[show abstract][hide abstract] ABSTRACT: BACKGROUND: The multidrug resistance (MDR) 1 gene encodes a 170-kDa membrane transporter called P-glycoprotein, which plays an important role in protecting cells against lipophilic xenobiotics by the way of an ATP-dependent cellular efflux mechanism. Three polymorphisms of MDR1, 3435C > T located in exon 26, 1236C > T in exon 12 and 2677G > T/A in exon 21 were the most extensively studied and were identified functionally important and ethnically diverse mapping to the gene region. Considering the potential influence of altering MDR1 activity, it is plausible that MDR1 polymorphisms might play a role in the development of cancer. Although the effects of MDR1 polymorphisms on susceptibility to human cancer have been investigated in many studies, the results still remain conflicting. METHODS: To resolve these conflicts, we performed a quantitative synthesis of the association between these three polymorphisms and cancer risk, including 52 studies (15789 cases and 20274 controls) for 3435C > T polymorphism, 10 studies (2101 cases and 2842 controls) for 1236C > T polymorphism and 18 studies (3585 cases and 4351 controls) for 2677G > T/A polymorphism. RESULTS: The stratified analyses for 3435C > T polymorphism, individuals with T-allele in 3435C > T had significantly higher ALL risks (TT versus CC: OR =1.286, 95% CI =1.123-1.474); significantly elevated risks were observed among Caucasian populations (TT versus CC: OR =1.276, 95% CI =1.112-1.464). When restricting the analysis to the source of controls, we found that HB (hospital-based) genetic models had higher risks (TT versus CC: OR =1.307, 95% CI =1.046-1.632), as well as in PB (population-based) genetic models (TT versus CC: OR =1.294, 95% CI =1.079-1.55).The T/A-allele frequency of 2677G > T/A polymorphism was associated with higher risk of cancer (TT + TA + AA vs. GG: OR =1.348, 95% CI =1.031-1.762), significantly elevated risks were observed among Asian populations (TT + TA + AA vs. GG: OR =1.642, 95% CI =1.340-2.012), and elevated risks could be associated with PB models (TT + TA + AA vs. GG: OR =1.641, 95% CI =1.018-2.646). CONCLUSIONS: Our meta-analysis suggested that 3435C > T polymorphism and 2677G > T/A polymorphism were associated with cancer risk when all studies were pooled together, while 1236C > T polymorphism not.
Cancer Cell International 05/2013; 13(1):46. · 2.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: To study the data of gene expression microarray by protein interaction network analysis, establish an interaction network of differentially expressed genes in invasive bladder cancer and verify the central nodes of the network.
A total of 152 differentially expressed genes in invasive bladder cancer detected by gene expression microarray were inputted into STRING database online for analysis and establishment of the interaction network. The interaction data were imported into Cytoscape 2.6.2 software for screening the central nodes of the network. KEGG database was exploited for pathway analysis and functional study of the central node genes. Real-time RT-PCR was used for verification, and the genes with maximal differential expressions were screened for exploring the molecular mechanism of carcinogenesis of invasive bladder cancer.
The protein products of 103 differentially expressed genes in bladder cancer had interactions, forming a complicated interaction network. Twenty-six nodes involved in several signal pathways were confirmed by Cytoscape as the central nodes of the network, among which UBE2C, VEGF, TGFBR2, and CAV1 nodes were verified by real-time RT-PCR as the genes with maximal differential expressions between the bladder cancer and normal tissues, and the 2(-delta delta Ct) of these genes were 9.45, 4.17, 0.13 and 0.18 (GAPHD as the internal control), respectively.
The interaction network of the differentially expressed genes, especially the central nodes of this network, can provide clues to the carcinogenesis, early diagnosis and molecular targeted therapy of invasive bladder cancer.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 08/2010; 30(8):1771-4.
[show abstract][hide abstract] ABSTRACT: To explore the correlation of the mutation of MTCYB and MTATP6 genes in sperm mitochondria with asthenospermia.
We extracted mtDNA from 80 semen samples of asthenospermia and 20 of normal sperm motility, amplified the MTCYB and MTATP6 genes by PCR, and analyzed their mutation by sequencing and BLAST matching.
The deletion of both MTCYB and MTATP6 were detected in 20 of the 80 asthenospermia samples, MTCYB deletion in 16 and MTATP6 deletion in 4, accounting for 20% and 5% respectively. Sequencing and BLAST matching revealed G8887A mutation in the MTATP6 gene in the asthenospermia samples, with a mutation rate of 20%, while no regular mutation was noted in MTCYB. Neither significant deletion nor mutation was observed in any of the 20 samples of normal sperm motility.
