p-Akt was decreased and the apoptosis-related genes, such as caspase-3, caspase-9, and Bax, were increased. These data serve to demonstrate the function of PTEN on apoptosis and gene regulatory in PI3K/Akt pathway in CMT cells. Collectively, our data link the tumor-suppressor activities of PTEN to the machinery controlling cell cycle through the modulation of signaling molecules whose signal target is the functional inactivation of the apoptosis gene product.

Regulation effect of β-catenin pathway on TGF-β1 induced pulmonary pro-fibrosis

To investigate the regulation effect of β-catenin pathway on transforming growth factor beta1 (TGF-β1) induced pulmonary pro-fibrosis. The rat alveolar typeⅡ cells (RLE-6TN) were divided into four groups: A1.control group; B1.TGF-β1 group was treated with 5 μg/L TGF-β1; C1.pcDNA+ TGF-β1 group was transiently transfected with eukaryotic expression vector pcDNA3.0 (pcDNA) and followed by TGF-β1 treatment (5 μg/L); D1.F-(β-TrCP)-Ecad+ TGF-β1 group was transiently transfected with β-catenin protein knockout vector [F-(β-TrCP)-Ecad] and followed by TGF-β1 treatment (5 μg/L). After 24 hours, cells were observed under the inverted phase contrast microscope, then the expressions of E-cadherin, α-smooth muscle actin (α-SMA) and fibronectin (Fn) in each group were measured by Western blot and the mRNA levels of Snail which was the downstream profibrotic transcription production in cell culture supernatants of each group were detected by real-time fluorescence quantification-polymerase chain reaction (RT-PCR) .The rat alveolar macrophages (CRL-2192) were divided into five groups: A2.control group; B2.Interferon gamma (IFN-γ) group was treated by 20 μg/L IFN-γ; C2.TGF-β1+ IFN-γ group was treated by 20 μg/L IFN-γ with 10 μg/L TGF-β1; D2.F-(β-TrCP)-Ecad+ TGF-β1+ IFN-γ group was transfected with F-(β-TrCP)-Ecad and other dispose was the same as group C2; E2.WTβ-catenin+ TGF-β1+ IFN-γ group was transfected with WTβ-catenin and other dispose was the same as group C2.After 24 hours, protein levels of β-catenin in group A2, B2, C2 were determined by Western blot.Inducible nitric oxide synthase (iNOS) mRNA levels of each group were detected by RT-PCR. The RLE-6TN cells of group B1, C1 showed a change in morphology to spindle-shaped cells, the cells of group D1 maintained a cobblestone morphology. Protein expressions of the fibroblast markers α-SMA and Fn, and mRNA expressions of the downstream profibrotic transcription production Snail of group B1, C1 were significantly higher than group A1, while protein expressions of the epithelial marker E-cadherin were significantly lower.The protein expressions of α-SMA, Fn and mRNA expressions Snail of group D1 were significantly lower than group C1 (0.352±0.076 vs 0.937±0.303, 0.319±0.072 vs 0.903±0.211, 3.675±0.642 vs 9.708±2.031), while the protein expressions of E-cadherin were significantly higher (1.482±0.227 vs 0.604±0.121) (all P<0.05). The steady state protein levels of β-catenin in CRL-2192 cells was low and β-catenin protein expressions of CRL-2192 cells in group A2, B2 and C2 had no significantly statistical differences.The mRNA expressions of iNOS of group B2 cells were significantly higher than group A2, C2, D2, E2 (64.95±4.47 vs 9.87±0.73, 21.32±2.41, 18.35±3.61, 22.87±3.14) (all P<0.01), the expressions of iNOS of group C2, D2, E2 were all higher than group A2 (all P<0.05), but there were no significant differences among group C2, D2 and E2. Inhibition of β-catenin pathway inhibits TGF-β1-induced epithelial-mesenchymal transition (EMT) and has no effect on its anti-inflammation effect.Therefore, β-catenin pathway regulates the pulmonary pro-fibrosis effect of TGF-β1.

A novel alternative splicing isoform of NF2 identified in human Schwann cells.

Vestibular schwannoma (VS) is a benign, slow-growing cranial tumor that originates from the hypertrophy of Schwann cells. The majority of sporadic VS are unilateral, and the mechanisms underlying VS tumorigenesis are not fully understood. The human neurofibromin 2 (NF2) gene encodes the tumor suppressor protein merlin and the NF2 transcript can be alternatively spliced to form numerous isoforms. The present study investigated human Schwann cells (HSCs) at the mRNA and protein level to understand the function of the alternative splicing (AS) isoform of NF2. The total RNA of HSCs was isolated and the full-length coding sequence of NF2 was amplified. The amplified products were excised from agarose gels, purified and sequenced. NF2 at a protein level was assayed by immunoprecipitation and western blot analysis. The full-length and spliced NF2 forms were amplified by polymerase chain reaction (PCR) from the HSC complementary DNA and ligated into eukaryotic expression vector pcDNA3.1(+). The plasmids were transfected into the HSC HEI-193 cell line and cell proliferation assays were performed using Cell Counting Kit-8. PCR analysis using HSC total RNA as a template revealed the presence of a shortened NF2 transcript, which was due to splicing at the 3'-end of the NF2 mRNA. Sequence analysis confirmed that this AS isoform omitted exons 11, 12, 13, 14, 15 and 16. Immunoprecipitation and western blot analysis demonstrated that the AS isoform was highly expressed in the HSCs at 38 kDa, while the wild-type (WT) isoform, which was expected at 66 kDa, was undetectable. Transfection and cell proliferation assays revealed that the WT isoform exhibited significant growth inhibition, while the AS isoform did not suppress cell growth. In conclusion, the present study detected AS NF2 isoforms in HSC for the first time, and investigated the function of the principle AS isoform. The present study suggests that although HSCs have an undetectable level of WT isoform of the NF2 protein merlin, they are not merlin-null, since they express the AS isoform. Although the AS merlin isoform has no suppressive effect on cell growth, certain mechanisms may exist that underlie this phenomenon, and this may be associated with the genesis and development of VS.

