Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China (LCDV-cn) genome

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene.

Regulatory Effects of X-linked Inhibitor of Apoptosis Protein and Pro-apoptotic Protein Smac on Apoptosis Resistance to Chemotherapy in Pancreatic Cancer Cells~*

Objective： To investigate the relation of X-linked inhibitor of apoptosis （XIAP） and second mitochondria-derived activator of caspase （Smac） signaling pathway to chemoresistance in human pancreatic cancer Panc-1 and BXPC-3 cells. Methods： Apoptosis and the changes of XIAP expression in permeabilized cells induced by cisplatin and 5-fluorouracil （FU） were measured by flow cytometry. The cytosolic expression of XIAP, Smac and caspase-3 was detected by Western blot. A recombinant plasmid vector pEGFP-N1/Smac was constructed and transfected into of Pancol cells. The effect of cytosolic overexpression of Smac on apoptosis of Panc-1 cells was evaluated by flow cytometry. Results： Panc-1 was more resistant to cisplatin or 5-FU induced apoptosis than BXPC-3. Western blot revealed that chemoresistant Panc-1 highly expressed XIAP, and increased cytosolic expression of Smac might be responsible for the marked down-regulation of XIAP in chemo-sensitive BXPC-3 cells after exposure to cisplatin or 5-FU. Furthermore, cytosolic overexpression of Smac could significantly down-regulate the levels of XIAP and promote the activity of caspase-3, as well as sensitize Panc-1 cells to anticancer drug-induced apoptosis. Conclusion： Anticancer drug-induced apoptosis requires mitochondrial release of Smac and downregulation of XIAP, which may be an important determinant of chemo-sensitivity in pancreatic cancer cells. Up-regulation of cytosolic expression of Smac may act as an effective modifying signal to overcome apoptosis resistance to chemotherapy in pancreatic cancer cells.

Targeting X-linked inhibitor of apoptosis protein inhibits pancreatic cancer cell growth through p-Akt depletion

AIM: To determine whether lentivirus-mediated shRNA targeting the X-linked inhibitor of apoptosis protein (XIAP) gene could be exploited in the treatment of pancreatic cancer. METHODS: Human pancreatic cancer cells Panc-1, Mia-paca2, Bxpc-3 and SW1990, infected with lentivirus, were analyzed by real-time polymerase chain reaction (PCR). Western blotting was used to examine XIAP protein levels, survivin and p-Akt to confirm the result of real-time PCR and determine the possible mechanism. The 3-(4,5-cimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay was used to measure IC50 to determine chemosensitivity to the chemotherapeutic drugs 5-fluorouracil (5-FU) and gemcitabine. A colony assay, MTT assay and a tumorigenicity experiment were used to study cell proliferation in vitro and in vivo . Caspase-3/7 activity, 4',6-diamidino-2-phenylindole-staining and flow cytometric measurements were used to study apoptosis in SW1990 cells. RESULTS: XIAP proteins were found to be differen- tially expressed among pancreatic cancer cell lines Panc-1, Mia-paca2, Bxpc-3 and SW1990. Data of real-time PCR and Western blotting showed that XIAP was reduced persistently and markedly by lentivirus-mediated shRNA. Downregulation of XIAP by transfection with XIAP shRNA resulted in decreased p-Akt expression. XIAP shRNA also inhibited the growth of pancreatic cancer cells in vitro and in vivo , enhanced drug-induced apoptosis and increased chemosensitivity to 5-FU and gemcitabine. Results also suggest that inhibition of XIAP and subsequent p-Akt depletion may have an anti-tumor effect through attenuating the ability of cancer cells to survive. CONCLUSION: Lentivirus-mediated gene therapy is an attractive strategy in the treatment of pancreatic cancer and justifies the use of lentivirus in pancreatic cancer gene therapy studies.

Expression of second mitochondria-derived activator of caspases, X-linked inhibitor of apoptosis protein, and caspase-3 in pituitary adenomas

