Keywords: l 9981; Gene chip; Gene expression; Nm23-H 1 gene
China Library Classification Number: R734.2；; Q8 13. 1
Document ID: a
Article number:1007-7847 (2006) 01-0077-05.
The nm23-H 1 gene has been proved to be a tumor metastasis suppressor gene. Transforming the cDNA of nm23-H 1 gene into human high metastatic large cell lung cancer cell line L998 1 lacking nm23-H 1 gene expression can reverse its malignant phenotype. However, the molecular mechanism of nm23-H 1 inhibiting the invasion and metastasis of lung cancer is still unclear. Our previous research showed that the inhibitory effect of nm23-H 1 gene on tumor invasion and metastasis can be achieved by regulating metastasis-related genes. In order to further study the molecular mechanism of nm23-H 1, we used gene chip to detect the transfection of nm23-Hl to L99865438.
1 materials and methods
1. 1 material
L, 1. 1 cell line
1)L998 1 (high metastatic large cell lung cancer cell line); 2) L998 1-nm23-H 1 (L998 1 cell line stably expressing nm23-H 1 gene). All of them were provided by the Key Laboratory of Lung Cancer Molecules of West China Hospital, Sichuan.
1. 1.2 Common reagents and equipment
Refined calf serum and RPMI t640 medium were purchased from Hy- clone Company. A stock solution of 1 mmol/L was prepared with NaOH solution of 1.20 mmol/L and stored at 4℃. Superscript TM II ribonuclease H- reverse transcriptase (1 nitrogen catalysis. No. 18064-0 14), dATP, dGTP, dCTP, dTYPOligod(T) primer (Bioasia Synthesis), Cy5 (Amersham, Cat,No. PA5502 1), Cy3 (amersham). No. PA5302 1), RNA inhibin (Takara, Cat. No. D23 1 1A), Ola rapid nucleotide detachable kit (OIAGEN, Cat, No.28306) Gene machine: OmniGrid 100Pixsys5500 chip printing is only purchased from Carte― sian company, and ScanArrayLite chip scanner and its analysis software Quantarray are purchased from GsiLumonics company.
1. 1.3 gene chip
The version number of the chip used in the project is 2.2 human cDNA gene expression profile chip, the product catalog number is SBC-R-HC- 100-22, and the chip numbers are 17874 and 17873 (Shanghai Biochip Engineering Company).
1.2 method
Total RNA was purified from 1.2. 1
Total RNA was purified by qiagen RNeasy Mini Kit. See rn easy Mini protocol 0 1 for detailed operation principles and methods.
Quality detection of 1, 2,2 total RNA
See the user's manual of human cD- NA expression spectrum chip, SBC-H-HC- 100-22 for specific operation steps, and refer to the sample quality inspection report for detailed results. According to the operating manual of Agi- lent 2 100 analyzer, the chip lab prepared gel, and carried out RNA electrophoresis operation according to the software prompts to evaluate RNA quality.
1.2.3 sample preparation
CDNA was digested with Sau3Al, then connected with linker, and then amplified with PCR universal primer. The PCR product was purified by PCR product purification kit and 500 ng was taken out. The experimental labeling method is cDNA direct labeling method. The RNA in the experimental group (L9981-nm23-h1) is labeled with cy3 fluorescence, while the RNA in the control group (L9981) is labeled with cy5 fluorescence. Adding primers labeled with Cy3/ Cy5 (100LLM01L) 21LL (the control group treated with NaOH was labeled with Cy5, and the treatment group treated with As20 was labeled with Cy3), then PCR amplification and purification were carried out under the same conditions, and the final concentration of PCR products was adjusted to 200 mg/L.
1, 2.4 hybridization
After denaturation at 95℃, the labeled samples were added to the surface of pre-hybridization chip array, covered with cover glass, placed in hybridization box, and hybridized at 42℃ 18h.
1.2.5 detection and analysis
Chip results were scanned by Agilent scanner, and scan resolution 10μm and PMT 100%% data were read by Ima- gene software. Finally, Genespring was used for normalization analysis, and the final ratio was cy3/cy5, that is, the experimental group/control group. The screening criteria of differential genes are (1) ratio >: Or =2 for up-regulated genes, and ratio = 0.5 for down-regulated genes.
Statistical analysis of 1.3
Cluster analysis method. Cluster analysis was carried out by using genespring and cluster analysis software.
Two results
2. 1 cell total RNA electrophoresis map
After the total RNA of two cell lines (L998 1, nm23-H 1 and L998 1) was extracted and purified, 1% agarose gel electrophoresis showed obvious bands of 28, 18 and 5S, and the band of 28S was significantly higher than that of/kloc-.
2.2 Gene Chip Hybridization Results and Analysis
Then the scanned images of Cy5 and Cy3 are completely overlapped. In the obtained hybridization map (Figure 2), red dots indicate low expression, green dots indicate high expression, and yellow dots indicate that the expression level has not changed. After software analysis, the scatter diagram of chip hybridization is obtained (Figure 3). The y axis of each point represents the relative intensity of Cy3 hybridization signal, and the x axis represents the relative intensity of Cy5 hybridization signal. The Cy5/Cy3 ratio of the point on the 45-degree straight line (shown by the solid line) is L, indicating that there is no difference, while the point far away from the 45-degree straight line indicates that there is a big difference, and the point falling outside the two dotted lines is the point where the Cy3/Cy5 ratio is greater than 2 or less than 0.5.
