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Copyright of Bulletin of Botanical Research is the property of Bulletin of Botanical Research Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

bHLH（basic helix-loop-helix）transcription factors play crucial roles in the biosynthesis of flavonoids in plants. In order to explore the function and molecular regulation mechanism of bHLH transcription factor in flavonoid biosynthesis in tartary buckwheat, RTPCR was used to clone the FtbHLH3, one bHLH transcription factor. Then the bioinformatics subcellular localization, transcriptional activation activity, gene expression, gene co-expression, and the correlation between gene expression level and total flavonoid content of it were conducted. As results, the CDS length of FtbHLH3 was total 783 bp and it encoded total 260 amino acids. Conserved domain and phylogenetic analysis showed that FtbHLH3 was a member of non-IIIf subfamily of bHLH transcription factor family. Subcellular localization and transcriptional activation assay suggested that FtbHLH3 was located in the nucleus and had no transcriptional activation activity. Gene co-expression analysis revealed that FtbHLH3 was co-expressed with the Arabidopsis TT8 homologous gene FtTT8 and 12 structural genes of flavonoid biosynthesis in the tartary buckwheat. The correlation analysis between gene expression level and total flavonoids content in different tissues indicated that there was a highly positive correlation between the expressions of FtbHLH3 and the contents of total flavonoids. All these results suggest that FtbHLH3 is a non-IIIf subfamily bHLH transcription factor that was located in the nucleus and has no transcriptional activation activity. It may regulate the flavonoids biosynthesis in tartary buckwheat through interacting with other transcription factors. [ABSTRACT FROM AUTHOR]

Light-induced greening of tubers seriously affects its safety and economic benefits, but the mechanism of light-induced chlorophyll synthesis in potato tubers remains unclear. In this study, the related metabolites of potato tubers with different light durations were analyzed. The results were as follows: When the light duration prolonged, the chlorophyll content of the tubers gradually increased. Moreover, the chlorophyll content increased significantly at 36 hours, with which the tuber skin turned green obviously. Transcriptome sequencing and bioinformatics analysis were carried out on the samples taken at 0 hour, 6 hours, and 36 hours, and 5646 differentially expressed genes (DEGs) were identified. According to the co-expression cluster analysis and quantitative RT-PCR verification results, 9 major structure genes of chlorophyll biosynthesis pathway (StGAS1, StCHLD, StCrd1, StHEMA, StGUN4, StPORA, StUROD, StCHLM, and StCHLG) and 6 transcription factors (StSBP, StLSD, StGATA, StWRKY, StMYB-like, and StMYB113) were significant induced. Predictions of the cis-acting elements of the promoter sequences of these 9 structural genes indicated that they all contained multiple MYB binding sites. Promoter element analysis and transcriptional activation validation further revealed that StMYB113 had a light-responsive element and could activate the expression of StUROD in tobacco. Therefore, StMYB113 may be light-responsive and be able to regulate potato tuber greening in the light. This study provides a reference for the research on the regulatory mechanism of light-induced chlorophyll synthesis in potato tubers, which is important for reducing the loss caused by greening of potato tubers. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Oil Crop Sciences is the property of Oil Crops Research Institute of Chinese Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

BACKGROUND: How to improve the proliferation of human dental pulp stem cells (hDPSCs) in vitro is of great importance in regenerative medicine. OBJECTIVE: To investigate the effect of TAZ, a transcriptional coactivator, on the proliferation of hDPSCs and its potential mechanisms. METHODS: hDPSCs were isolated from human dental pulps and cultured in vitro. The expression of TAZ in hDPSCs was detected by immunofluorescence method. Then, we knocked down the expression of TAZ in hDPSCs, and the proliferation of hDPSCs was measured by MTT and BrdU kit analysis respectively. The levels of CTGF and Cry61, which are the downstream target genes of TAZ, were detected by RT-qPCR and western blot assay. We also investigated the effect of TAZ on the levels of transforming growth factor-β (TGF-β) signaling proteins, Smad3 and Smad 4 by using western blot. RESULTS AND CONCLUSION: TAZ protein was expressed in hDPSCs. After the transfection by siTAZ for 24 hours, the mRNA and protein levels of TAZ were both significantly decreased. After the transfection by siTAZ for 24 and 48 hours, the proliferation of hDPSCs was obviously inhibited. Meanwhile, the mRNA and protein levels of CTGF and Cry61 were decreased by siTAZ transfection. The further research showed that TAZ silence also inhibited the expression of Smad3 and Smad 4, which belonged to the TGF-β signaling pathway. To conclude, TAZ may modulate the proliferation of hDPSCs through regulating the expression of CTGF and Cry61, and TGF-β-dependent signaling pathways may be involved in this regulation. [ABSTRACT FROM AUTHOR]

Objectives: Application of ultrashort wave (USW) to rats with cerebral ischemia and reperfusion injury could inhibit the decrease of expression of secretory pathway Ca 2+ -ATPase 1 (SPCA1), an important participant in Golgi stress, reduce the damage of Golgi apparatus and the apoptosis of neuronal cells, thereby alleviating cerebral ischemia-reperfusion injury. This study aims to investigate the effect of USW on oxygen-glucose deprivation/reperfusion (OGD/R) injury and the expression of SPCA1 at the cellular level., Methods: N2a cells were randomly divided into a control (Con) group, an OGD/R group, and an USW group. The cells in the Con group were cultured without exposure to OGD. The cells in the OGD/R group were treated with OGD/R. The cells in the USW group were treated with USW after OGD/R. Cell morphology was observed under the inverted phase-contrast optical microscope, cell activity was detected by cell counting kit-8 (CCK-8), apoptosis was detected by flow cytometry, and SPCA1 expression was detected by Western blotting., Results: Most of the cells in the Con group showed spindle shape with a clear outline and good adhesion. In the OGD/R group, cells were wrinkled, with blurred outline, poor adhesion, and lots of suspended dead cells appeared; compared with the OGD/R group, the cell morphology and adherence were improved, with clearer outlines and fewer dead cells in the USW group. Compared with the Con group, the OGD/R group showed decreased cell activity, increased apoptotic rate, and down-regulating SPCA1 expression with significant differences (all P <0.001); compared with the OGD/R group, the USW group showed increased cell activity, decreased apoptotic rate, and up-regulating SPCA1 expression with significant differences ( P <0.01 or P <0.001)., Conclusions: USW alleviates the injury of cellular OGD/R, and its protective effect may be related to its up-regulation of SPCA1 expression.

Objectives: To analyze the expressions and distributions of hypoxia-inducible factor-1α (HIF-1α), CD147, and glucose transporter 1 (GLUT1) in epidermis from psoriasis vulgaris and normal people, and to explore the associations among these proteins and their roles in hypoxic HaCaT cell line., Methods: The expression levels of HIF-1α, CD147, and GLUT1 were determined by immunohistochemistry staining in skin biopsies from 48 psoriasis vularis patients and 33 healthy subjects. Cobalt chloride (CoCl 2 ) was added into the culture media of HaCaT cells to mimic hypoxia while RNA interference and transfection technologies were used to explore the association among these proteins by quantitative real-time polymerase chain reaction and Western blotting. Glycolytic capacity was detected by ATP and lactate measurements., Results: HIF-1α, CD147, and GLUT1 were highly expressed and the glycolytic capacity was increased in lesions of psoriasis vulgaris; HIF-1α upregulated the expression of CD147 and GLUT1, increased the lactate production and decreased the ATP level in CoCl 2 -treated HaCaT cells, while CD147 and GLUT1 directly or indirectly bound to each other., Conclusions: Glycolytic capacity increases in the injured keratinocytes of psoriasis vulgaris, suggesting that HIF-1α, CD147, and GLUT1 are associated with glycolysis, which can be considered as the promising targets for psoriasis therapy.

