Abstract Objective To study the ginsenoside Rh2 regulation of normal mice with lung cancer gene expression differences. The use of oligonucleotide microarray technology, the detection of of Rh2 on normal and tumor-bearing mice gene expression regulation, and differences in gene clustering and statistical analysis. To further validate the microarray results, we used real-time quantitative PCR was used to test the effect of Rh2 on gene expression. Results Compared with normal mice, Rh2 on tumor-bearing mice gene expression greater impact on both the regulated organ; Rh2 on normal and tumor mouse gene expression regulation there is no clear correlation; Rh2 energy The tumor-bearing mice effectively antagonize local tumor inoculation caused abnormal gene expression of various organs. The regulation of gene expression of the conclusions Rh2 on tumor-bearing mice with tumor-specific.

Key words ginsenoside Rh2; gene chip; gene expression profiles

Tumor Specific Gene Regulation by Ginseng Saponin Rh2 in Mice Bearing Lung Cancer

    ZHANG Wen  jing, BAO Li  hua, YU Chun  ying, ZHANG Jun  yi, ZHAO An, XU Chang  jiang, JIA Wei  guo

    Shanghai Innovative Research Center of TCM, Shanghai 201203, China

    Corresponding Author: JIA Wei  guo, E  mail: wjia@interchange.ubc.caAbstract: Objective To study gene differential expression profiles by ginsenoside Rh2 in normal and tumor  bearing mice.Methods Gene expression profiles after treatment with Rh2 in normal and tumor  bearing mice were determined by Oligo chip technology as well as the clustering and statistic analysis of single gene.To further validate the microarray results, we used quantitative real  time RT  PCR to examine the effect of Rh2 on individual genes.Results As compared with normal mice, treatment with Rh2 changed gene expression more potently in mice bearing tumor.There  s no obvious correlation on the gene expression profiles between the normal and tumor  bearing mice.Treatment with Rh2 was found to be effectively resistant abnormal gene expression profiles in some major organs that resulted from the local implantation of tumor.Conclusion The data showed that there is specific regulation of gene expression profiles related to tumor by Rh2 in mice bearing tumor.

    Key words: Rh2; Oligo chip assay; Gene expression profile
Ginseng (Panax ginseng CA meyer) is a perennial herb of the Araliaceae is a precious Chinese medicine for thousands of years of application history. The effect is not only earlier for countless physicians of the ancient and modern, certainly, but also for the Institute of modern science to prove. Earliest spoke highly of ginseng pharmacology function devaluation “Shen Nong’s Herbal Classic". The book made it clear that ginseng “Lord completed five internal organs, Ann ended fright, in addition to evil, eyesight, fun puzzle.

    Ginseng contain saponins, ginseng polysaccharides, polypeptides, amino acids and vitamins and other substances. Ginsenoside is the main active ingredient in the ginseng, mainly including protopanaxdiol and the original Panaxatriol, group. The Ginsenoside the physiological activity, edible and medicinal value. A large number of research results, The Ginsenoside have anti-fatigue [1,2], improve memory [3,4], anti-aging, improve immune function [5] and anti-tumor and other role. Especially worth mentioning is that the anti-tumor effect of ginsenosides. Experiments show that, the ginseng extract the prevention and treatment of esophageal cancer, gastric cancer, lung cancer, liver cancer and other cancers [6]. Original panaxdiolsaponins Rh2, the study found that it has a strong role in inhibiting the growth of tumor cells [7].

    Gene chip technology has been widely applied to the study of gene expression and regulation, organisms (organizations) have different functional gene expression under different time conditions, anti-RNA was extracted from the samples and then transcribed into cDNA containing behalf of the organisms all functional gene chip hybridization (or organization), you can get the sample function gene expression. E.g., in the human genome encoding about 100,000 different genes, to be detected by DNA chip technology, and just one can be obtained almost all of the expression.

    The use of gene chip technology to study oral ginsenoside Rh2 gene regulation of normal and tumor-bearing mice. The results show that: Compared with normal mice, Rh2 on the gene expression of the tumor-bearing mice a greater impact, both the regulated organ; gene expression in various organs of normal and tumor-bearing mice Rh2 treatment there is no clear correlation between the spectral; Rh2 effectively antagonize the tumor-bearing mice tumors causing abnormal gene expression in various organs, and that regulation of gene expression of Rh2 on tumor-bearing mice with tumor-specific.

    1 Materials and methods

    The 1.1 test animals C57BL / 6 mice, female, 18 ~~ 20g, purchased from Hayes Lake experimental animals LLC.

