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Mirabilis himalaica (Edgew.) Heimerl is among the most important genuine medicinal plants in Tibet. However, the biosynthesis mechanisms of the active compounds in this species are unclear, severely limiting its application. To clarify the molecular biosynthesis mechanism of the key representative active compounds, specifically rotenoid, which is of special medicinal value for M. himalaica, RNA sequencing and TOF-MS technologies were used to construct transcriptomic and metabolomic libraries from the roots, stems, and leaves of M. himalaica plants collected from their natural habitat. As a result, each of the transcriptomic libraries from the different tissues was sequenced, generating more than 10 Gb of clean data ultimately assembled into 147,142 unigenes. In the three tissues, metabolomic analysis identified 522 candidate compounds, of which 170 metabolites involved in 114 metabolic pathways were mapped to the KEGG. Of these genes, 61 encoding enzymes were identified to function at key steps of the pathways related to rotenoid biosynthesis, where 14 intermediate metabolites were also located. An integrated analysis of metabolic and transcriptomic data revealed that most of the intermediate metabolites and enzymes related to rotenoid biosynthesis were synthesized in the roots, stems and leaves of M. himalaica, which suggested that the use of non-medicinal tissues to extract compounds was feasible. In addition, the CHS and CHI genes were found to play important roles in rotenoid biosynthesis, especially, since CHS might be an important rate-limiting enzyme. This study provides a hypothetical basis for the screening of new active metabolites and the metabolic engineering of rotenoid in M. himalaica.

As a major tumor suppressor gene, the role of PinX1 in breast cancer and its molecular mechanism remain unclear. In this study, overexpression of PinX1 was generated in 3 breast cancer cell lines, and knockdown of PinX1 was performed in a nontumorigenic breast cell line. The regulation of PinX1 on cell proliferation and cell cycle was observed. A microarray-based lncRNA and mRNA expression profile screening was also performed. We found a lower growth rate, G0/G1 phase arrest, and S phase inhibition in the PinX1 overexpressed breast cancer cells, while a higher growth rate, decreased G0/G1 phase, and increased S phase rate in the PinX1 knocked-down nontumorigenic breast cell. A total of 977 mRNAs and 631 lncRNAs were identified as differentially expressed transcripts between PinX1 overexpressed and control MCF-7 cells. Further analysis identified the involvement of these mRNAs in 52 cancer related pathways and various other biological processes. 11 enhancer-like lncRNAs and 25 lincRNAs with their adjacent mRNA pairs were identified as coregulated transcripts. Our results confirmed the role of PinX1 as a major tumor suppressor gene in breast cancer cell lines and provided information for further research on the molecular mechanisms of PinX1 in tumorigenesis.

Objective: To investigate the genes associated with temporal epilepsy and explore the molecular mechanism of epilepsy., Methods: The microarray data of temporal epilepsy were downloaded from the Gene Expression Omnibus (GEO) database and analyzed by bioinformatics methods using String, KEGG and Panther databases., Results: Of all the 71 differentially expressed genes, 51 were found to encode proteins with interactions; the main biological pathways involved included neuroactive ligand-receptor interactions, MAPK signaling pathway, and calcium signaling pathway etc., Conclusion: The pathogenesis of epilepsy involves multiple genes, and investigations of these genes may provide valuable insights into the mechanism of epilepsy.

Objective: To predict the function of KIAA0101 gene over-expressed in human non-small cell lung cancer by bioinformatics methods., Methods: The gene expression profiles of the lung cancer tissues and the adjacent normal tissues were compared by dChip software analysis, and the differential genes coexpressed with KIAA0101 gene were identified. The biological functions of these genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), Gene Ontology (GO) and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), and the common transcription factors of these genes were predicted using Gene Annotation Tool to Help Explain Relationships (GATHER)., Results: Nine genes were found to have at least two-fold overexpressions in the lung cancer tissues in comparison with the expression level in the adjacent normal tissues, and showed similar pattern of expression variations in the lung cancer tissue. Most of these genes had the E2F1 binding sites in the promoter region., Conclusion: KIAA0101 gene may participate in the cell cycle regulation of the non-small cell lung cancer, and the expression levels of the 9 genes identified may be regulated by the transcription factor E2F1.