Human bocavirus 1 (HBoV1) non-structural protein NS1 is a multifunctional protein important for virus replication and induction of apoptosis in host cell. To better understand the function of the NS1 protein, it is urgent to address reducing the toxicity of NS1 to host cells. In the present study, we established a stable cell line that regulates expression of NS1 of HBoV1. The recombinant lentivirus plasmid containing a regulatable promoter fused with ns1 gene was constructed and transfected into HEK 293T cells using transfection reagent. The HEK 293T cell lines stably expressing NS1-100 and NS1-70 proteins were established by screening resistant cells with puromycin and inducing NS1 expression with doxycycline. The expression of NS1 protein was determined by fluorescent labeling protein and Western blotting. HBoV1 promoter was transfected into stably expressing NS1 cell line and its trans-transcriptional activity was analyzed. The results showed that NS1 protein was expressed stably in the established cell lines and had a strong activation activity on the HBoV1 promoter driving luciferase gene. Taken together, this study provides a solid basis for further research on the function of NS1 and the pathogenesis of human bocavirus.

The genetic compensation response (GCR) was firstly described in zebrafish to explain the phenotypic discrepancies between gene-knockout and gene-knockdown, whereby a deleterious mutation, but not gene-knockdown, can lead to the transcriptional upregulation of related genes, which can assume the function of the mutated gene. This phenomenon was also found in other model systems including mice and Arabidopsis. However, the underlying molecular mechanism of the GCR remains elusive until two papers were published in Nature on April 3, 2019: one from our lab and the other from Stainier's lab. Using different genetic mutants of various genes in zebrafish or culture cells of mice, both of us reveal that the upregulation of compensatory genes is only triggered by mutations that generate a premature termination codon (PTC); the compensatory genes share nucleotide sequence homology to the mutated genes; nonsense mRNA mediated decay pathway (NMD) is essential for the induction of GCR, and the increased transcription of the compensatory genes is accompanied by an enhancement of H3K4 trimethylation (H3K4me3) at their transcription start site (TSS) regions. In this review, we summarize the mechanisms of the GCR proposed in the two studies.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, has become a major human infectious disease. The existing first-line and second-line TB drugs have poor treatment outcomes in patients with MDR-TB and XDR-TB. There is an urgent need for new and better drugs to treat tuberculosis due to lengthy and complex treatment regimens and a rising problem of drug resistance. Microbial-derived natural products have revealed enormous reservoirs of as yet untapped lead compounds. In this review, we discuss the strategies that have been developed in bacteria and fungi to isolation of non-culturable microorganisms and activation of silent biosynthetic gene clusters involved in the study of microbial-derived natural products. This review also highlights recent advances in microbial-derived natural products with anti-tuberculosis activity using these methods.

Objective: To investigate the mechanism for the synergistic effect of interferon regulatory factor 4 (IRF4) and microphthalmia-associated transcription factor (MITF) on tyrosinase (TYR) promoter.
 Methods: The synergistic transcriptional effect, subcellular localization, and protein-protein interaction for IRF4 and MITF were observed by luciferase assay, immunofluorescence, GST-pull down, and co-immunoprecipitation, respectively.
 Results: IRF4 and MITF proteins were co-expressed in the cell nucleus. IRF4 augmented the transcriptional function of MITF (but not the mutant MITF) to activate the expression of the TYR promoter, but with no effect on other MITF-specific target promoters. IRF4 alone did not affect TYR promoter significantly. No direct interaction between the two proteins was noted.
 Conclusion: IRF4 and MITF exert a specifically synergistic effect on activation of TYR promoter through IRF4-mediated upregulation of transcriptional function of MITF. This synergistic effect is mainly regulated by MITF; DNA might be involved in the interaction between the two proteins.

Objective: To identify the novel HAND1 mutation associated with congenital ventricular septal defect (VSD) and to perform the functional analysis.
 Methods: A total of 125 patients with congenital VSD and 210 control individuals were recruited, and their clinical data and blood samples were collected. The genomic DNA from each study subject was isolated, and all the coding exons of HAND1 were amplified. The amplicons from HAND1 were sequenced to identify a sequence variation. The functional characteristics of the mutant HAND1 were analyzed by a dual-luciferase reporter assay system.
 Results: A novel heterozygous HAND1 mutation c.355G>T, equivalent to E119X, was identified in a patient with sporadic VSD. This nonsense mutation was absent in the 210 control subjects. Functional analysis revealed that the mutant HAND1 lost the ability to transactivate a target gene.
 Conclusion: A novel HAND1 mutation with VSD is identified in this study.

Objective: To investigate the interaction between nuclear transcriptional factor E26 transformation specific 1 (Ets1) and peroxiredoxin 1 (Prx1) in nicotine-induced oral precancerous lesion cells. Methods: Human oral precancerous lesion dysplastic oral keratinocyte (DOK) cells were cultured and divided into nicotine group, control group, knockdown group and knockdown control group. The nicotine group, knockdown group and knockdown control group were treated with 1 μmol/L nicotine for 7 days while the control group was untreated. Western blotting, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) were performed to detect Prx1 and Ets1 protein expression, Prx1 and Ets1 protein interaction, combined activity of Ets1 with PRDX1 gene promoter region in nicotine group and control group DOK cells. In nicotine group, DOK cells were transfected with siRNA or lentivirus to knockdown Ets1 and Prx1 expression. Prx1 and Ets1 protein expression was examined by Western blotting. Results: Nicotine increased the expression of Prx1 and Ets1 protein in DOK cells. The relative expression of Prx1 and Ets1 was 0.71±0.02, 0.12±0.01 in nicotine group and 0.53±0.06, 0.01±0.01 in control group ( P= 0.009, P= 0.000). Co-IP showed that Prx1 could form protein complex with Ets1. The expression of Prx1 and Ets1 complex protein was increased in nicotine group. ChIP revealed that nicotine upregulated the combination of transcriptional factor Ets1 with PRDX1 gene promoter region, and the enrichment fold was 80.9±19.7 in nicotine group and 13.8±1.2 in control group ( P= 0.004). Ets1 and Prx1 protein expression was knocked down. The relative expression of Ets1 and Prx1 was 0.60±0.06, 0.48±0.03 in knockdown group and 0.83±0.08, 0.80±0.06 in knockdown control group ( P= 0.016, P= 0.002). Ets1 knockdown suppressed the expression of Prx1 ( P= 0.002). Conversely, Prx1 knockdown also inhibited the expression of Ets1 significantly ( P= 0.000). Conclusions: In oral precancerous lesion cells, Ets1 directly regulates Prx1 expression and nicotine might promote the development of oral precancerous lesion by magnifying the positive feedback signal pathway between Ets1 and Prx1.

The present study was aimed to investigate the effect of acute hypoxia on telomere length of rat gastric mucosa tissue and possible mechanism. Forty male Wistar rats were randomly divided into control group (resided in Lanzhou, 1 500 m) and experimental group (hypoxia chamber, 5 000 m). The experimental group was further divided into 3 subgroups and exposed to hypoxia for 1, 3, 7 d (n = 10), respectively. The morphological changes of the gastric mucosa tissue were observed by HE staining. By means of real-time PCR, ELISA and chemical immunofluorescence methods, the telomere length, the mRNA and protein levels of telomerase reverse transcriptase (TERT), hypoxia-inducible factor 1α (HIF-1α) and HIF-2α, and reactive oxygen species (ROS) level in gastric mucosa tissue were measured, respectively. The results showed that, with the extension of hypoxia-exposure time, the injury in gastric mucosa cells progressively became worse, and telomere length was increased gradually, along with intracellular ROS generation. The changes of TERT and HIF-1α expressions induced by acute hypoxia were in the same trend as that of telomere length. There were positive correlations between TERT mRNA expression and telomere length and between TERT and HIF-1α expressions, but not between TERT and HIF-2α mRNA expressions. These results suggest that under acute severe hypoxia environment, ROS could damage the gastric mucosa tissue cells, meanwhile the expressions of TERT and telomerase activity may be up-regulated by HIF-1α, which can elongate the telomere length and protect gastric mucosa tissue against fatal injury.