    1.2 Reagents

    Trizol kit (Invitrogen); Rneasy Mini reagent cartridge (QIAGEN); 20 x SSPE lotion (Invitrogen); 20% of N  lauroyl sarcosinate solution N  Lauroylsarcosine Solution (SIGMA); by reverse transcriptase MMLV-RT (Promega ); oligonucleotide mixture dNTP Mix (of ATP, GTP, CTP, UTP) (Amersham); AA  UTP (Ambion); sodium bromide Sodium bricarbonate (SIGMA); hydroxylamine Hydroxylamine (SIGMA); Cy3-NHSs Easter and Cy5 NHS easter (Amersham); stability and drying the solution Stabilization and Drying Solution (Agilent); mice oligonucleotide chip Mouse oligo microarray (Agilent)

    1.3 The main instrument

    Agilent the scanner, Chrom4 fluorescent quantitative PCR instrument.

    1.4 Method

    1.4.1 mouse tumor model and derived

    Total of 80 mice were randomly divided into four groups (n = 20), in which two groups of tumor-bearing mice, were inoculated subcutaneously in the right axillary 0.2ml of 8 × 106Lewis lung cancer (3LL) cell suspension. The two groups of normal mice (non-tumor-bearing mice) were orally solvent 0.5% sodium carboxymethyl cellulose (CMC  Na), as a negative control group (NS), 100mg / (kg · d) Rh2 (0.5% CMC  Na solution to dubbed 4mg/ml solution) by gavage, referred to as the NR group; tumor-bearing mice orally solvent 0.5% CMC  Na, referred to as TS group; Rh2 orally to the same dose , referred to as a TR pack. 24 h after tumor inoculation gavage once daily for 21 consecutive days. Were taken after the end of the trial, each mouse’s brain, heart, lung, liver, spleen, thymus, and tumor tissue, 10 mice each organ homogenate were extracted RNA, to get the same amount of RNA is mixed in the same group, The reverse-transcribed into cDNA after for microarray.

    1.4.2 microarray

    (1) Total RNA was extracted and purified RNA was extracted using Trizol reagent cartridge. The Determination of OD260 and OD280 values ​​identification of nucleic purity; purification of total RNA using the RNeasy  Kit.

    (2) cDNA synthesis to take 2μg of total RNA, the T7 promoter to Primer 5μl 0.1μg/μl, made up to volume with water to 11.5μl, 65 ° C incubated 10min the ice bath was 5min in the system added 4μl 5 × First Strand the Buffer, 2μl 0.1M DTT, 1μl 10mM dNTP, 1μl MMLV  RT, 0.5μl RNase OUT, 40 ℃ insulation 2h.

    (3) fluorescence-labeled cDNA synthesis, purification of the synthesized cDNA at 65 ° C incubated 15min, the ice bath was 5min. Add Transcription mix (containing 5.7μl Rnase  free Water, 20μl 4 × Transcription Buffer, 6μl 0.1M DTT, 16μl dNTP Mix (dATP, dGTP, dCTP, dUTP), 6.4μl 50% PEG, 0.5μl Rnase OUT, 0.6μl Inorganic Pyrophosphatase, 4μl 25mM aa  UTP, 0.8μl T7 RNA Polymerase and mix, 40 ℃ insulation 2h and subsequently placed on ice. cDNA purified according to the RNeasy Mini Protocol.

    (4) cDNA probe after mark, cDNA probe purification take 4μg cDNA, and concentrated to 6.6μl, 10 μl of DMSO (Sigma), and mix, adding 3.4μl 0.3M sodium bicarbonate buffer (pH 9.0), and mix. Added to the mixture of 20μl cDNA fluorescent dye and mix at room temperature for 1h, adding 9 μl of 4M Hydroxylamine mixing, at room temperature for 15min. CDNA probe purification according to the RNeasy Mini Protocol.

    (5) cDNA probe quantitative Cy3 probe concentration (pmol / μl) = A552/0.15, Cy5 probe concentration (pmol / μl) = A650/0.25.

    (6) chip hybridization of DNA fragmentation (100pmol Cy3-cDNA, 50pmol Cy5 cDNA, 50μl 10 × Control target The Add RNase  free Water additional volume to 240 μl), in which 10 μl Fragmentation the Buffer, mix gently, 60 ℃ insulation 30min (no more than 30min) added 250μl of Hybridization Buffer, mix gently, take 490μl the hybridization melting dropping the coverslip, covered with chip and chip sealed hybridization cassette, 60 ° C rolling hybridization 16h.

    (7) chip washing the chip into the washings (700ml DEPC- H2O 300ml 20 x SSPE, washed 1min, 0.25ml 20% N the  Lauroylsarcosine) in the chip into the lotion 2 (997ml DEPC- H2O, 3ml 20 × SSPE, 0.25ml 20% N  Lauroylsarcosine) washed 1min chip into lotion 3 (Stabilization and Drying Solution) washing 30s.