Cell cycle progression is regulated by both transcriptional and post-transcriptional mechanisms. MicroRNAs (miRNAs) emerge as a new class of small non-coding RNA regulators of cell cycle as recent evidence suggests. It is hypothesized that expression of specific miRNAs oscillates orderly along with cell cycle progression. However, the oscillated expression patterns of many candidate miRNAs have yet to be determined. Here, we describe miRNA expression profiling in double-thymidine synchronized HeLa cells as cell cycle progresses. Twenty-five differentially expressed miRNAs were classified into five groups based on their cell cycle-dependent expression patterns. The cyclic expression of six miRNAs (miR-221, let-7a, miR-21, miR-34a, miR-24, miR-376b) was validated by real-time quantitative RT-PCR (qRT-PCR). These results suggest that specific miRNAs, along with other key factors are required for maintaining and regulating proper cell cycle progression. The study deepens our understanding on cell cycle regulation. [BMB reports 2009; 42(9): 593-598].

Objective: To better understand the molecular pathogenesis of cervical cancer, and provide novel means for clinical diagnosis and treatment of this malignancy., Methods: The gene chip data of cervical cancer were obtained from GEO database and statistically analyzed using BRB-ArrayTools to identify the genes related to cervical cancer with bioinformatics analysis using Panther software., Results: Thirty-seven differentially expressed genes were identified in cervical, cancer samples, including 23 up-regulated and 14 down-regulated genes. These genes were associated with the cell skeletons transporters and such processes as cell signal transduction, transcriptional control, cell adhesion, and cell apoptosis., Conclusion: Bioinformatics analysis can help with effective analysis of the gene chip data. The pathogenesis of cervical cancer involves abnormal expression of multiple genes, and these data may benefit further investigations of the early diagnosis and treatment of the malignancy.

Objective: To analyze alterations in the gene expression profiles of Velcade-treated K562 cells using bioinformatics methods., Methods: The total RNAs of Velcade-treated and control K562 cells were amplified and labeled with fluorescent dyes. The labeled RNAs were hybridized to Agilent Human 1A Microarray, and the raw expression data were processed with Agilent Feature Extraction Software. GeneSifter and GATHER were used for data analysis of the differentially expressed genes to perform gene ontology classification, KEGG pathway analysis, functional protein association network construction and literature mining., Results: Totally 228 differentially expressed genes were identified in the Velcade-treated K562 cells. including 84 up-regulated and 144 down-regulated genes. Chymase 1 gene had the greatest down-regulation by 10.80 folds (log ratio), and interferon alpha-21 gene was also down-regulated by 2.31 folds. Gene ontology classification suggested enhanced aging and leukocyte activity. KEGG pathway analysis showed significant impact of Velcade on JAK-STAT signaling pathway, cytotoxicity mediated by natural killer cells, and antigen processing and presentation pathways. Protein-protein interaction analysis revealed that ubiquitin-dependent protein catabolism, antigen presentation and immune response, as well as JAK-STAT signaling pathway were the major elements of the protein network. Literature mining showed that the differentially expressed genes were strongly associated with terms such as leukemia, apoptosis, cell cycle, proteasome, inhibitor, aging and IkappaB, etc., Conclusions: Velcade may inhibit the cell survival pathways such as NF-kappaB and JAK-STAT signaling pathways to enhance the cytotoxicity and inducing tumor cell apoptosis. Velcade might also be involved in antigen processing and presentation, immune response and inflammation. Chymase 1 gene is probably the key target of Velcade.

Background & Objective: Epstein-Barr virus (EBV) infection plays a key role in the pathogenesis of nasopharyngeal carcinoma (NPC). This study was to explore the effects of the recurrent infection by EBV reactivation on the genomic expression profile of NPC., Methods: The microarray expression data from different cell lines subjected to primary infection of EBV+ vs. EBV- targets in NPC and recurrent EBV reactivation were collected from public data depository. Cross comparison, t-test analysis as well as filtering by flag, expression level and fold change were used to analyze the data and identify differential genes. Moreover, a set of web-based applications, such as DAVID (database for annotation, visualization and integrated discovery), pSTIING (protein, signaling, transcriptional interactions and inflammation networks gateway), GATHER (gene annotation tool to help explain relationships) and TELiS (transcription element listening system), were used to analyze and predict the probable expression profile of the differential genes., Results: As compared with the genes expressed during primary infection of EBV, 25 genes, including DUSP1, TOP1, HOXA9, DEK, PABPC1 and IMPDH2, were differentially expressed during EBV reactivation. Many of them were oncogenic. The differential genes together with related transcriptional factors were interacted mainly through 2 mechanisms: one mainly included TOP1, DUSP1, DUSP6, and RPS28; the other one was a circuit of PITX1, CD9, HOXA9 and IMPDH2., Conclusion: The differential genes might participate in EBV reactivation by changing their expression level through two mechanisms, which contributes to the final development of NPC.