Melatonin has a significant inhibitory effect on various cancers, especially on breast cancer. In estrogen receptor (ER)-positive human breast cancer, anti-cancer effects of melatonin on breast cancer cells and transplanted tumors mainly achieve by suppressing ER mRNA expression and ER transcriptional activity via the MT1 receptor. In addition, melatonin regulates the transactivation of other members of the nuclear receptor super-family, estrogen metabolizing enzymes, and the expression of related genes. Furthermore, melatonin also suppresses tumor aerobic metabolism, critical cell-signaling pathways relevant to cell proliferation, survival, metastasis, and drug resistance. Melatonin demonstrates both cytostatic and cytotoxic activity in breast cancer cells that appears to be cell-type specific. Studies on animal and human models indicate that disruption of the circadian nocturnal melatonin signal promotes the growth, metabolism, and signaling of human breast cancer, resulting in invalid hormone therapy and chemotherapeutic resistance in breast tumors.

Objective: To explore the role of the special AT rich sequence binding protein-1 (SATB1) and ribonucleotide reductase M2 (RRM2) in enhancing malignant progression of non-Hodgkin lymphoma (NHL). 
 Methods: A total of 42 NHL and 42 chronic lymphadenitis patients were recruited. The protein expressions of SATB1 and RRM2 in cervical lymph nodes were determined by Western blot. After overexpression of SATB1, siSATB1 or siRRM2, the mRNA levels of SATB1 and RRM2 in cells were analyzed via RT-PCR, the cell proliferation was evaluated via MTT and EdU assays, while the migration and invasion of cells were assessed by transwell assays.
 Results: Compared with chronic lymphadenitis, the expressions of SATB1 and RRM2 in NHL patients were up-regulated. There was positive correlation between SATB1 and RRM2 in NHL patients. RRM2 mRNA level was up-regulated after transfection of SATB1 and down-regulated after transfection of siSATB1. Overexpression of SATB1 increased tumor cell proliferation, migration and invasion, while knockdown of RRM2 reversed those phenomena.
 Conclusion: SATB1 functions as an oncogene and promotes tumor cell proliferation, migration and invasion by up-regulation of RRM2 in NHL.

Objective: To study the effect of chronic hypoxia on pulmonary arterial remodeling and Krüppel-like zinc-finger transcription factor 5 (KLF5) protein expression in pulmonary artery smooth muscles in a hypoxia-induced pulmonary hypertension model. Methods: Totally 20 adult SD rats (200-250 g) were divided into a normoxia group and a chronic hypoxia group by the random number table. Rats in the chronic hypoxia group were put in an automatic hypoxia box for 21 days. After that, right ventricular systolic pressure (RVSP), mean right ventricular pressure (mRVP) and RV/(LV+ S) were measured. Lung tissue sections were made. The lumen area, ratio of wall thickness to radius of pulmonary artery were gauged by using the Image Pro Plus software. Primary PASMCs were cultured in oxygen-deficient environment (4% O 2 )or normal oxygen environment (21% O 2 )for 60 hours respectively, and then total protein or RNA were extracted for Western blotting and Q-PCR analysis.KLF5 protein expression in rat pulmonary arterial smooth muscle and pulmonary arterial smooth muscle cells was detected by Western blot. Results: Compared with the normoxia group(28.3±0.4), (11.3±1.0)mmHg (1 mmHg=0.133 kPa), the RVSP and mRVP in the chronic hypoxia group [(43.9±1.3), (26.5±2.3)mmHg] were significantly increased( P <0.05). The Rv/(LV+ S)of the chronic hypoxia group was(0.48±0.03), markedly higher than that of the normoxia group(0.27±0.01, P <0.05). The luminal area/total area of artery in the chronic hypoxia group decreased to (46.1±6.6)% compared with that in the normoxia group [(68.73±3.06)%, P <0.05]. The wall thickness/arterial radius(WT%)of the chronic hypoxia group increased up to (5.64±0.32)% as compared with (3.7±0.4)% of the normoxia group ( P <0.05). The level of KLF5 protein in pulmonary arterial smooth muscles of the chronic hypoxia group was (21.6±7.2) times that of the normoxia group ( P <0.05). Conclusion: Hypoxia induced the increase of RVSP, mRVP, RV/(LV+ S), accompanied with pulmonary arterial remodeling. The underlying mechanism of the artery change may be related to up-regulated expression of KLF5.

Objective: To investigate the expression of microRNA-107 (miR-107) and its functional role in hepatocellular carcinoma(HCC)., Methods: The gene chip data of HCC obtained from the Gene Expression Omnibus (GEO) database and the Cancer Genome Atlas (TCGA) database were used to analyze the expression levels of miR-107 in liver cancer. Twenty-two pairs of fresh surgical specimens of HCC and adjacent tissues and 53 paraffin-embedded specimens of HCC were examined for miR-107 expression by qRT-PCR. The correlation of the expression levels of miR-107 with the clinicopathologic characteristics of the patients were analyzed. The role of miR-107 in regulating the proliferation of hepatocellular carcinoma cells were determined by MTT assay in Huh7 cells transfected with a miR-107 mimic or inhibitor., Results: The expression levels of miR-107 were significantly up-regulated in HCC tissues as compared to the adjacent tissues (P<0.05) in positive correlation with the tumor size (P<0.032). Transfection with miR-107 mimics significantly promoted the cell proliferation (P<0.0001) while miR-107 inhibitor inhibited the cell proliferation (P<0.0001)., Conclusion: The expression of miR-107 is up- regulated in HCC tissues and its expression levels are correlated with HCC cell proliferation, suggesting its role as a potential oncogene in liver cancer.

This study was designed to identify inducers of ATP-binding cassette transporter A1(ABCA1) and CD36 and lysosomal integral membrane protein-II analogous-1(CLA-1) and to evaluate the in vitro effect of the active compound on lipid metabolism. Among 20 000 compounds screened, E23869 was found as a positive hit using cell-based high throughput screening models. The up-regulating activities of E23869 in ABCA1p-LUC and CLA-1p-LUC Hep G2 cells were 196% and 198%, respectively. The EC(50) values of E23869 in ABCA1p- LUC and CLA-1p-LUC Hep G2 cells were 0.25 μmol·L(-1) and 0.66 μmol·L(-1), respectively. E23869 significantly upregulated the protein levels of ABCA1, scavenger receptor class B type I(SR-BI)/CLA-1 and ATP-binding cassette transporter G1(ABCG1) in both macrophages RAW264.7 and L02 cells by Western blotting analysis. Foam cell assay showed that E23869 inhibited lipids accumulations in macrophages RAW264.7. Cholesterol efflux assay showed that E23869 induced HDL-mediated cholesterol efflux in macrophages RAW264.7. Moreover, E23869 up-regulated ABCA1, SR-BI/CLA-1 and ABCG1 expressions through activation of PPARα and PPARγ. In addition, E23869 weakly promoted in vitro differentiation of mouse preadipocytes 3T3-L1. In conclusion, E23869 up-regulated ABCA1, SR-BI/CLA-1 and ABCG1 expressions to promote cholesterol efflux, which is a good leading compound for regulation of lipid metabolism.