Both the deletion and mutation of MTCYB and MTATP6 genes in sperm mitochondria might affect sperm motility in adults.
Zhonghua nan ke xue = National journal of andrology 05/2008; 14(4):321-3.
[show abstract][hide abstract] ABSTRACT: To perform the detection of spermatozoal gene expression in order to accelerate the study of spermatozoal molecular biology.
To collect the healthy adults sperm and lymphocytes respectively, and then to extract the total RNAs from them by RNeasy mini kit (QIAGEN) or Trizol reagent. Corresponding cDNAs were produced, digested, ligated, finally labeled with Cy3 (sperm) and CyS (lymphocyte) in the course of RD amplifying reactions. Hybridization with self-made microarrays contained 560 probes was carried out after the labeled cDNAs pured by PCR Product Purification Kit.
Among the 560 probes, 72 genes were up-regulated, 321 genes were down-regulated, the others had no different expression. Furthermore, genes associated with replication, transcription, translation and regulative functions were non-different expression or down-regulated, and those belonged to the spermatogenesis associated, sperm associated antigen were up-regulated, but those involved in the glycolysis were up-regulated, in the oxidative phosphorylation were down-regulated.
It had successfully confirmed that there were a plenty of genes expressed in sperm, furthermore the genes expressed were accorded to spermatozoal functions and characteristics.
Zhonghua nan ke xue = National journal of andrology 06/2006; 12(5):401-4, 407.
[show abstract][hide abstract] ABSTRACT: To investigate the differentially expressed genes in keloids in comparison with normal skin using cDNA microarray.
The cDNA microarray consisting of 8064 clones of human genes was employed to detect and screen the differentially expressed genes in keloid and normal skin tissues. Semi-quantitative RT-PCR was applied to verify the results of gene microarray.
Totally 277 differentially expressed genes were identified in keloids in comparison with normal skin tissue, including 163 up-regulated genes and 114 down-regulated ones according to the designed data filter criteria. These differentially expressed genes belonged to 26 different functional gene families involving different biological processes. RT-PCR yielded results were consistent with those of microarray study.
A variety of genes are involved in the formation of keloids. The 277 differentially expressed genes comprise the differential gene expression profile of keloids and describe the general changes in the gene expressions in keloid at transcriptional level. Further analysis of the identified genes might help reveal the molecular mechanism of abnormal scarring.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 04/2006; 26(3):308-12.
[show abstract][hide abstract] ABSTRACT: To screen differentially expressed genes in cytochalasin B (CB)-induced denucleated K562 cells by restriction display (RD) technique.
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.
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.
AQP1 gene might be in close association with the regulation of denucleation processes and CB-induced proliferation inhibition of K562 cells.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 03/2006; 26(2):162-5.
[show abstract][hide abstract] ABSTRACT: To study the inhibitory effect of small interfering RNA (siRNA) targeting c-myc gene in K562 cells.
siRNAs targeting the site 1357 of c-myc mRNA was designed and synthesized. In vitro cultured K562 cells were transfected with lipofectamine 2000 and the inhibitory effect was detected by reverse transcriptase (RT)-PCR, cell count, MTT assay and fluorescence-activated cell sorting.
Compared with the negative and blank control group, the transfection group showed marked decrease in the c-myc expression and the K562 cells exhibited increased apoptosis rate.
RNA interference can effectively inhibit c-myc expression and induce apoptosis in K562 cells.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 07/2005; 25(6):647-50.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
Ai zheng = Aizheng = Chinese journal of cancer 06/2005; 24(5):631-3.
[show abstract][hide abstract] ABSTRACT: To clone the obesity gene of Chinese and express human leptin in E.coli.
The obesity gene was amplified from the total RNA isolated from cultured human adipocytes of Chinese by reverse transcriptional PCR, inserted into TA-vector and cloned into the expression plasmid pBV220 after sequence identification.
DNA sequencing confirmed that the isolated obesity gene was identical to the previously reported sequence. The recombinant plasmid pBV220-OB was constructed and leptin successfully expressed in E.coli.
Successful cloning and expression of human obesity gene in E.coli may facilitate further research of the mechanism of fat metabolism and adipocyte differentiation.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 05/2005; 25(4):395-8.
