The Studies of Chlorogenic Acid Antitumor Mechanism by Gene Chip Detection: The Immune Pathway Gene Expression

abstract: ...Persistently increasing incident of cancer in human beings has served to emphasize the importance of studies on mechanism of antitumor substances. Chlorogenic acid (CA), extracted from folium cortex eucommiae, has been confirmed to have lots of biological activities encompassing inhibition of tumor. However, the anticancer mechanism of CA remains unclear. Here, we have utilized a whole mouse genome oligo microarray (444K) to analyze gene expression level of female BALB/c mice (implanted with EMT-6 sarcoma cells) after treatment with low, medium, and high-dose CA (5mg/kg, 10mg/kg, and 20mg/kg), docetaxel, interferon, and normal saline separately at 6 time points (3rd, 6th, 9th, 12th, 15th, and 18th days after administration). Differentially expressed genes screened out by time-series analysis, GO analysis, and pathway analysis, and four immune-related genes were selected for further confirmation using RT-qPCR. The results demonstrated that CA is able to change gene expression and that the responsive genes (CaN, NFATC2, NFATC2ip, and NFATC3) involved in immune pathways had been significantly upregulated by CA. Expression of immune factors such as IL-2R and IFN- can be improved by CA to promote activation and proliferation of T cells, macrophages, and NK cells, thus enhancing their surveillance and killing abilities, further suppressing the growth rate of tumor cells.

... genes (CaN, NFATC2, NFATC2ip, and NFATC3) involved in immune pathways had been significantly upregulated by CA. Expression of immune factors such as IL-2R and IFN- can be improved by CA to promote activation and proliferation of T cells, macrophages, and NK cells, thus enhancing their surveillance and killing abilities, further suppressing the growth rate of tumor cells. 1. Introduction Cancer dormant cell theory takes the view that activation of resting cells is the key factor causing cancer

... gene expressing analysis into SBC Analysis System (V2010.05). 2.6. RT-qPCR RNA extracted from experimental group of CA, positive control group of docetaxel, and interferon, negative control group were used to measure expression of selected genes of interest by RT-qPCR. Genes CaN, Nfatc2, Nfatc3, and Nfatc2ip showed a different expression level following treatment with low, medium, and high-dose CA, respectively. Primers used for RT-qPCR validation were designed according to each gene sequence and

... and at 85C for 5min at a 20 L total reaction volume containing the following reagents: 2 L RNA, 1 L oligo, 2 L (10mM) dNTP mixture, 1 L RiboLock RNase Inhibitor, 4 L 5 reaction buffer, 9 L water, and 1 L RevertAid M-MuLV. PCR was performed using a Sino Bio Taq 2 Master Mix. The RT-qPCR products were analyzed by 1.0% agarose gel electrophoresis. PCR reaction should be taken at a temperature gradient in the range of annealing temperature of CaN, Nfatc2, Nfatc3, and Nfatc2ip. RT-qPCR analysis

..., Nfatc3, Pbx2, Rest, Smarca5, and Nfatc2. After doing some queries on the function of genes covered in statistically significant pathways and GO, we found out immune related genes and common genes of pathway and GO. Nfatc2, Nfatc3, Nfatc2ip, and CaN were found through combing raw analysis data of gene expression profile, as shown in Table 5 . 3.2. Validation of Differentially Expressed Genes by Fluorescence Quantitative PCR Relative expression level of the target genes Nfatc2, Nfatc3, Nfatc2ip, and CaN on average is shown in Table 6 . Relative expression level of Nfatc2, Nfatc3, Nfatc2ip, and CaN in high-dose CA group, Nfatc2ip and CaN in medium-dose CA group, and CaN in low-dose CA group significantly improved compared with negative control group. The exact expression quantity is shown in Figure 2 . As shown in Figure 2 , relative expression level of Nfatc2ip, Nfatc3, Nfatc2, and CaN in high-dose CA group improved compared with negative control group, which corresponds to the result of microarray analysis. 3.3. Discussion of Genes Nfatc2ip, Nfatc3, Nfatc2, and CaN Here, a whole mouse genome oligo microarray (444K) was used to analyze gene expression level of female BALB/c mice and to compare the expression of corresponding target genes of each group on the time series. Systematic error or accidental error can affect the accuracy of microarray analysis to some extent and the errors may come from the processes of RNA extraction, RNA reverse transcription, and hybridization as well as the

....21, expression of critical genes in T-cell receptor signaling pathway, B-cell receptor signaling pathway, and natural killer cell mediated cytotoxicity had changed and immune related genes Nfatc2ip, Nfatc3, Nfatc2, and CaN were found. NFAT (nuclear factor of activated T cells) which consists of 4 components NFATc1, NFATc2, NFATc3, and NFATc4 has been proved important in lymphocyte activation and development. Nfatc2 existing in cytoplasm translocates to the nucleus upon T-cell receptor stimulation

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Gene: NFATC2IP - nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 interacting protein - (NCBI)

Protein : 2jxx

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