Objective: The NKX3.1 homeobox gene is closely associated with the development and progression of prostate cancer. This study was to explore NKX3.1-related down-stream node genes and their possible regulating mechanisms in prostate cancer., Methods: By multi-omics analysis of the TCGA data on prostate cancer,we screened 5 node genes in the down-stream signaling pathways that were possibly related to NKX3.1.We achieved the overexpression of NKX3.1 in prostate cancer by transfecting the prostate cancer PC-3 cell lines with the NKX3.1 expression vector and determined the expression levels of the node genes by real-time PCR., Results: Based on the results of multi-omics analysis,MAZ,LPAR3,TUBB2A,CAMKK2 and CPT1B were identified as the node genes involved in the NKX3.1-related signaling pathways in prostate cancer. The NKX3.1 overexpression experiments showed that the CAMKK2 and CPT1B genes were up-regulated 3. 439 and 4. 641 times respectively and the MAZ gene down-regulated 5.236 times in the prostate cancer PC-3 cells with the overexpression of NKX3.1., Conclusion: NKX3.1 may suppress the development and progression of prostate cancer by down-regulating the expression of MAZ and up-regulating those of CAMKK2 and CPT1B,and it may also be involved in the regulation of the metabolic process of prostate cancer through the CAMKK2 down-stream signaling pathway and CPT1B.

Lipin-1 can promote the synthesis of triglyceride and fatty acid oxidation during lipid metabolism. Ethanol can up-regulate the expression of lipin-1 via the AMPK-SREBP-1-lipin-1 signaling pathway and increase lipin1β/αratio via the SITR1-SFRS10-LPIN1 signaling pathway. In the cytoplasm, lipin-1βfunctions as a Mg2+-dependent phosphatidic acid phosphohydrolase to accelerate the synthesis of triglyceride. In the nucleus, lipin-1αacts as a transcriptional coactivator and reduces mitochondrial fatty acid oxidation by inhibiting the activities of PPARαand PGC-1α, and therefore promotes lipid deposition in hepatocytes and causes hepatic steatosis.

Objective: To construct a luciferase reporter gene vector of p-selectin gene promoter and determine its transcriptional activity for screening the effect of drugs on the transcriptional activity of p-selectin promoter., Methods: Primers were designed based on human p-selectin promoter sequence from UCSC software. The p-selectin promoter from human genome DNA was then amplified. After digestion of pGL3-Basic vector and p-selectin promoter with Kpn I and Xho I, p-selectin promoter was inserted into pGL3-basic vector. The recombinant plasmid, namely pGL3-p-selectin-promoter, was transiently cotransfected into 293F cells with pRL-SV40 as the control vector, and the activity of the dual luciferase was detected. The transcription activity of serially truncated segments of the p-selectin promoter reporter gene was quantified by luciferase expression. 293F cells transfected with pGL3-p-selectin-promoter reporter gene and dual luciferase were stimulated with LPS, TNF-α and As2O3, and the transcriptional activity of p-selectin promoter were assessed., Results: pGL3-p-selectin-promoter was constructed successfully as verified by restriction digestion and sequence analysis. The luciferase activity was higher in pGL3-p-selectin-promoter/pRL-SV40 group than in pGL3-basic/pRL-SV40 group (0.8573±0.4703 vs 0.03955±0.05894). pGL3- 1826 bp was actively transcribed compared with pGL3-1092 bp and pGL3-3738 bp. LPS, TNF-α and As2O3 significantly enhanced the transcriptional activity of p-selectin promoter., Conclusion: pGL3-p-selectin-promoter can be transcribed and activated in 293F cells. This study provided an important basis for acquiring transcriptional factors and screening inflammatory factors and drugs.

Objective: To determine the expression of Skp2 in different prostate cancer (PCa) cell lines and tissues, and explore its influence on the androgen receptor (AR) signaling pathway and development of castration-resistant prostate cancer (CRPC)., Methods: The expression levels of Skp2 and AR in different PCa cell lines were detected by Western blot. After knockdown of Skp2 in the C4-2 and 22RV1 cells transfected with shRNA, the expressions of AR and P27 were determined and the activity of ARR3-Luc measured by dual-luciferase reporter gene assay following treatment with dihydrotestosterone (DHT). The expressions of AR and Skp2 in human naïve PCa or CRPC specimens were detected by immunohistochemical staining followed by analysis of their differences and correlation., Results: The Skp2 protein expression level was significantly higher in the C4-2 or 22RV1 cells than in the LNCaP cells. DHT treatment increased the expression of Skp2 in the C4-2 cells, but knock-down of Skp2 significantly up-regulated the expression of the well-known downstream protein P27 and down-regulated that of AR. Consistently, DHT treatment increased the activity of ARR3-Luc, while knockdown of Skp2 remarkably decreased it in the C4-2 and 22RV1 cells (P < 0.05). In addition, significantly higher expressions of Skp2 and AR were observed in the CRPC than in the naïve specimens (P < 0.05), with a positive correlation between the two proteins (r = 0.658 1, P < 0.05)., Conclusion: Skp2 can enhance the expression and transcription activity of the AR protein in CRPC cells or tissues and is promising to be a critical molecular therapeutic target.

Objective: To study the phenotypic alteration of intestinal dendritic cells (DC) in a rat model of irritable bowel syndrome (IBS) and the change of mitogen-activated protein kinase (MAPK) signaling pathway, in order to explore the potential mechanism of ERK1/2 pathway mediation in abnormal DC immune response., Methods: IBS rat model was established by combining neonatal maternal separation and colorectal distension in 10 SD rats, and 10 healthy rats served as controls. Visceral sensitivity was evaluated with abdominal withdrawal reflex (AWR). Mesenteric lymph node DC (MLNDC) was isolated and purified by magnetic label-based technique after modeling. Expression of surface major histocompatibility complex (MHC)-Ⅱin control rats was determined by flow cytometric analyses. Western-blot was used to determine the expression of MHC-Ⅱ, p-p38, p38, phosphorylated extracellular regulated protein kinase (p-ERK1/2), ERK1/2, phosphorylated c-Jun N-terminal kinase (p-JNK), and JNK in MLNDC., Results: Visceral sensitivity was significantly higher in the IBS group than in the control group. The purity of the OX62 positivity MLNDC following magnetic sorting was about 85.57%±7.67%. MLNDC in the control group expressed high level of MHC-Ⅱ. The expression of MHC-Ⅱ and p-ERK1/2 in MLNDC in the IBS group were higher than those in the control group (1.05±0.13 vs 0.67±0.18, t=-2.973, P=0.041; 3.21±0.48 vs 2.34±0.85, t=-3.130, P=0.035); while there was no significant difference in the expressions of p-JNK and p-p38 compared with the control groups (0.95±0.17 vs 0.76±0.36, t=0.808, P=0.464; 1.07±1.13 vs 1.19±0.91, t=0.137, P=0.897)., Conclusion: The intestinal DC in IBS rats show a upregulated expression of MHC-Ⅱ, which may be related to the activation of intracellular ERK1/2 pathway.

Objective: To study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome., Methods: Eukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays., Results: Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus., Conclusion: This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.