    (8) chip scanning and data analysis using the the Agilent Scanner to obtain images. Scan pixel values: 10 μm, the value of the PMT (%): 100, the image is usually 16  bit tiff file. Images directly using the image analysis software Feature Extraction Agilent systems corresponding to the quantitative analysis processing. Generally considered the ratio values ​​in the range of from 0.5 to 2.0 within the gene does not exist significant expression differences, while outside this range, the gene is considered to significantly change the expression appears.

    1.4.3 Real  time PCR

    Real  time PCR experiments in accordance with the SYBR Green kit (Bio  Rad), the equipment used for the production MJ Research Chrom4 Real-time PCR instrument. β  actin used as an internal standard, the amplification of mouse β  actin primers of  GCTAC AGCTT CACCA CCACA the 3 of  /  CATCG TACTC CTGCT TGCTG .

    The amplified mouse Ifi202b the Cidea, Cdk8, MMP9, Calm4 the primers were 5  GGTGTGG GATAAAG AACAGCA 3  / 5  TCAAT GCCAC CACTT GTTTG 3 , 5  GCCTG CAGGA ACTTA TCAGC 3  / 5  TGCTT GCAGA CTGGG ACATA 3 , 5  GATTC CCTCA ACAAC CCAGA 3  / 5  CGGCT TTCCT TCATT CTGTT 3 , 5  GTGGG TGTAC ACAGG CAAGA 3  / 5  ACTCC TTATC CACGC GAATG 3 , 5  TGATG GCAAG ATCAG CTTTG 3  / 5  CACGG ATCAT GTCCT CCAG 3 .

    cDNA preparation described in the same chip detection “method. The PCR reaction is done in 20μl system, including: the 10μl IQTM SYBR  Green Supermix (Bio-rad), 0.1μl cDNA, 0.3μm corresponding primer. PCR conditions: 94 ° C melting 30s; 40 cycles (94 ° C melting 30s, 55 ℃ annealing 30s, 72 ℃ extends 30s); extending at 72 ° C for 10min. Collected after the extension step of each cycle the fluorescence signal, calculation results using ΔΔCT method.

    2 Results

    2.1 Rh2 gene expression in a number of organs and tissues of normal mice regulation

    Application Agilent mouse genome oligonucleotide chip (chip product catalog number: G4121A, 60 mer Oligo Microarray, a total of 20,868 genes) regulation of gene expression detected of Rh2 on normal mouse organs and tissues. NS group, the control group, the NR group for the detection of the experimental group, in order to understand the potential regulatory role of the gene Rh2 on normal tissues and organs.

    Rh2 on normal mouse organs gene expression is less affected, and less performance in its regulation of various organs of the number of differentially expressed genes. On normal mouse gene expression regulation is mainly concentrated in the spleen and lung, while their brain, heart, thymus, liver gene also have a certain adjustment, as shown in Figure 1.

    2.2 subcutaneous local transplant tumor gene expression regulation of certain organs and tissues of normal mice

    Agilent mouse whole genome oligonucleotide microarray subcutaneous local transplant tumor gene expression regulation of normal animals. NS group, the control group, the detection of the TS group and the experimental group.

    Left raised in the various organs of the brain, heart, lung, thymus, liver, spleen, and down-regulated genes

    The right is the number of differentially expressed genes in the various organs accounted for all of the organ as a percentage of the number of differentially expressed genes

    Figure 1 Rh2 normal mice regulation of gene expression

    Subcutaneously transplanted tumor growth as a local lesions can greatly affect the gene expression of the various organs of the animals, the main function of the body’s organs, heart, lung, liver and immune organs thymus and spleen gene expression to a large extent impact, while for the control of the gene expression of the brain of the nerve function regulator, shown in Figure 2. These results indicate a localized tumor lesions have significant effects on the overall distal end of the gene expression of the various organs, which indicates the local lesion may disrupt the normal function of the whole, and traditional Chinese medicine to treat localized disease with the overall concept guiding ideology has consistency.

    2.3 Rh2 gene expression in a number of organs and tissues of the tumor-bearing mice regulation

    Agilent mouse genome oligonucleotide microarray Rh2 regulating gene expression of several organs and tissues of the tumor-bearing mice. TS group for the control group, the TR group detection, in order to understand the of Rh2 on various tissues and organs of the tumor-bearing mice gene for the experimental group.

    Rh2 on tumor-bearing mice regulation of gene expression and its great differences in the regulation of gene expression of normal mice, greatly increased the number of First gene regulation, and is a major regulator of organ differ. Rh2 regulation occurs mainly in the heart, thymus and brain gene expression in tumor-bearing mice, while the lung, liver, spleen, as shown in Figure 3. Shows that Rh2 significantly change the gene expression of tumor-bearing mice brain and heart, while the normal mouse brain and heart.