Whim Syndrome Caused By a 5 Base Pair Deletion in the C-Terminus of Chemokine Receptor CXCR4

▪Introduction: WHIM syndrome is a rare autosomal dominant, primary immunodeficiency disease characterized by warts, hypogammaglobulinemia, infections and myelokathexis (impaired neutrophil egress from bone marrow resulting in neutropenia). Mutations in the C-terminus of chemokine receptor CXCR4 cause almost all known cases of WHIM syndrome (4 truncations, 3 frame-shifts and 1 single amino acid charge-change), and result in impaired ligand-induced receptor internalization and desensitization. This enhances receptor signaling, and exaggerates normal CXCR4 function to retain neutrophils and other leukocytes in bone marrow, resulting in myelokathexis and increased susceptibility to certain infections.Methods: A 2 year old boy with recurrent infections and neutropenia was referred to the NIH Clinical Center. Four anonymized healthy donors were recruited as homozygous CXCR4WT controls. Genomic DNA was isolated from peripheral blood and the CXCR4 gene was sequenced using the Sanger method. The eukaryotic expression vector pcDNA3.1(-) was used to make 3 recombinant plasmids encoding the wild type gene, (CXCR4WT) the most common disease mutation (CXCR4R334X) and the index patient's mutation (CXCR4L329fs). K562 cells were transfected with plasmids by nucleofection. Blood leukocyte subsets and CXCR4 expression on PBMCs from healthy donors and the patient and stably transfected K562 cells with or without CXCL12 stimulation were examined by FACS analysis. Calcium flux in response to CXCL12 was analyzed in receptor-transfected and parental cell lines as well as in primary leukocytes.Results: The boy had recurrent infections, hypogammaglobulinemia, panleukopenia and myelokathexis, consistent with WHIM syndrome but both parents were healthy. Genetic analysis revealed a novel heterozygous mutation in CXCR4, designated CXCR4L329fs, involving a 5 base pair deletion from nucleotides 986-990 in the open reading frame. This deletion causes a frame-shift at codon 329 that introduces 12 missense amino acids and deletes the conserved C-terminal 24 amino acids. Interestingly a different mutation that causes a frame-shift at the same codon has been recently described as a somatic mutation in one case of Waldenström's macroglobulinemia, which deletes nucleotide 984-986 and inserts a T. The CXCR4L329fs mutation in our patient is the largest truncation mutant reported to date in WHIM syndrome, and was not identified in unaffected family members or in the general population, suggesting that it is a de novo mutation. As with the most common WHIM mutation, CXCR4R334X, this new mutation also caused decreased receptor downregulation in response to CXCL12 in both PBMCs from the patient as well as in K562 cells transfected with the cloned receptor and increased CXCR4 calcium flux signaling in stably transfected K562 cells.Conclusion:CXCR4L329fs is a novel de novo gain-of-function mutation of the C-terminus of CXCR4 that causes WHIM syndrome. DisclosuresLiu:US National Institutes of Health: Employment, Patents & Royalties: pending. Murphy:US National Institutes of Health: Employment, Patents & Royalties: pending. McDermott:US National Institutes of Health: Employment, Patents & Royalties: pending.

Construction and inhibitory effect of MEG3 expression plasmid vector on the proliferation of human pancreatic carcinoma SW1990 cells

Objective To construct a maternally expressed gene 3 (MEG3) expression plasmid vector, and to obtain MEG3 over-expressed human pancreatic carcinoma SW1990 cells by transfection, and to analyze the effect of MEG3 overexpression on the proliferation of human pancreatic carcinoma SW1990 cells. Methods A complete gene sequence based on the sequence of MEG3 in the GenBank was designed and inser-ted into the eukaryotic expression vector pcDNA3.0 to construct recombinant plasmid pcDNA3.0-MEG3. It was identified by sequencing and transfected into human pancreatic carcinoma SW1990 cells. The expression of MEG3 in SW1990 cells was confirmed by RT-PCR. The effect of MEG3 on proliferation was evaluated by MTT assay. In this study, the SW1990 cells transfected by plasmid pcDNA3.0 were named negative control group, and the usual SW1990 cells were named blank control group. Results A MEG3 expression plasmid vector-pcDNA3.0-MEG3 was constructed successfully. And pcDNA3.0-MEG3 vector was transfected into SW1990 cells successfully. The expression of MEG3 at mRNA in MEG3-SW1990 cells increased significantly, about 895 times (F=73.592, P<0.01). The results of MTT assay indicated that over-expressed MEG3 could obviously inhibit SW1990 cells proliferation in vitro. After SW1990 cells transfected with pcDNA3.0-MEG3 for 72 hours, the absorbance value was 0.81±0.06, with a statistically significance (F=33.489, P<0.01) compared with negative control group (1.17±0.07) and blank control group (1.08±0.03). Conclusion A MEG3 expre-ssion plasmid vector-pcDNA3.0-MEG3 is constructed successfully. It is confirmed that MEG3 and its product have obvious inhibitory effects for the proliferation of human pancreatic carcinoma SW1990 cells. Key words: Pancreatic neoplasms; RNA; Gene expression regulation; Maternally expressed gene 3