Objective: To evaluate the effect of physiological dose 17-β-estrodiol (E2) replacement therapy on vascular dementia caused by cerebral chronic hypoperfusion., Methods: The rats with bilateral common carotid artery occlusion (BCCAO) received E2 treatment starting from 3 days or 3 months after the operation. IgG leakage into the brain parenchyma and the changes of microvascular ultrastructure following BCCAO were examined using immunohistochemistry and electron microscopy, respectively; Western blotting was used to detect the expression of vascular endothelial growth factor (VEGF) protein., Results: Compared with the sham-operated groups, the rats at 3 days and 3 months after BCCAO showed extensive vascular damages surrounded by IgG immunoreactivity in both the cortical and hippocampal CA1 regions. Stronger IgG immunoreactivity in the hippocampal CA1 region was observed at 3 days after BCCAO than at 3 months, but no significant IgG leakage was found in rats with continuous E2 treatment. Electron microscopy revealed severe edema around the blood vessels, mild vascular dilation, and endothelial cell damages at both 3 days and 3 months after BCCAO. E2 treatment markedly reduced the microvascular ultrastructural damages. Western blot analysis showed a significant increase in VEGF expression in the CA1 region at 6 h and 1 day after BCCAO followed by an obvious reduction till reaching the lowest level at 3 days; VEGF expression remained low even at 3 months after BCCAO and was significantly increased by E2 treatment., Conclusions: Vascular structural damage occurs early after BCCAO and can last for 3 months. E2 replacement therapy at physiological doses can reduce the incidence of BCCAO-induced vascular dementia by up-regulating VEGF expression.

To study the regulation of androgen receptor (AR) expression in human prostate cancer LNCaP cells by triptolide (TP) and the possible mechanism, by using qRT-PCR and Western blot, the AR mRNA and protein levels in TP treated LNCaP cells were detected, and the AR protein level in TP and NF-κB inhibitor treated LNCaP cells was also detected; a series of pGL3-AR promoter reporter gene vectors were built using restriction-free cloning method, and the vectors were employed to investigate the effects of TP on the transcriptional activity of AR promoter in LNCaP cells; the upstream proteins which may play regulatory roles were detected using western blot assay. After treated LNCaP cells with TP for 48 h, AR mRNA and protein expressions decreased with increasing TP concentration. The expression of AR target gene PART1 and prostate specific antigen (PSA) was also downregulated by TP treatment; a series of pGL3-AR promoter reporter vectors were constructed and validated by sequencing and luciferase activity; the results of dual luciferase reporter assay showed that TP downregulated AR at the transcriptional level; PI3K/AKT/NF-κB pathway which is associated with AR promoter activity was drowregulated by TP. In conclusion, our results demonstrated that the transcriptional activity of AR in LNCAP cells was downregulated by TP, and PI3K/AKT/NF-κB pathway may be involved in the regulation mechanism.

The study aimed to investigate the effects of small ubiquitin-related modifier (SUMO) specific protease 1 (SENP1) on human PXR-mediated MDR1 transcriptional activity and mRNA expression. Empty vector and expression plasmids, including PXR, SENP1 and SENP1 mutant (SENP1m) were transiently transfected into HepG2 and LS174T cells using Lipo2000. Transcriptional activity was detected by dual luciferase reporter gene assay, and mRNA level was measured using real-time polymerase chain reaction. The results showed that SENP1 could remarkably reduce the rifampicin (RIF)-induced MDR1 reporter activity and mRNA level in hPXR over expressed HepG2 and LS174T cells (P < 0.05), whereas adding SENP1m restored the RIF-induced increases (P < 0.05). These results indicated that SENP1 could repress the RIF-induced hPXR-mediated MDR1 transcriptional activity and mRNA expression.

Objective: To investigate the effect of up-regulation of Rap1GAP on the invasion ability of leukemic HL-60 cells in vitro, and to establish leukemia mouse model to verify the effects in vivo., Methods: Quantitative RT-PCR and Western blot methods were used to detect the expression of Rap1GAP in Venus/HL-60 (vehicle control) and Rap1GAP/HL-60 cells (R1 andR2). Transwell method was used to examine the invasion ability in vitro. Quantitative RT-PCR and gelatin zymograph were used to study the expression of MMP-2 and MMP-9. Four-week-old BALB/c nu/nu mice were pre-treated and inoculated with leukemic cells from different groups, several index including survival time were then monitored., Results: Rap1GAP mRNA level of R1 and R2 increased about 16-17 folds as compared to the control cells. The invasion rate of R1 and R2 are (55 ± 5)% and (59 ± 4)%, which are significantly higher than (14 ± 4)% of the control cells. The mRNA level of MMP-9 was up-regulated about 12.0 folds in R1 and R2 cells compared to the corresponding control cells. The median survival times of R1 and R2 mice are (32.00 ± 1.85) d and (33.37 ± 2.50) d, respectively, which are shorter than (43.62 ± 2.32) d of the control group. Three mice of R1 and R2 groups showed leukemic cells infiltration in meninges tissue, and the genes of Rap1GAP and MMP-9 were amplified by PCR method., Conclusion: Up-regulated expression of Rap1GAP increased the invasion ability of HL-60 cells accompanied with enhancement of MMP-9 expression in vitro, and the experiment in mouse model also confirmed that Rap1GAP enhanced the invasion of HL-60 cells in vivo.

Objective: To investigate the role of endoplasmic reticulum stress in the apoptosis of testicular germ cells in hyperlipidemic rats., Methods: We randomly assigned 42 four-week-old male Wistar rats into a normal control group (n = 12) and a high-fat group (n = 30) to be fed on a normal diet and a high-fat diet, respectively, for 10 weeks. Then we measured the concentrations of triglyceride (TG) and total cholesterol (TC) in the serum using an automatic biochemistry analyzer, detected the apoptosis of testicular germ cells by TUNEL staining, and determined the protein and mRNA expressions of GRP78 and. caspase-12 in the testis tissue by immunohistochemistry and RT-PCR, respectively., Results: The concentrations of TG and TC were significantly increased in the animals of the high-fat group ([3.00 ± 0.92] and [3.04 ± 0.39] mmol/L) as compared with the control rats ([1.43 ± 0.41] and [1.55 ± 0.23] mmol/L) (P < 0.01), and so was the apoptosis index of the testicular germ cells ([37.17 ± 2.74]% vs [5.16 ± 0.81]%, P < 0.01). The high-fat group, in comparison with the control, also showed remarkably upregulated protein and mRNA expressions of GRP78 (0.32 ± 0.03 and 0.86 ± 0.05 vs 0.19 ± 0.01 and 0.37 ± 0.03, P < 0.01) and caspase-12 (0.34 ± 0.02 and 0.87 ± 0.01 vs 0.12 ± 0.01 and 0.34 ± 0.03, P < 0.01) in the testis tissue., Conclusion: The apoptosis of testicular germ cells is increased in hyperlipidemic rats, which may be attributed to endoplasmic reticulum stress.

Objective: To screen and verify differentially expressed genes in prostate cancer., Methods: Using DNA microarray, we screened differentially expressed genes in prostate cancer tissue and its adjacent tissue followed by verification by PCR., Results: A total of 1 444 genes were found to be differentially expressed (differentiation ≥ 1.5-fold; P≤ 0.05) in the prostate cancer tissue, of which 769 (53%) were up-regulated and 675 (47%) down-regulated. Fifty percent of the differentially expressed genes showed a 1.5- to 2-fold differentiation, including 396 up-regulated and 182 down-regulated ones. Additionally, 308 up-regulated and 334 down-regulated genes exhibited a >2- to 5-fold, 46 up-regulated and 78 down-regulated genes a > 5- to 10-fold, and 19 up-regulated and 81 down-regulated genes a > 10-fold differentiation. Verification by subjecting 15 most significantly up-regulated and another 15 most markedly down-regulated genes to quantitative real-time PCR (qRT-PCR) showed that most of the genes had a transcriptional profile similar to that in the microarray data, with a Pearson correction coefficient of 0.83 between the microarray data and qRT-PCR results. Totally, 10 significantly differentially expressed genes were identified., Conclusion: DNA microarray analysis provides reliable information on differentially expressed genes in prostate cancer and benign tissues. The 10 significantly differentially expressed genes verified by qRT-PCR could possibly become new bio-markers and specific molecules for tumor identification.