Objective: To investigate the effect of the total saponin of Panax ginseng (TSPG) on gene expression profile of K562 cells using microarray technique., Methods: The total RNA were extracted and purified from K562 cells treated by 200 microg/ml TSPG for 3 days, and untreated K562 cells cultured in parallel served as the control. cRNAs were synthesized and labeled with Cy3 and Cy5 respectively. The labeled cRNA fragments were hybridized with Agilent human 1B 60 mer oligonucleotide microarray, which was then scanned to reveal the changes of gene expression profile in relation to TSPG treatment., Results: Totally 362 differentially expressed genes were identified in TSPG-treated K562 cells, including 20 up-regulated ones (consisting of metabolism-associated genes, signal transduction-associated genes and cell receptor-associated genes etc) and 342 down-regulated ones (consisting of immunity and defense-associated genes, DNA-binding and transcription genes, metabolism-associated genes and cell cycle-associated genes etc). Changes in expressions of FOSL1, E2F2, CCNE2 and ODZ1 were confirmed by semi-quantitative RT-PCR., Conclusions: TSPG may induce changes in the gene expression profile in k562 cells possibly relevant to the anti-tumor mechanism of TSPG.

Objective: To investigate the effect of microRNA on the gene expression profile of human leukemia K562 cells using microarray technique., Methods: miR-181a RNA duplexes were designed and synthesized according to the mature sequence of miR-181a. Forty-eight hours after transfection of in vitro cultured K562 cells using Oligofectamine, gene expression profiles of the cells were studied and analyzed using Agilent Human 1A Oligo microarray., Results: Totalling 228 differentially expressed genes were identified from the 20,173 screened genes, including 59 up-regulated ones (consisting of metabolism-associated genes, tumor suppressor genes, signal transduction-associated genes, immunity and defense-associated genes etc), and 169 down-regulated ones (consisting of oncogenes, DNA-binding and transcription genes, metabolism-associated genes, signal transduction-associated genes, cell cycle and development-associated genes etc.) in the transfected K562 cells as compared with the control K562 cells. Changes in expressions of CTCF, ZAP70, SEMA4C and RALA were confirmed by semi-quantitative reverse transcription-polymerase chain reaction., Conclusions: miR-181a transfection for 48 h induces gene expression profile changes in K562 cells, indicating the functionality of the miR-181a. These differentially expressed genes are related to the functions of the microRNA, and may also be the basis of the regulation model of posttranscriptional gene silencing. These findings provide an evidence for further study of the machineries and functions of the microRNA in mammalian cells.

Objective: To study the effect of the S-bioallethrin on human lymphocytes by microarray technique., Methods: The changes of normal human lymphocytes treated with S-bioallethrin were examined with light microscope, flow cytometry, electron microscope, DNA ladder and microarray techniques., Results: Morphological study showed that the lymphocytes underwent apoptosis after S-bioallethrin exposure, which as further confirmed by the expression changes of 346 genes., Conclusion: S-bioallethrin can induce apoptosis of normal human lymphocytes and changes in their gene expression profiles.

Objective: To screen differentially expressed genes in cytochalasin B (CB)-induced denucleated K562 cells by restriction display (RD) technique., Methods: The total RNA was isolated and purified from K562 cells before and after CB (10 mug/ml) treatment. The mRNA from both treated and untreated K562 cells were reversely transcribed into cDNA, and the differentially expressed genes were separated using RD technique combined with polyacrylamide gel electrophoresis and sliver staining, followed by cloning, sequencing and homology analysis against GenBank database of these genes., Results: Seven differentially expressed genes were identified in CB-treated cells including aquaporin 1 (AQP1) gene, which was verified to be up-regulated after CB treatment by RT-PCR., Conclusion: AQP1 gene might be in close association with the regulation of denucleation processes and CB-induced proliferation inhibition of K562 cells.