Studies concerning correlations between pituitary adenomas and cell apoptosis have mainly focused on upstream apoptosis signaling, but seldom on downstream mediators. In the present study, second mitochondria-derived activator of caspases （Smac）, X-linked inhibitor of apoptosis protein （XIAP）, and caspase-3 protein were qualitatively analyzed using immunohistochemistry, and quantified by western blot. Smac, XIAP, and caspase-3 mRNA expressions were detected by reverse transcription-PCR. Results showed that XIAP protein and mRNA expressions were greater in the invasive pituitary adenoma group compared with the noninvasive pituitary adenoma group. However, Smac and caspase-3 protein and mRNA expressions were lower in the invasive pituitary adenoma group compared with the noninvasive pituitary adenoma group. In the invasive pituitary adenomas, Smac expression was positively correlated with caspase-3 protein and mRNA expression （Protein： r = 0.55, P 0.01; mRNA： r = 0.50, P 0.01）. Smac and caspase-3 expressions were negatively correlated with XIAP protein and mRNA expression （Protein： r = -0.56, -0.64, P 0.01; mRNA： r = -0.69, -0.67, P 0.01）. However, no significant differences in correlation among Smac, XIAP, and caspase-3 were detectable in noninvasive pituitary adenomas. These data indicated that high expression of XIAP and low expression of Smac and caspase-3 suppressed cell apoptosis and led to enhanced invasiveness of pituitary adenomas. Thus, Smac, XIAP, and caspase-3 may be useful markers in determining the invasive behavior of pituitary adenomas.

Expression of X-linked Inhibitor of Apoptosis Protein and Its Effect on Chemotherapeutic Sensitivity of Bladder Carcinoma

The expression of X-linked inhibitor of apoptosis protein (XIAP) gene and its effect on chemotherapeutic sensitivity in bladder carcinoma was explored. By using immunohistochemistry, the expression of XIAP was detected in 47 bladder carcinomas and 5 normal bladder tissues. The XIAP gene was transfected into bladder cancer cell line T24 by liposome and the positive clone was screened by G418. Cellular XIAP mRNA level was detected by RT-PCR. Low-dose mitocycin C was administered to induce the apoptosis of T24 cells. The in vitro growth of bladder carcinoma cells was analyzed by MTT colorimetry, and the apoptosis rate was assayed by TUNEL methods. It was found XIAP was moderately expressed in bladder carcinomas with the the positive rate being 78.73% (37/47), but the positive rate was not correlated with carcinoma stages and grades (P<0.05). XIAP mRNA level in transfected T24 cells was significantly increased by 3.8 times as compared with that in the cells not transfected with XIAP. After treatment with low-dose mitomycin C (0.005 and 0.05 mg/mL), the growth rate in XIAP no-transfected control group was increased by (11.60±0.25)% and (16.51±0.87)% (P<0.05), and the apoptosis rate was decreased by (10.1±0.2)% and (11.9±0.2%) (P<0.05) respectively as compared with XIAP transfected group. It was concluded that XIAP was expressed in most of bladder carcimoma samples. Overexpression of XIAP in T24 could significantly reduce the MMC-induced apoptosis of bladder carcinoma, suggesting its effect on the chemothera- peutic sensitivity of T24 cells.

Early-onset refractory diarrhea due to immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome associated with a novel mutation in the FOXP3 gene: A case report

BACKGROUND Immune dysregulation,polyendocrinopthy,enteropathy,X-linked(IPEX)syndrome is a rare X-linked recessive disease caused by mutations in the forkhead box protein 3(FOXP3)gene,which is a master transcriptional regulator for the development and function of CD4+CD25+regulatory T(Treg)cells.The dysfunction of these cells leads to multiple system autoimmune diseases.We present a case of IPEX due to a mutation not reported in the literature before.CASE SUMMARY We report a male patient with IPEX syndrome who presented with refractory diarrhea and malabsorption leading to failure to thrive,as well as with hypothyroidism and nephrotic syndrome.Laboratory investigation showed increased total IgE and Treg cells,decreased free triiodothyronine(FT3)and free thyroxine(FT4),and proteinuria.Multiple dietary and supportive treatments were introduced but did not improve the diarrhea during his hospital stay.Ultimately,whole exome sequencing revealed that the patient was hemizygous for the exon 5,c.542G>A(p.Ser181Asn)mutation of the FOXP3 gene,which has not been previously reported.The patient remains on prednisone and euthyrox while awaiting hematopoietic stem cell transplantation at the time of the compilation of this case report.CONCLUSION We report a novel FOXP3 gene mutation involved in IPEX.A high level of suspicion should be maintained in an early-onset refractory diarrhea patient.

Stress Responsive Zinc-finger Protein Gene of Populus euphratica in Tobacco Enhances Salt Tolerance

The Populus euphratica stress responsive zinc-finger protein gene PSTZ, which encodes a protein including typical Cys2/His2 zinc finger structure, was isolated by reverse transcription-polymerase chain reaction from P. euphratica. Northern hybridization revealed that its expression was induced under drought and salt stress conditions. To examine its function, cDNA of the PSTZ gene, driven by the cauliflower mosaic virus 35S promoter, was cloned into a plant expression vector pBin438 and introduced into tobacco plants. Transgenic tobacco showed an enhanced salt tolerance, suggesting that PSTZ may play a role in plant responsiveness to salt stress.