Objective: To investigate the effect of transforming growth factor-β1 (TGF-β1) on epithelial-mesenchymal transition in type II alveolar epithelial cells and the relationship with Calpain., Methods: The type II alveolar epithelial cell line-A549 cells cultured in vitro were divided into 4 groups: the control group, the TGF-βl group, the TGF-β1 + PD150606 group, the PD150606 group. Given the corresponding processing,Western-blot and RT-PCR were utilized to evaluate the expression of Calpain- 1,E-cad and α-SMA., Results: Compared with the control group, the protein and mRNA expression of Calpain-1 and α-SMA in the TGF-β1 group was markedly increased, but the protein and mRNA expression of E-cad was significantly decreased. While, after calpain inhibitor PD150606 was added, the protein and mRNA expression of Calpain-1 and α-SMA was markedly decreased and the protein and mRNA expression of E-cad was significantly increased in the TGF-βl + PD150606 group, but there was no difference between TGF-β1 + PD150606 group and control group. But with Calpain inhibitor PD150606 alone, the expression of Calpain-1,E-cad and α-SMA was no difference with the control group., Conclusions: TGF-βl could induce epithelial-mesenchymal transition in A549 cells by up-regulating expression of calpain which could be inhibited by calpain inhibitor PD150606.

Objective: To study the protective effect and mechanism of augmenter of liver regeneration (ALR) on mice with acute liver injury., Methods: Thirty BALB/c mice were randomly divided into normal control group, acute liver injury group and ALR intervention group. Acute liver injury group was given intraperitoneal injection of the mixture (2 mL/kg body mass once) of CCl4 (500 mL/L) and mineral oil. ALR intervention group was given ALR plasmid by tail intravenous injection 8 hours before CCl4 injection. The control group was injected the same amount of normal saline. The pathological changes of liver tissue were detected by HE staining. The serum was collected for detecting alanine transaminase (ALT) and aspartate transaminase (AST). The frequency of regulatory T cells (Tregs) in liver tissue was analyzed by flow cytometry. The mRNA levels of Foxp3, ALR, interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were measured by real-time quantitative PCR (qRT-PCR)., Results: The expression level of ALR mRNA in ALR intervention group was significantly higher than that in acute liver injury group and in normal control group; no statistically significant difference was found between the latter two groups. The frequency of Tregs in liver tissue had significant differences among these groups; CD25⁺Foxp3⁺Treg/CD4⁺T level was higher in ALR intervention group [(5.90 ± 0.10)%] than in acute liver injury group [(4.23 ± 0.46)%] and in normal control group [(2.93 ± 0.74)%]; and it was higher in acute liver injury group than in normal control group. The expression level of Foxp3 mRNA in liver tissue had the same trend as the results of flow cytometry. It was higher in ALR intervention group than in acute liver injury group and in the normal control group. The mRNA levels of IL-6 and TNF-α in ALR intervention group decreased as compared with acute liver injury group; the mRNA levels of IL-6 and TNF-α in acute liver injury group significantly increased as compared with the control group., Conclusion: ALR can protect mice against acute liver injury by up-regulating the expression of Tregs, which may be related to Tregs-mediated down-regulation of inflammatory cytokines IL-6 and TNF-α.

In-vitro assay methods were established to evaluate transactivation and binding activity of compounds on peroxisome proliferator-activated receptor y (PPARγ). Firstly, plasmids were constructed for transactivation assay of PPARγ response element (PPRE) triggered reporter gene expression, and for cell-based binding activity assay of the chimeric receptor, which was fused with PPARγ ligand binding domain (LBD) and yeast transcriptional activator Gal4. Secondly, by using PPARy competitive binding assay based on time resolved-fluorescence resonance energy transfer (TR-FRET), affinities of compounds and drugs to PPARγ were evaluated. In application of these above methods, the PPARγ activating potency and characteristics of different compounds were evaluated, and a novel benzeneselfonamide derivative, ZLJ01, was found to have comparable binding activity and affinity with the well-known PPARy agonist, but lack of PPRE mediated transactivation activity. In preliminary study on in-vitro hypoglycemic activity, ZLJ1 was found to promote insulin-stimulated glucose uptake by liver cells. Therefore, we believe that combining transactivation and binding activity as well as affinity evaluation, the system could be used to screen non-agonist PPARγ ligand as anovel PPARγ modulator

Objective: To investigate the expression of microRNA-128a (miR-128a) and its role in hepatocellular carcinoma (HCC)., Methods: Nineteen pairs of fresh surgical specimens of HCC and adjacent tissues were examined for miR-128a expression using qRT-PCR. A miR-128a mimics or inhibitor was transfected into HCC cells, and the cell viability was analyzed by MTT assay. RND3, one of the potential targets of miR-128a, was predicted by bioinformatics software and demonstrated by dual luciferase reporter assay. The expression of RND3 after transfection was detected using qRT-PCR and Western blotting, and the cell cycle-related proteins were determined with Western blotting., Results: The expression of miR-128a were significantly up-regulated in HCC tissues as compared to the adjacent tissues (P<0.05). In cultured HCC cells, miR-128a promoted the cell proliferation and resulted in down-regulated RND3 mRNA and protein expressions by targeting RND3' 3'UTR (P<0.05) and also in the down-regulation of cyclin B1, cyclin D1 and CDK4 protein expressions., Conclusion: miR-128a is up-regulated in HCC and promotes HCC cell proliferation by targeting RND3.

Objective: To explore the efficacy of photodynamic antimicrobial therapy in the treatment of pressure sore with pathogen infection., Methods: A total of 42 pressure sore patients with pathogen infection were divided randomly into experimental and control groups (n = 21 each). Fufanghuangbai liquid was used for external application with control group. In the experimental group, wound was treated with Fufanghuangbai liquid wet dressing and irradiated by semiconductor laser 30 min late. The distance from semiconductor laser probe to wound site was 10-15 cm, 20 min twice daily, continuous exposure to 7 days for 1 course. The results of bacterial culture and epidermal growth factor (EGF) expression of wound granulation tissue were observed before and after treatment. And the changes of healing rate of pressure sore were measured at post-treatment in each group., Results: The positive rates of bacterial culture, rates of change around wound inflammation, healing rate of days 7 and 14, the high expression of EGF on healing wound granulation tissue was 9.75%, (32.2% ± 5.8%), (89.1% ± 5.6%), (12.4% ± 2.9%), (34.7% ± 3.6%), 14/21 in the treatment group versus 51.2%, (17.8% ± 2.0%), (57.3% ± 2.6%), (5.1% ± 1.1%), (10.5% ± 2.4%), 2/21 in the control group respectively. The inter-group differences were statistically significant (P < 0.05)., Conclusion: Photodynamic antimicrobial therapy is an effective method for pressure sore with pathogen infection. Wound healing is promoted through an up-regulation of EGF.

