| [Objective]:Colorectal cancer (CRC) is one of the most common cancers in the world, with more than one million new cases every year. Preoperative chemoradiotherapy (CRT) is the standard treatment for colorectal cancer (CRC). However, with the wide use of chemoradiotherapy in colorectal cancer patients, the chemoradioresistance became more and more obvious. It is proved that 5-FU based chemoradioresistance may ressult from the abnormity of gene expression. Here, we established chemoradioresistant colorectal cancer cell line in vitro, analysised the expression profile variation of mRNA in it and filtrate the key genes leading to chemoradioresistance to explore the genetic mechanisms of chemoradioresistante. This project will provide scientific evidences for the reversal of chemoradioresistence and improvement of clinical efficiency of chemoradiotherapy in CRC.[Methods]:1. Simulate clinical high doses therapy to establish HCT116 chemoradioresistant colorectal cancer cell line in vitro.4Gy 6Mv X-ray and 10μmol/15-Fu were choosen for chemoradiotherapy in vitro.2. Clongenic survival assay was applied to confirm the chemoradioresistance of HCT116 CRR cell;3. Microarray analysis was applied to identify differentially expressed mRNA between HCT116 parental cell and HCT116 CRR cell;4. GO categories consist of three families:biological process, cellular component and molecular function derived from Gene Ontology and pathway analysis were performed.5. Analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) database applied to determine the biological pathways that there was a significant enrichment of mRNAs with differential expression.6. To validate the microarray results, randomly selected 6 differentially expressed mRNAs between CRR-HCT116 and parental HCT116 cells to confirm their expression levels by qRT-PCR.7. All data were analyzed by SPSS17.0 software and statistics were expressed as mean ± standard deviation (x±s), and single factor analysis of variance (ONE WAY ANOVA) and t test were used to get the statistical significance.[Results]:1. HCT116 chemoradioresistant colorectal cancer cell line was established by 10 times of chemoradiotherapy;2. The chemoradioresistance of HCT116 CRR cell was validated by Clongenic survival assay;3. After quantile normalization of the raw data, the expression profiles of 21 811 mRNAs were obtained from parental HCT116 and CRR-HCT116 cells, a total of 11.0 %(2397/21811) mRNAs were significantly differently expressed in CRR-HCT116 compared with parental HCT116 (fold change> 2).4. GO analyses categoried differentially expressed mRNAs as three GO families: biological process, cellular component and molecular function. And showed a variety of biological functions of differentially expressed mRNAs;5. KEGG pathway analysis indicated that 57 (38 up-regulated and 19 down-regulated) pathways were involved in the HCT116 chemoradioresistant;6. All of the differentially expressed mRNAs Validated by the qRT-PCR except HOBX7 showed the same trends of up- and down-regulation as the microarray data.[Conclusion]:1. The application of the clinical high doses therapy is a method through which we can induce the cell line with chemoradioresistance. Compared with its parental cell line, HCT116 cell has a higher chemoradioresistance.This project will provide scientific evidences for the reversal of chemoradioresistence and improvement of clinical efficiency of chemoradiotherapy in colorectal cancer.2. We established the profile of differentially expressed mRNAs related to chemoradioresistance in colorectal cancer cell. Its chemoradioresistance may be attributed to several mRNAs in some mRNA cluster/family. Bioinformatics analyses provide us possible biological processes and pathways for further study.3. The results of qRT-PCR were consistent well with the microarray results. |
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