    2.4 compare Rh2 on normal mice and tumor-bearing mice regulation of gene expression in various organs of normal mice organs regulation of gene expression is different from the microarray data analysis, are shown in Table 1. The Rh2 up-regulated genes in tumor-bearing mice only 1.45% in the normal mice were also Rh2 raised. Both normal and tumor-bearing mice was only Rh2 lowered 2.44% of the genes of the tumor-bearing mice also Rh2 down-regulated genes. Description of gene regulation in normal mice and tumor-bearing mice Rh2 no significant correlation. Tip Rh2-dependent regulation of gene expression in tumor-bearing mice with tumor. Table 1 compares the Rh2 on tumor-bearing animals and the regulation of gene expression in normal animals (NR / NS vs TR / TS)

    2.5 by Rh2 treatment of tumor-bearing mice before and after (TR / TS), and in mice after the subcutaneous tumor inoculation (TS / NS) of various organs of the correlation of the regulation of gene expression microarray data analysis

    Organs of mice Rh2 treatment of tumor-bearing mice before and after various organs of gene expression changes were inoculated subcutaneously with tumor gene expression changes compare (TR / TS vs TS / NS, see Table 2, we found Rh2 tumor-bearing gene regulation of the brain and thymus of mice with local tumor inoculation led gene regulation that have great relevance. Rh2 has obvious reply to adjustment due to tumor inoculation lead to brain and thymus tissue gene expression abnormal thymus 29.85% down due to tumor gene Rh2 treatment raised; thymus have 18.60% due to tumor raised the gene Rh2 treatment lowered. interesting is, Rh2 in the brain by tumor impact and raised the genes have a more significant role, but Rh2 on brain tumor effects down the gene seems little, the performance of the regulation of these genes in 29.13% down Rh2 treatment due to tumor upregulated genes, only 2.25% of down-regulated genes are tumor because Rh2 tends recovery. changes may Rh2 treatment appropriate regulation of gene expression of Rh2 on tumor-bearing mice highly tumor-specific. Table 2 compares the gene expression of normal mice Rh2 on tumor-bearing mice and tumor adjust

    2.6 Rh2 treatment of tumor-bearing mice after tumor gene expression

    Found Rh2 treatment of tumor-bearing mice tumor gene expression gene microarray the Rh2 raised 48 gene in the tumor tissue, 105 genes were down. Among these upregulated genes, including inhibit transcription regulation factors and inhibition of cell proliferation function of genes and apoptosis-inducing gene, whereas in the down-regulated genes include the genes of the cell cycle regulatory gene, and cells related to the transfer. The results of the study showed that Rh2 in the tumor tissue by regulating tumor cell growth, apoptosis, and the transfer of genes related to some extent inhibited tumor growth and metastasis.

    2.7 Real time PCR technology validation Rh2 treatment of tumor-bearing mice tumor gene expression differences

    Using SYBR Green real-time PCR relative quantification technology Actin as reference gene chip detection of tumor growth related gene RNA expression levels of detection. The ΔΔCT method results, shown in Table 3, you can see the 21 days of treatment the tumor Ifi202b raised 65.8 times, Cidea 15.3 times higher, Cdk8 down about six times, MMP9 down about 5 times. Be seen that the experimental results of real-time PCR and microarray maintain a high degree of consistency. Table 3 with chip technology detects Rh2 treatment tumor gene expression differences in tumor-bearing mice by Real time PCR technology to verify

    3 Discussion

    Based on the above results, we can see Rh2 on tumor-bearing mice regulation of gene expression highly tumor-specific: First Rh2 on normal organs of mice, gene expression was less affected, the regulated gene few, regulation focused gene expression in the spleen and lungs, and Rh2 on tumor-bearing mice organs impact is relatively large, greatly increased the number of the regulated gene, regulating organ mainly concentrated in the heart, thymus and brain; Second Rh2 on tumor-bearing mice and normal mice organs in the regulation of gene expression is different, only 4% of the same genes; once again, quite a lot due to tumor growth and abnormal expression of genes in the thymus and the brain effectively antagonistic; Finally, tumor-bearing mice by Rh2 treatment, a considerable number of gene expression in the tumor tissue subject to regulation, the role of the principle that inhibition of cell growth, induction of apoptosis affect tumor metastasis. Rh2 on regulation of gene expression in tumor-bearing mice highly tumor-specific. Rh2 small effects on the gene expression of normal organs, explain the clinical observation of the toxic side effects of ginsenoside years show. And organs of the body caused by the local tumor cultivation gene expression significantly change, may be the role of the tumor cells or surrounding the tumor tissue and the immune system by the release of many factors, Rh2 on these organs due to the tumor resulting ( especially the brain, heart and thymus) gene expression abnormalities recovery, to verify the functionality of traditional Chinese medicine in clinical use ginseng the Rotary evil Reformed.