2.3 Gene Chip Screening Results
Before and after transfection of nm23-H 1 gene into L998 1 cells, there were ***l792 genes, of which the expression increased (up-regulated) to 1 156, and decreased (down-regulated) to 642. Among the up-regulated genes, 1038 genes have unknown functions. Among them, cell cycle and apoptosis-related gene 3 1, oncogene 0, tumor suppressor gene 18, metastasis-related gene 12, cytokine and transcription factor 13, cell signal and protein-related gene 38, extracellular matrix and cytoskeleton protein 9, among which the function is 54 1. However, there are 10 1 genes, including 12 genes related to cell cycle and apoptosis, 1 1 genes related to oncogenes, 3 genes related to tumor suppressor genes and 14 genes related to metastasis.
3 discussion
The nm23-H 1 gene is a tumor metastasis suppressor gene, and its low expression and deletion are closely related to the invasion and metastasis of lung cancer. Our previous study found that the low expression, mutation and allele deletion of nm23-Hi gene protein and mBNA are closely related to the high metastasis and poor prognosis of lung cancer. There is loss of heterozygosity of nm23-HI allele in human high metastatic large cell lung cancer L998 1 cells with high metastatic potential. L998 1 cells have strong ability of cloning and invasion in vitro, and the spontaneous lung metastasis ability of nude mice is 100%. Previous studies have shown that introducing nm23-HI gene into L998 1 cell line can reverse its malignant phenotype, which shows that the proliferation ability, clonal formation ability, invasion ability and spontaneous lung metastasis ability in nude mice are significantly reduced. Tsinghua Zhou et al found that lung cancer with nm23-H 1 gene deletion is often accompanied by abnormal expression of some tumor invasion-related molecules. It is speculated that nm23-H 1 gene may be the upstream regulatory gene of the "lung cancer metastasis inhibition cascade" related genes, and its inhibitory effect on tumor metastasis potential is achieved by regulating the downstream genes. However, the molecular mechanism of nm23-HI gene reversing the malignant phenotype of lung cancer is still unclear.
Gene chip technology provides an ideal technical support for us to study the molecular mechanism of invasion and metastasis of lung cancer caused by structural and functional changes of nm23-H 1. Gene expression microarray has the characteristics of Qualcomm, large scale, high efficiency and high sensitivity, and can analyze the differences of mRNA abundance between two or more groups of different sources. By calculating the ratio of hybridization signals and statistical analysis, we can obtain differentially expressed gene information.
In this experiment, tumor gene expression microarray was used to analyze the expression changes of related genes before and after transfection of nm23-H 1 gene into L998 1 cells. The application of gene expression microarray provides genomic evidence for studying the molecular mechanism of nm23-H 1 gene, which breaks through the limitation of single gene isolation research in the past. In particular, introducing foreign genes into cells that do not express the genes avoids the histological differences in sample sampling, making the experimental results more reliable and easier to analyze. In this study, we used SBC-R-HC- 100-22cDNA gene expression profile chip containing 14 000 genes to transfect nm23-Hl gene in L98 1 cells. By comparing the difference information of their gene expression, the genes related to nm23-H 1 gene expression were searched. In this study, * * screened out 1792 differentially expressed genes. After nm23-Hl gene transfection, there were 1 156 genes, including 1035 genes. There are 642 genes whose gene expression is down-regulated, of which 54 1 is unknown and 10 1 is functional. Known genes up-regulated and down-regulated include: cell cycle and apoptosis genes, oncogenes, tumor suppressor genes, metastasis-related genes, cytokinesis and transcription factors, cell signal and protein-related genes, extracellular matrix and cytoskeleton proteins. Among the up-regulated genes, there are many genes related to tumor metastasis, such as E-Cad, β-catenin, nm23 and TIMP- 1, 2, etc. The down-regulated genes related to tumor metastasis mainly include Ras, VEGF, PDGF-A, ERK-/, 2, c-src, etc. These results are consistent with our previous research results.
However, this study also found that the high expression of nm23-H 1 gene can also increase the expression of some tumor suppressor genes, enhance the activities of cytokines and transcription factors, improve the activity of extracellular matrix enzymes, and affect the synthesis of cytoskeleton proteins. Therefore, it can be inferred that nm23-H 1 gene can inhibit the invasion and metastasis of tumor cells by inhibiting the proliferation ability and clone formation rate of cells, reducing the activity of matrix metalloproteinases, and inhibiting the expression of angiogenic genes, so as to inhibit the invasion and metastasis of tumors. It can be seen that nm23-Hl gene reverses the malignant phenotype of L998 1 cells through the regulation of downstream related genes, which is the key gene and upstream gene in the forward regulation of "metastasis inhibition cascade of lung cancer".
In a word, the invasion and metastasis of tumor is the result of the interaction of many genes and factors. One or two key genes cooperate with each other in time and space, which promotes the canceration, invasion and metastasis of cells. Therefore, when studying the molecular mechanism of tumor invasion and metastasis, we should also analyze multiple genes, not just a few single genes. Gene chip provides a powerful research tool for studying the occurrence and development of tumors and the interaction between genes. This article is the full text of the original. Users who don't have a PDF browser should download and install the original text first.