Objective: Subtractive hybridization technology is a common method to screen and clone differentially expressed genes. This study was to construct subtracted cDNA library of leukemia cell line K562, and screen for differentially expressed genes., Methods: cDNA fragments of K562 cells (tester), prepared by restriction display (RD), were subtracted with the Sau3A I-digested cDNA fragments of normal lymphocytes (driver). The subtracted cDNA fragments were re-amplified, and cloned into pMD18-T vectors. Positive clones were selected by blue-white screening. The inserts in plasmid were amplified by polymerase chain reaction (PCR), and some of which were sequenced., Results: The subtracted library contained 360 positive clones with cDNA fragments distributed mainly from 200 to 800 bp. The 50 randomly sequenced clones were derived from 42 known genes., Conclusion: Specific subtracted cDNA library of K562 cells was successfully constructed with reliable quality, and may be used to further screen and clone differentially expressed genes of K562 cells.

Objective: To collect the normal spermatozoal gene expression sequence tags with the restriction display technique for constructing a microarray to understand spermatozoal gene expression profiles., Methods: The total RNA extracted from normal human spermatozoa were reversely transcribed into cDNAs, which were digested by Sau3AI and linked to universal adapters (adapter 1) at both ends. According to the sequence of the adapter, a pair of primers (universal primers 1) was designed, followed by PCR with primers 1 and the PCR products were transferred into E.coli. The positive clones were collected after identification to serve as the probes for constructing the gene expression microarray of spermatozoa by printing those probes on the slides. The accomplished microarrays were examined by Cy3-labeled normal spermatozoal samples., Results: Altogether 1 859 probes were collected, from which 368 were picked out randomly for constructing the microarray., Conclusions: Human spermatozoa contain a rich repertoire of RNAs, and the probes we prepared possess good incredibility and speciality.

Objective: To investigate differential gene expression in apoptotic cells induced by As(2)O(3), and identify novel apoptosis-related genes., Method: Apoptosis of K562 cells cultured in RPMI 1640 medium supplemented with 10 % calf serum was induced by As(2)O(3). Total RNA of the apoptotic and normal cells were then extracted, purified and subject to reverse transcription into first-strand cDNA, labeled with Cy3/Cy5. Placenta DNA microarrays containing 348 DNA fragments were used to analyze the changes in gene expressions in the cells treated with As(2)O(3)., Result: Eleven differentially expressed genes were identified in the apoptotic cells in comparison with the normal cells, 3 of which were associated with apoptosis, while the others were related to cell growth and proliferation., Conclusion: The placenta DNA microarrays we constructed may well apply to the analysis of the differentially expressed genes.

Objective: To collect the gene fragments of the gene chips for investigating the expression profile of K562 cells., Methods: The mRNA was extracted from cultured K562 cells to obtain cDNA by reverse transcription-PCR. The gene fragment products of different groups were acquired and isolated by reverse display PCR (RD-PCR)., Result: The probes for the gene chip were successfully prepared., Conclusion: RD-PCR adapted in this study suits the needs for collecting the probes for the gene chips.

Objective: To prepare human placenta genechip for analyzing differential gene expressions., Methods: The target gene amplified from Hybrid Hunter cDNA library was dotted onto the slides by the arrayer (PixSys 5500). Total RNA from K562 cells treated with or without arsenic trioxide was extracted, the mRNA purified and cDNA synthesized. Fluorescent labeling of the samples was performed by restriction display PCR, followed by hybridization with the microarray that was subsequently washed and scanned, with the derived signals analyzed by computer., Results: A reliable and specific method for preparing and assessing gene expression profile microarray was established, with which a total of 45 differentially expressed gene fragments were isolated., Conclusion: The genechips can be helpful in the analysis of differential gene expressions.

Objective: To investigate the gene expression profile in the mature spermatocytes and the molecular mechanism of the male infertility syndromes., Methods: Restriction display PCR (RD-PCR), a new method of differential display technique, was used to collect plenty of gene fragments expressed in either normal or abnormal spermatocytes, the gene expression profile of which were demonstrated by these fragments(gene expression sequence tags, ESTs), in the form of cDNA fragments length polymorphism. These ESTs will be used to make DNA microarray as probes to further explore the gene expression profile in spermatocytes and to diagnose the male infertility., Results: Lots of ESTs were expressed in either normal or abnormal spermatocytes and some differentially expressed ESTs have been collected and purified., Conclusion: Either for known or unknown genes, RD-PCR is very effective in rapid acquisition of large amount of ESTs with proper and similar length fit for making microarray as the probes.