Objective: To evaluate whether atorvastatin inhibits oxidized low-density lipoproteins (Ox-LDL)-stimulated foam cell formation from THP-1 macrophages by regulating the activation of peroxisome proliferator-activated receptor γ (PPARγ) and nuclear factor-κB (NF-κB). Methods THP-1 macrophages were pretreated with 10, 20, or 40 µmol/L atorvastatin for 2 h, and after washing with PBS twice, the cells were incubated with 60 µg/ml of Ox-LDL for 48 h. The quantity of intracellular lipid of the cells was detected with Oil red O staining and enzymatic fluorometric method. The expression of the scavenger receptors of CD36 and SRA were analyzed with Western blotting. We also examined the effect of atorvastatin on adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and the activation of PPARγ and p-iκB, and further assessed the capacity of the macrophages to bind to Dil-oxLDL., Results: Atorvastatin potently inhibited ox-LDL-induced macrophage-derived foam cell formation, down-regulated the expression of CD36 and SRA, and up-regulated the expression of ABCA1. Atorvastatin markedly suppressed the activation of PPARγ and p-iκB in ox-LDL-stimulated THP-1 macrophages (P<0.05) and significantly decreased the Dil-oxLDL-binding capacity of the macrophages (P<0.05)., Conclusion: Atorvastatin as an effective anti-atherosclerosis agent can suppress the activation of PPARγ and p-iκB to reduce lipid accumulation in macrophages.

Zinc-finger proteins have been widely studied due to their highly conserved structures and DNA-binding specificity of zinc-finger domains. However, researches on the zinc-finger proteins from microorganisms, especially those from prokaryotes, are still very limited. This review focuses on the latest progress on microbial zinc-finger proteins, especially those from prokaryotes and the application of artificial zinc-finger proteins in the breeding of robust strains. Artificial zinc-finger proteins with transcriptional activation or repression domain can regulate the global gene transcription of microbial cells to acquire improved phenotypes, such as stress tolerance to heat, ethanol, butanol, and osmotic pressure. Using the zinc-finger domain as DNA scaffold in the construction of enzymatic system can enhance the catalytic efficiency and subsequently the production of specific metabolites. Currently, zinc-finger domains used in the construction of artificial transcription factor are usually isolated from mammalian cells. In the near future, novel transcription factors can be designed for strain development based on the natural zinc-finger domains from different microbes, which may be used to regulate the global gene expression of microbial cells more efficiently.

Objective: To screen differentially expressed genes and gene pathways in L02 cell line stably expressing hepatitis C virus (HCV) Ib type nonstructural protein 4B (NS4B) mediated by lentiviral system, and to provide a basis for further research of molecular biological mechanism of NS4B gene in chronic hepatitis C and hepatocarcinogenesis., Methods: NS4B stably overexpressed L02 cell line and negative control stable L02 cell line, designated as L02-NS4B and L02-mkate2 respectively, were resurrected and amplified in vitro. The differentially expressed genes between L02-NS4B and L02-mkate2 were determined by gene expression microarray from Human Gene 1.0ST. The significant pathways of the differential genes were selected by the Fisher's exact test and χ2 test according to kyoto encyclopedia of genes and genomes (KEGG) database. The differential expression levels of 5 selected genes including protein kinase C delta binding protein (PRKCDBP), tumor protein p53 (TP53), v-akt murine thymoma viral oncogene homolog 1 (AKT1), baculoviral IAP repeat containing 3 (BIRC3) and B-cell lymphoma 2-like1 (BCL2L1) from cDNA microarray data were further verified by real-time quantitative polymerase chain reaction (real-time QPCR)., Results: Between L02-NS4B and L02-mkate2, the genes with flurescence intensity ratio >1.2 or <0.8 were considered as differentially expressed genes. A total of 2 682 differentially expressed genes in the known 28 869 human genes were detected in L02-NS4B, 1 446 genes were upregulated and 1 236 genes were downregulated. A total of 41 involved pathways of up-regulated differential genes were identified by KEGG database, mainly including apoptosis, extracellular matrix receptor interaction, cell cycle, pathways in cancer and Toll-like receptor signaling pathway; and 20 involved pathways of down-regulated differential genes were identified, mainly including pathways in cancer, Wnt signaling pathway and cell cycle pathway. Of the 5 upregulated genes selected from cDNA microarray data, 3 genes showed the same differential expression pattern by real-time QPCR as that shown in cDNA microarray data, namely AKT1, BIRC3 and BCL2L1. The confirmation rate of real-time QPCR was 60%., Conclusion: HCVNS4B can up-regulate or down-regulate the expression of many genes in L02 cells, thus affecting multiple signaling pathways relevant to cell apoptosis, cell cycle and carcinogenesis.

Objective: To explore the proliferation and invasive effects of inhibitors of kinase 4(INK4)(P15(ink4b) and P16(ink4a)/CDKN2) gene protein activation on RKO human colorectal cell in vivo and in vitro., Methods: RKO human colorectal cell line was exposed to the specific DNA methyltransferase inhibitor 5-Aza-CdR and INK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression was detected by Western blotting. Soft agar cloning experiment and Transwell chamber assay were used to detect the proliferative and invasive ability in vitro. Tumorigenicity in nude mice was analyzed in vivo., Results: INK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression of RKO human colorectal cells after exposure to 1×10(-7), 5×10(-7) and 1×10(-6) mol/L 5-Aza-CdR concentrations(A, B, C groups) were 1.13, 1.38, 1.92 folds and 1.11, 1.45, 2.14 folds compared to positive control group respectively. Soft agar cloning experiment showed the number of cell colony significantly decreased from 36.8±5.1(positive control group) to 32.4±7.2, 21.3±5.4 and 19.5±6.4 (3 experiment groups, all P<0.05) respectively. Transwell chamber assay showed that migrated cell number in positive control group(67.4±7.2) was significantly higher than those of 3 experimental groups(35.3±4.6, 29.5±7.3 and 25.3±6.2, respectively). The tumor volume of metastasis model in nude mice was inhibited in experimental groups, but not significantly lower compared to control group (P>0.05). There were significant differences of tumor weight and inhibition rate between control group and 3 experimental groups in nude mice respectively(all P<0.01)., Conclusion: INK4(P15(ink4b) and P16(ink4a)/CDKN2) protein activation can inhibit tumor proliferation, migration and suppress the tumor formation ability.

Objective: To construct a luciferase reporter vector containing the response element of transcription protein AP2α for screening the effect of bone morphogenetic proteins (BMPs) on the transcriptional activity of AP2α., Methods: Four tandem-linked response elements of AP2α were cloned to the pBGLuc luciferase reporter gene plasmid, which was digested with Bam HI and Mlu I to construct pBGLuc-AP2α-RE vector. The recombinant adenovirus Ad-AP2α and its dominant negative mutant Ad-dnAP2α were used to infect mouse mesenchymal stem cells C3H10; the changes in cellular AP2α mRNA and protein expressions were detected by real-time PCR and Western blotting, and electrophoretic mobility shift assay (EMSA) was carried out to assess the DNA-binding ability of AP2α. C3H10 cells were transfected with pBGLuc-AP2α-RE vector, and AP2α transcriptional activity was measured using luciferase reporter gene assay. In pBGLuc-AP2α-RE vector-transfected C3H10 cells infected with Ad-BMPs, luciferase reporter gene assay was performed to screen the effect of BMPs on AP2α transcriptional activity., Results: The results of PCR, enzyme digestion and sequencing all confirmed correct cloning of AP2α-RE into pBGLuc-AP2α-RE luciferase reporter vector, and Ad-AP2α infection significantly increased AP2α expression and its DNA binding ability. The dominant negative mutants expressed the corresponding mutants, and EMSA results showed that Ad-dnAP2α-δbHLH significantly lowered while Ad-dnAP2α-δTAD enhanced the DNA-binding ability of AP2α. AP2α over-expression promoted AP2α transcriptional activity, which was suppressed by the two dominant negative mutants. AP2α transcriptional activity increased in the cells infected with the recombinant adenovirus BMPs, especially in cells with BMP9 infection., Conclusions: The luciferase reporter vector containing the response element of AP2α we constructed allows detection of AP2α transcriptional activity. BMP9 can significantly enhance AP2α transcriptional activity.