[show abstract][hide abstract] ABSTRACT: To study the inhibitory effect of small interference RNA(siRNA) of cyclin E gene on the growth of K562 cells.
siRNA targeting the 940 bp site of the cyclin E mRNA were designed and generated by PCR amplification. The PCR products containing U6 promoter and the siRNA were then transfected into K562 cells via Lipofectamine2000. The cells transfected with non-functional siRNA served as the negative control group and those only treated with serum-free RPMI1640 as the blank control group. Cell counting, reverse transcriptase (RT)-PCR and flow cytometry were employed to evaluate the effect of RNA interference.
Compared with the negative and blank control groups, the viable cell count in the interference group was decreased by approximately 80%, the ratio of G(1)-phase cells increased by nearly 30%, and growth arrest was observed. Cyclin E mRNA expression in the cells of the interference group was significantly lowered by about 70%; as compared with that of the negative and blank control groups, whereas the latter two groups had similar expression levels.
RNA interference induces obvious inhibition of cyclin E gene expression, which consequently affects the proliferation of K562 cells.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 05/2005; 25(4):361-5.
[show abstract][hide abstract] ABSTRACT: To study the method for using a 70-mer oligo microarray as the probe to isolate target genes from the cDNA restriction fragments.
Samples of Saccharomyces cerevisiae mRNA was extracted after heat shock culture and reversely transcribed into the double-stranded cDNAs, which were prepared into restriction cDNA fragments using restriction display (RD) method. The microarray was printed using a single 70-mer specific oligo designed to according to the SSA1 gene of yeast. The cDNA restriction fragments were labeled by PCR method with the Cy5 universal primer before hybridization with the microarray. The microarray was stripped after washing and scanning, and the strip solution was collected for another round of PCR amplification using the universal primer without fluorescence. The PCR product was then cloned into PUC18 T vector and transformed into to E.coli JM109 cells for amplification, and the plasmids were extracted and sequenced for identification.
BLAST results showed that the target gene was cloned successfully.
The target gene can be isolated directly using the 70-mer oligo microarray as the probe from the cDNA fragments prepared by RD method, without the necessity of building a cDNA library. This method can also be used in further research to acquire the differentially expressed genes after the oligo microarray hybridization.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 08/2004; 24(7):738-41.
[show abstract][hide abstract] ABSTRACT: To investigate the expression of cyclin-dependent kinase-2 (CDK-2) gene in SH-SY5Y cells.
The expression of CDK-2 gene was examined with reverse transcriptional (RT)-PCR, and the PCR products underwent electrophoresis on non-denaturing poly-acrylamide gel (PAG) followed by silver staining. The separated and purified DNAs were ligated into pMD18-T vector, and the positive clones identified by sequence analysis.
Two DNA bands were displayed on PAG, and the one with smaller molecular weight was less intensively stained. The sequences of the two clones indicated that both were products of CDK-2 gene.
Two kinds of CDK-2 gene products are co-expressed in the SH-SY5Y cells, one of which lacks the fifth exon and is expressed at a low level.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 04/2004; 24(3):349-51.
[show abstract][hide abstract] ABSTRACT: To amplify and clone the N gene of severe acute respiratory syndrome-associated coronavirus.
Using primer Premier 5.0 software, two pairs of nested PCR primers were designed to amplify the N gene. After purification, the amplified products were cloned into pMD18-T vectors, and the positive clones with the inserted fragments were identified by sequence analysis.
The amplified products was about 1 375 bp in length, and sequence analysis demonstrated that the N gene fragments had been successfully inserted into pMD18-T vectors.
The successful amplification and cloning of N gene facilitates further investigation of the expression of the N protein and study of its structure and functions.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 02/2004; 24(1):39-41.
[show abstract][hide abstract] ABSTRACT: To explore an effective means for the detection of Severe Acute Respiratory Syndrome (SARS)- associated coronavirus.
The RNAs of the virus contained in the sputum samples from established SARS patients were extracted and reversely transcripted, followed by nested PCR using the reversely transcripted cDNA as the template. The PCR products were cloned then into the pMD18-T vectors, followed by sequence analysis.
Specific fragments were amplified from the sputum samples of SARS patients, which were confirmed by DNA cloning and sequencing to belong to SARS-associated coronavirus. The Result of Blast shows only the difference in one nucleic acid from the TOR2 strain of SARS-associated coronavirus.
Sequence analysis has confirmed the existence of SARS-associated coronavirus in the sputum samples of SARS patients, and nested RT-PCR is a quick, easy, and convenient way for the detection of the virus.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 06/2003; 23(5):421-3.
[show abstract][hide abstract] ABSTRACT: To study the signal-to-noise ratio (SNR) of two restricted fluorescence labeling methods for examining gene expression profile by microarray hybridization.