Mutation Analysis of Gap Junction Protein Beta 1 and Genotype-Phenotype Correlation in X-linked Charcot-Marie- Tooth Disease in Chinese Patients

Background： Among patients with Charcot-Marie-Tooth disease （CMT）, the X-linked variant （CMTX） caused by gap junction protein beta 1 （GJB1） gene mutation is the second most frequent type, accounting for approximately 90% of all CMTX. More than 400 mutations have been identified in the GJB1 gene that encodes connexin 32 （CX32）. CX32 is thought to form gap junctions that promote the diffusion pathway between cells. GJB1 mutations interfere with the formation of the functional channel and impair the maintenance of peripheral myelin, and novel mutations are continually discovered. Methods： We included 79 unrelated patients clinically diagnosed with CMT at the Department of Neurology of the Chinese People＇s Liberation Army General Hospital from December 20, 2012, to December 31, 2015. Clinical examination, nerve conduction studies, and molecular and bioinformatics analyses were performed to identify patients with CMTX 1. Results： Nine GJBI mutations （c.283G〉A, c.77C〉T, c.643C〉T, c.515C〉T, c.191G〉A, c.610C〉T, c.490C〉T, c.491G〉A, and c.44G〉A） were discovered in nine patients. Median motor nerve conduction velocities of all nine patients were 〈 38 m/s, resembling CMT Type 1. Three novel mutations, c.643C〉T, c.191G〉A, and c.610C〉T, were revealed and bioinformatics analyses indicated high pathogenicity. Conclusions： The three novel missense mutations within the GJB1 gene broaden the mutational diversity ofCMT1X. Molecular analysis of family members and bioinformatics analyses of the afflicted patients confirmed the pathogenicity of these mutations.

Expression and function of long non-coding RNA DLX6-AS1 in endometrial cancer

Background:LncRNA DLX6-AS1 has been uncovered to exert effects on various cancers.Nevertheless,the impacts of DLX6-AS1 on endometrial cancer(EC)development remained obscure.The study explored the influence of DLX6-AS1 on EC progression via the microRNA(miR)-374a-3p/zinc-finger protein(ZFX)axis.Methods:EC cell lines were collected and DLX6-AS1,miR-374a-3p,and ZFX levels in EC cell lines were detected.The EC cells were transfected with DLX6-AS1,miR-374a-3p,and ZFX constructs to examine the biological functions of EC cells.The xenograft model was established for detecting tumor growth.Rescue experiments were conducted to verify the interaction of DLX6-AS1,miR-374a-3p,and ZFX in EC cells.Results:DLX6-AS1 and ZFX levels were elevated,while miR-374a-3p exhibited a reduced level in EC cells.Silencing DLX6-AS1 and elevated miR-374a-3p expressions repressed the biological activities of EC cells.Reduced DLX6-AS1 repressed tumor development.MiR-374a-3p silencing reversed the impacts of DLX6-AS1 silencing,while ZFX overexpression abrogated the impacts of miR-374a-3p elevation on EC cell growth.Mechanically,DLX6-AS1 was found to bind to miR-374a-3p,and miR-374a-3p targeted ZFX.Conclusion:DLX6-AS1 depletion restricts the malignant phenotype of EC cells.The study might provide novel therapeutic biomarkers for EC treatment.

DEXH-Box protein DHX30 is required for optimal function of the zinc-finger antiviral protein

The zinc-finger antiviral protein(ZAP)is a host factor that specifically inhibits the replication of certain viruses by eliminating viral mRNAs in the cytoplasm.In previous studies,we demonstrated that ZAP directly binds to the viral mRNAs and recruits the RNA exosome to degrade the target RNA.In this article,we provide evidence that a DEXH box RNA helicase,DHX30,is required for optimal antiviral activity of ZAP.Pull-down and co-immunoprecipitation assays demonstrated that DHX30 and ZAP interacted with each other via their N terminal domains.Downregulation of DHX30 with shRNAs reduced ZAP’s antiviral activity.These data implicate that DHX30 is a cellular factor involved in the antiviral function of ZAP.