The 11 kDa protein, a small nonstructural protein of parvovirus B19, may play important roles in viral replication cycle. To investigate the effect of 11 kDa protein on the NF-kappaB signaling pathway, we first prepared the poly-antiserum using GST-11 kDa fusion protein purified via prokaryotic expression system, and demonstrated that the 11 kDa protein mainly localized in cytoplasm when expressed in Hela cells. Meanwhile, luciferase activity assay and Western blotting assay showed that 11 kDa up-regulated the transcriptional activity of NF-kappaB and induced the degradation of IkappaB-alpha in Hela cells. Moreover, the 11 kDa protein activated the IL6 promoter, which is probably through the NF-kappaB pathway. Taken together, these results suggested that 11 kDa protein may contribute to activating inflammatory factors through participating in the cell signaling pathway.

Purpose: To evaluate the effect of estradiol on the expressions of estrogen receptor in mandibular condylar cartilage cell (MCC)., Methods: The MCC were harvested from neonatal rats. The experimental groups was cultured in medium with different concentration(10(-3),10(-6),10(-9),l0(-12) mmol/L) of estradiol(E2), while the control group was cultured in medium with ethanol. The ERα mRNA and ERβ mRNA in cells were detected with RT-PCR.The data was processed with SPSS10.0 software package., Resluts: Both ERα and ERβ expression were observed in MCC. Exogenous 17β-E2 raised the gene expression of ERβ and lowered the expression of ERα. Under the concentration of 10-9 mmol/L(physiological condition), the up-regulation of ERα mRNA was found most obviously., Conclusions: E2 can raise the expression of ERα and decrease the expression of ERβ, and the effect of up grading is the most obvious under physiological concentration.

iASPP can prompt the cell proliferation and inhibit the apoptosis of many cells. There are putative binding sites of transcription factor GATA-2 upstream of iASPP transcription start site. GATA-2 plays an important role in the proliferation and differentiation of hematopoietic stem cells (HSC) and progenitors. This study was aimed to explore the role of GATA-2 protein in iASPP gene transcription. Firstly, the expression of iASPP and GATA-2 protein in some leukemia cell lines was detected by Western blot. Second, The expressive vector of pCMV5-GATA2 and the luciferase reporter vectors containing possible binding sites of GATA-2 were constructed and co-transfected into HEK293 and CV-1 cells. Then the luciferase activity was assayed by luminometer. Also, ChIP assays were performed to further confirm the specific binding of GATA-2 to iASPP promoter. The results showed that GATA-2 was overexpressed in most cell lines with high level of iASPP. GATA-2 exhibited a significant effect on luciferase activity of reporter gene iASPP and in a dose-dependant manner. The relative luciferase activity was up-regulated to about two-fold of the empty vector control when the transfection dose of pCMV5-GATA2 plasmid was increased to 100 ng. While the effect was more significant in CV-1 cells and showed a 6.7-fold increase. The ChIP assay demonstrated the in vivo specific binding of GATA-2 to iASPP. The binding sites of GATA2 were located between nt -361 ∼ -334 in upstream of iASPP gene transcription start site. It is concluded that transcription factor GATA-2 can bind with the cis-regulatory region of the iASPP promoter and up-regulate iASPP expression.

Objective: To identify the transcriptional regulation of exosporium basal layer structural gene exsB in Bacillus thuringiensis., Methods: We analyzed exsB and its promoter sequence in Bacillus cereus group by sequence alignment. We performed beta-galactosidase assay of exsB-lacZ gene fusion to analyze transcriptional activation of exsB promoter; we used Electrophoretic mobility shift assays to detect binding of GerE and exsB promoter., Results: exsB was the high similarity in Bacillus cereus group strains. Beta-galactosidase assay showed that exsB promoter had the strong transcriptional activation on the late sporulation phase. Deletion of gerE or sigK gene decreased the activation of exsB promoter. Electrophoretic mobility shift assays showed that GerE could bind with exsB promoter., Conclusion: The exsB gene is regulated by SigmaK and GerE on the late sporulation phase.

Usually, secreted or transmembrane proteins complete their three-dimension folding within endoplasmic reticulum (ER). Under the conditions of nutrient depletion, cell differentiation, or other stress statuses, misfolded or unfolded proteins aggregate within ER, and consequently cause ER stress and Unfolded Protein Response (UPR). In response to ER stress, BiP (Binding immunoglobulin protein) dissociates with IRE1a (Inositol-requiring kinase 1) and binds to unfolded proteins as a molecular chaperone in helping maintain their correct structure. Co-related to BiP's dissociation, IRE1a oglimerizes and activated its endoribonuclease domain by transautophosphorylation. Activated IRE1a then, by cleaving mRNA of Xbp1 and activating its transcription activity, triggers UPR. In this paper, in order to determine effect of BiP on transcription activity of IRE1a, we cloned promoter region of IRE1a into reporter gene analysis vector and found that BiP could upregulate promoter activity of IRE1a. Then, we constructed another 6 truncated promoter reporter vectors of IRE1a and pinpoint the core promoter activity region. Furthermore, both our RT-PCR and Western blot results showed that BiP could upregulate mRNA transcription level and protein expression level of IRE1a. Base on these findings, we can propose that, in order to alleviate ER stress caused by the misfolded or malfolded proteins, BiP could upregulate expression of IRE1a by increase its promoter activity. This study may suggest a novel signal pathway on IRE1a regulation in ER stress.

Vpr, an auxiliary protein of HIV-1(Human immunodeficiency virus type 1), exerts important functions to promote viral replication and AIDS progression. In this study, we performed a yeast two-hybrid screening assay using human cDNA library to further investigate the molecular mechanism of various functions of Vpr RelB, a key protein in NF-kappaB signaling pathway, was identified as a Vpr interaction protein by co-immunoprecipitation. Further investigations indicated that RelB not only promoted the Vpr-mediated activation of NF-kappaB reporter gene, but also enhanced the transactivation of HIV LTR. Moreover, the results showed that RelB promoted Vpr-induced cell cycle G2/M arrest. Collectively, these results indicated that RelB might interact with Vpr and regulate its transcriptional activation and cell cycle arrest.

Objective: To detect and compare the transcriptional activities of prostate-specific membrane antigen (PSMA) promoter and enhancer and survivin promoter in different human prostate cancer cell lines, and to search for some evidence for the targeting gene therapy of human prostate cancer., Methods: The fragments of the PSMA promoter and enhancer and survivin promoter were amplified by PCR and inserted into pGL3-Basic. The recombinant plasmids were transiently transfected into human prostate cancer cell lines and normal Chang liver cells, and, their transcriptional activities in various cells were determined by measuring the expression of luciferase., Results: The survivin promoter exhibited a higher transcriptional activity than PSMA promoter and enhancer in tumor cell lines, and the S2pro promoter showed the highest activity, reaching one third of that of the CMV promoter., Conclusion: The survivin promoter is highly activated in prostate cancer cell lines and may serve as a new tool for the transcriptional targeting gene therapy of prostate cancer.

To study the coumarins of Anemone raddeana Regel, the compounds were separated by silica gel column chromatography and HPLC. Their structures were identified by their physicochemical property and spectral analysis. Two new compounds were isolated and identified as 4, 7-dimethoxyl-5-methyl-6-hydroxy coumarin (1) and 4, 7-dimethoxyl-5-formyl-6-hydroxycoumarin (2). The bioassays indicated that compounds 1 and 2 could significantly inhibit the proliferation of cancer cell, and showed the agonist effect on the transactivity of retinoic acid receptor-alpha (RARalpha). In addition, the two compounds had inhibitory effect against human leukocyte elastase (HLE).