Samples of Saccharomyces cerevisiae mRNA was labeled by traditional reverse transcription method and 2 restriction fluorescent labeling methods using respectively Cy-universal primer and extension incorporated Cy-dNTP. The labeled samples were examined by the microarray, followed by washing and scanning under the same conditions.
The two restriction labeling methods showed superior results with lowered background and enhanced SNR and sensitivity, and Cy-universal primer labeling presented the best results.
SNR can be enhanced by the restriction labeling methods, which improve the applicability of microarray technology.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 03/2003; 23(2):124-6.
[show abstract][hide abstract] ABSTRACT: The advanced technique of DNA microarray makes it possible to monitor the expression of thousands of genes simultaneously in one hybridization experiment. This technique accelerates demonstration of anti-tumor drug mechanisms and discovery of new drug targets. This study was designed to investigate the differential gene expression of K562 cells after cytochalasin B treatment using cDNA microarray.
Restriction display polymerase chain reaction (RD-PCR) products of 277 human genes were spotted on a glass slide in microarray. K562 cells grew in RPMI 1640 medium with 10 microg/ml cytochalasin B. After 24 hours, the total RNA was isolated from K562 cells, and mRNA was purified. Both mRNA from the treated K562 cells and the controlled K562 cells were reversely transcribed into cDNA and labeled with two different fluorescence dyes: Cy5 or Cy3, using a method of restriction digestion and PCR labeling (RD-PCR). The probes were hybridized to the cDNA microarrays. After high-stringent washing,the cDNA microarray was scanned for the fluorescent signals and showed difference between the two cells.
Among the 277 target genes, 18 down-regulated genes were identified after cytochalasin B treatment.
There is a consistent tendency toward lower-expressed genes in partial K562 cells after cytochalasin B treatment. Most down-regulated genes were correlated with cell proliferation, signal transduction, and transcription factor.
Ai zheng = Aizheng = Chinese journal of cancer 03/2003; 22(3):239-43.
[show abstract][hide abstract] ABSTRACT: To observe the effect of cytochalasin B on the denucleation of K562 cells.
K562 cells were grown in RPMI 1640 medium supplemented with 10% calf serum and 4 microL cytochalasin B (CB). Denucleation was induced in the cultured cells by CB, and the cells were examined by phase contrast microscopy and Giemsa staining respectively.
The denucleation of K562 cells induced by CB was clearly observed, and the cell proliferation was obviously inhibited.
The denucleation efficiency of K562 cells is positively correlated with CB concentrations and the duration of CB treatment. CB at low doses may inhibit the cell proliferation and at high doses causes cell death.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 01/2003; 22(12):1087-9.
[show abstract][hide abstract] ABSTRACT: To construct and identify the cDNA library of E.coli mRNA with poly(A) tracts.
The cDNA library of E.coli was constructed by restriction display-PCR (RD-PCR) technique, followed by sequencing and bioinformatics analyses.
cDNA library of E.coli mRNA with poly(A) tracts was successfully constructed, and 66 gene fragments were sequenced.
The constructed cDNA library of E.coli mRNA with poly(A) tracts contains a low rate of repetition and is of high quality.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 12/2002; 22(11):1005-7.
[show abstract][hide abstract] ABSTRACT: To investigate differential gene expression in apoptotic cells induced by As(2)O(3), and identify novel apoptosis-related genes.
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).
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.
The placenta DNA microarrays we constructed may well apply to the analysis of the differentially expressed genes.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 10/2002; 22(9):772-5.
[show abstract][hide abstract] ABSTRACT: To isolate gene fragments from SH-SY5Y cells by way of restriction display polymerase chain reaction (RD-PCR).
Total mRNA was extracted from SH-SY5Y cells followed by synthesis of the single-strand cDNA with Oligo (dT18) as the anchored primer, and the second strand was synthesized by nick translation. The double strands were cleft with restriction enzyme Sau3A I and the fragments ligated with a universal adapter to be amplified with the universal primers and selected primers. The products were then ligated into the pMD18-T vector and sequenced.
One of the sequenced clones was retrieved in the National Center for Biotechnology Information (NCBI) databases with Blast program. The results showed that the sequence possessed great similarity to one fragment of the 17th chromosome in the genome. Sequence analysis with GenScan software indicated that the EST might be one section of an unknown gene.
RD-PCR provides simple and efficient approach for isolating EST from cells, and cDNA clone sequencing combined with bioinformatics analysis may be helpful in identifying new genes.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 05/2002; 22(4):309-11.