Structural insights into a novel histone demethylase PHF8

Dynamic regulation of histone methylation/demethylation plays an important role during development. Mutations and truncations in human plant homeodomain （PHD） finger protein 8 （PHF8） are associated with X-linked mental retardation and facial anomalies, such as a long face, broad nasal tip, cleft lip/cleft palate and large hands, yet its molecular function and structural basis remain unclear. Here, we report the crystal structures of the catalytic core of PHF8 with or without α-ketoglutarate （α-KG） at high resolution. Biochemical and structural studies reveal that PHF8 is a novel histone demethylase specific for di- and mono-methylated histone H3 lysine 9 （H3K9me2/1）, but not for H3K9me3. Our analyses also reveal how human PHF8 discriminates between methylation states and achieves sequence specificity for methylated H3K9. The in vitro demethylation assay also showed that the F279S mutant observed in clinical patients possesses no demethylation activity, suggesting that loss of enzymatic activity is crucial for pathogenesis of PHF8 patients. Taken together, these results will shed light on the molecular mechanism underlying PHF8-associated developmental and neurological diseases.

Clinical and Genetic Features of Chinese X-linked Charcot-Marie-Tooth Type 1 Disease

Background： X-linked Charcot-Marie-Tooth type 1 （CMT1 X） disease is one of the most common forms of inherited neuropathy caused by mutations in the gap junction beta-1 protein （GJB1） gene （also known as connexin 32）. This study presented the clinical and genetic features of a series of Chinese patients with GJB1 gene mutations. Methods： A total of 22 patients from unrelated families, who were referred to Department of Neurology, Peking University First Hospital from January 2005 to January 2016, were identified with GJBI mutations. Their clinical records and laboratory findings were retrospectively collected and reviewed. Mutations in the GJB1 gene were analyzed by targeted next-generation sequencing （NGS）. Nucleotide alternations were confirnled with Sanger sequencing. Results： The CMT1X patients predominantly showed distal muscle weakness of lower limbs with mild sensory disturbance. The mean age of onset was 15.6 ± 8.7 years （ranging from 1 year to 42 years）. The sudden onset of cerebral symptoms appeared in four patients （ 18.2%）： two were initial symptoms. One case had constant central nervous system （CNS） signs. There were 19 different heterozygous mutations, including 15 known mutations and tbur novel mutations （c. II5G〉T, c.380T〉A, c.263C〉A, and c.818_819insGGGCT）. Among the 22 Chinese patients with CMT1X, the frequency of the GJB1 mutation was 4.5% in transmembrane domain 1 （TM1）, 4.5% in TM2, 27.7% in TM3, 9.1% in TM4, 4.5% in extracellular 1 （EC1）, 27.3% in EC2, 9.1% in intracellular loop, 13.6% in the N-terminal domain, and 4.5% in the C-ternlinal domain. CMTIX with CNS impairment appeared in five （22.7%） of these patients. Conclusions： This study indicated that CNS impairment was not rare in Chinese CMT1X patients. Mutations in the EC2 domain of the GJBI gene were hotspot in Chinese CMT1X patients.

ZNF217 expression correlates with the biological behavior of human ovarian cancer cells

The aim of the study was to investigate the correlation of zinc-finger protein 217 （ZNF217） gone ex- pression with the biological behavior of human ovarian cancer HO-8910 cells. Methods： The expression of ZNF217 in ovarian carcinoma cell line：s was detected by RT-PCR and Western blot, respectively. The biological behaviors of the transfectants were investigated by MTT, in vitro Boyden chamber and in vivo invasion assay, respectively. Results： RT-PCR and Western blotting revealed that transfection of ZNF217 into the HO-8910 cells significantly increased their proliferation along with mark- edly enhanced in vitro and in vivo invasion and metastatic abilities. MTT assay showed that the proliferation ability of pEGFP- N1-ZNF217/HO-8910 cells was significantly higher than that of pEGFP-N1/HO-8910 cells and HO-8910 cells （P 〈 0.001）. The Boyden chamber assay showed that the numbers of migrating pEGFP-N1-ZNF217/HO-8910, pEGFP-N1/HO-8910 and HO-8910 cells were （141.25 ± 13.91） cells/200 x field, （82.50 ± 11.73） cells/200 × field and （81.75 ± 12.12） cells/200 x field, respectively, with a significant difference between them （F = 29.274, P 〈 0.001）. The nude mouse experiment showed that the in vivo tumor formation ability of pEGFP-N1-ZNF217/HO-8910 cells was significantly higher than that of pEGFP-N1/HO-8910 cells （P 〈 0.001）. Conclusion： Based on these clinical and laboratory observations, we conclude that ZNF217 may contribute to ovarian cancer invasion and metastasis, and associated with worse clinical outcomes. We evaluated ZNF217＇s role as a biomarker of ovarian carcinogenesis and tumor progression in patient samples and explored possible molecular mechanisms in promoting tumor growth and invasion.

XIAP expression and its predictive significance of prognosis in stage IIB osteosarcomas

Objective： To evaluate X-linked inhibitor of apoptosis protein （XIAP） expression in biopsy specimens from the patients with stage lib osteosarcomas before chemotherapy to study XIAP expression and its predictive significance of progosis in stage lib osteosarcomas. Methods： The expression of XIAP was retrospectively detected by SP immunohistochem- istry in 31 cases biopsy specimens from stage lib osteosarcomas before chemotherapy, and the relationship between XIAP expression and clinicopathologic features of the patients with stage lib osteosarcomas was analyzed. Results： The osteo- sarcoma specimens showed strong cell nucleus immunoreaction of XIAP while normal bone specimens showed weak. High XIAP positive expression independently predicted the patient＇s poor outcome and had no relationship with clinicopathologic variables such as age, gender, location, tumor size, rate of tumor necrosis to preoperative chemotherapy, local recurrence （P 〉 0.05）. Significant correlations were found between high XIAP expression and metastasis （P -- 0.022）, and also between high XIAP expression and final survival （P = 0.011）. Conclusion： XIAP plays an important role in the development of stage liB osteosarcomas and XIAP expression of biopsy specimens before chemotherapy can be seen as a promising prognostic marker in early predicting the outcome of patients suffering from stage lib osteosarcomas,

Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA

The zinc-finger antiviral protein(ZAP)is a host factor that specifically inhibits the replication of certain viruses,including murine leukemia virus,Sindbis virus and Ebola virus,by targeting the viral mRNAs for degradation.ZAP directly binds to the target viral mRNA and recruits the cellular RNA degradation machinery to degrade the RNA.No significant sequence similarity or obvious common motifs have been found in the so far identified target viral mRNAs.The minimum length of the target sequence is about 500 nt long.Short workable ZAP-binding RNAs should facilitate further studies on the ZAP-RNA interaction and characterization of such RNAs may provide some insights into the underlying mechanism.In this study,we used the SELEX method to isolate ZAP-binding RNA aptamers.After 21 rounds of selection,ZAP-binding aptamers were isolated.Sequence analysis revealed that they are G-rich RNAs with predicted stem-loop structures containing conserved“GGGUGG”and“GAGGG”motifs in the loop region.Insertion of the aptamer sequence into a luciferase reporter failed to render the reporter sensitive to ZAP.However,overexpression of the aptamers modestly but significantly reduced ZAP’s antiviral activity.Substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity,suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA.The aptamers identified in this report should provide useful tools to further investigate the details of the interaction between ZAP and the target RNAs.

Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines

To investigate the regulation of tumor sup-pressor XAF1 gene expression in digestive system cancers,we studied XAF1 gene promoter transcription activity and mRNA level in digestive system cancer cell lines(human hepatoma cell line HepG2,human colon cancer cell line LoVo,and human gastric cancer cell line AGS)and nontumor cell lines(human embryonic liver cell line L02(L02 cells)and human embryonic kidney 293 cells[HEK293 cells])as controls.1395-bp-promoter fragment of XAF1 gene was amplified by polymerase chain reaction(PCR)and cloned into pGL3-basic vector and pEGFP-1 vector to assay its promoter transcription activity.The plasmids were transfected into a variety of cell lines by lipofectamine 2000.The promoter transcription activity was determined by dual-luciferase report assay,and enhanced greenfluorescent protein(EGFP)-positive cells were detected byfluorescence microscope.The expression of XAF1 mRNA in HEK293 and L02 were significantly higher than that in any of the three digestive system cancer cell lines.The dual-luciferase reporter assay showed that the promoter transcription activity in digestive system tumor cell lines transfected with pGL3-XAF1p promoter was apparently lower than that of both HEK293 and L02 cells.Expression of greenfluorescent protein(GFP)under the control of XAF1 promoter in the three digestive system cancer cell lines was lower than that of both HEK293 and L02 cells.The activities of pGL3-XAF1p in the three digestive system cancer cell lines after treatment with heat stress were significantly lower than those in the unstressed cells.The results suggested that remarkably down-regulated XAF1 mRNA expression in digestive system cancer cell lines may be due to loss of transcription activity of XAF1 promoter.