| BackgroundColorectal cancer is the third common malignant tumor in the world. In China, the morbidity and mortality of colorectal cancer is rising. Rectal cancer is the most common type in colorectal cancer, and accounts for about 60%. Metastasis is one of the most main biological characteristics of malignant tumors. Tumor cells migrate to other parts of metastases from primary lesions, and maintain the growth of recurrent tumor cells. Once the tumor cells have metastasis, the prognosis of patients will be poor. Most cancer patients do not die from primary carcinoma but died of metastatic carcinoma. Therefore, how to prevent rectal cancer metastasis is the key to successfully treat this cancer. Although surgery has always been the principal treatment means of rectal cancer, the postoperative recurrence rate is high. At present, the preoperative radiotherapy has become a standard treatment for phase â…¡/â…¢ rectal cancer. Radiotherapy can maximize the dose of radiation exposure to the lesions, kill tumor cells, and make the surrounding normal tissues and critical organs from unnecessary exposure or as little as possible. Preoperative radiotherapy can make reduction of tumor staging and tumor size, or even completely disappear the tumor on pathology, so as to improve the local radical cure rate of surgery. Preoperative radiotherapy can also reduce intraoperative tumor growing opportunities, decrease the postoperative recurrence of the tumor, and improve the long-term survival of patients. However, what molecular changes have occurred in preoperative radiotherapy of rectal cancer cells is still unclear.Gene chip technology is a kind of high flux and fast whole genome analysis technology, and it is one of the powerful tools of the study on gene function. This technology has been widely used in various fields of medical research. Gene expression profile is formed when an organism or a cell makes a response to the environment, genetic or biochemical reaction of signal. In the gene expression profile, gene expression was activated or inhibited several times or even thousands of times. Gene expression profile chip can be used to detect gene expression level, by comparing the difference between the gene expressions in different conditions. Being accurate and simple, it can be used to analyze the pathogenesis of diseases, gene function and pathways assiciated with diseases. The data analysis of gene chip usually needs a certain process flow, namely bioinformatics analysis. Bioinformatics is an interdiscipline that integrates a variety of technologies such as statistics, bioinformatics, and computer science. This technology fist filters massive biochip data, by using the existing analysis tools and public databases. Then it finds key biological molecules and underlying mechanisms via some methods such as sequence alignment, statistical analysis, clustering, function or pathway analysis and visualization mapping. Thus, this technology can analysis diseases on molecular level, and enrich the understanding of people on the occurrence, treatment and prognosis of diseases. With the completion of genome sequencing of various model organisms, bioinformatics has entered the functional genomics era. At present, the study on the molecular effect of preoperative radiotherapy for rectal cancer using gene expression profiling is limited.Objective and significance(1) One of objectives in this study is to identify key genes and functional pathways in rectal cancer samples with preoperative radiotherapy, via gene expression profiling of rectal cancer samples with and without preoperative radiotherapy. These results may be helpful to recover molecular mechanisms of preoperative radiotherapy on rectal cancer.(2) Another objective in this study is to validate the above predictions, and detect the differences of rectal cancer cell proliferation and metastasis ability between samples with gene silence and ones without gene silence, as well as the effect of different dose X-ray on rectal cancer cell proliferation and metastasis ability. These findings can provide experimental bases for clinical radiotherapy of rectal cancer.Methods(1) The gene expression profile data of rectal cancer samples with and without preoperative radiotherapy were downloaded from the public database GEO. The differentially expressed genes (DEGs) in the samples with preoperative radiotherapy were screened by limma package in Bioconductor, and|logFC (fold change)|> 1 and p-value< 0.05 were seleted as the criteria. Then their functions were predicted by GO and KEGG enrichment analyses. Besides, the protein-protein interactions (PPI) network was constructed for DEGs using Cytoscape. Furthermore, microRNAs (miRNAs) and transcription factors (TFs) regulating DEGs were identified using UCSC and several miRNA databases, and miRNA-DEG and TF-DEG regulatory networks were respectively constructed by Cytoscape.(2) The gene that was correlated with cell proliferation and metastasis and had the highest|logFC (fold change)| and lowest p-value, were chosen to be validated by experiments. The rectal cancer cell line SW620 which was high metastatic and high malignant was selected as the experimental material. The effect of different dose X-ray on rectal cancer cell proliferation and metastasis ability was detected by MTT and Transwell methods. Besides, the effect of different dose X-ray on the selected gene expression level was detected by RT-PCR. Furthermore, the selected gene was silenced by siRNA methods, and silence effects were evaluated by RT-PCR and western blot. The rectal cancer cell proliferation and metastasis ability after gene silence was detected.Results(1) A total of 606 DEGs were identified from the rectal cancer samples with preoperative radiotherapy, including 271 up-regulated ones and 335 down-regulated ones. Among them, MMP1 has the highest|logFC| and lowest p-value.(2) According to GO enrichment analysis, the up-regulated DEGs (e.g. SLC6A3, SLC30A4, RYR2 and NEDD4L) were mainly enriched in ion transport, response to inorganic substance and metal ion. Meanwhile, the down-regulated DEGs were mainly related to cell-cell signaling (e.g. SLC6A4 and PDX1), regulation of cell proliferation (e.g. PTGS2 and CDH5) and collagen metabolism (e.g. MMP10, COL1A1, MMP3 and MMP1).(3) According to KEGG enrichment analysis, the up-regulated DEGs were mainly enriched in five pathways, including steroid hormone biosynthesis, calcium signaling pathway, androgen and estrogen metabolism, neuroactive ligand-receptor interaction and maturity onset diabetes of the young. Among them, HSD3B2, UGT2A3, SULT1E1 and UGT2B15 were significantly correlated with steroid hormone biosynthesis, as well as androgen and estrogen metabolism; CYSLTR2, CHRM1 and HTR6 were markedly enriched in calcium signaling pathway and neuroactive ligand-receptor interaction pathway. The down-regulated DEGs were mainly related to ECM-receptor interaction (e.g. COL4A2, COL4A1 and COL6A3) as well as complement and coagulation cascades (e.g. SERPINE1, SERPIND1 and PLAU).(4) The PPI network consisted of 241 nodes and 410 interactions. Twenty nodes had higher connective degree more than 10. Among them, the connective degree of COL1A2 and COL1 A1 was both 18, and the connective degree of MMP1 was 11.(5) Using UCSC database, a total of five TFs were identified from DEGs, including PAX6, PLAU, FOXL1, NKX2-2 and FOSL1. In the DEG-TF regulatory network, there were 77 regulatory relationships. Except for modulating some DEGs (e.g. MMP1 and COL1 A1), PLAU also regulated NKX2-2 and PAX6.(6) Based on the analysis of seven miRNA database, a total of 177 regulatory relationships between miRNAs and DEGs, containing 145 miRNAs and 40 DEGs. For example, hsa-miR-29c regulated COL1A1, COL1A2, COL4A1 and COL4A2. MMP1 was modulated by hsa-miR-222, and MMP3 was regulated by hsa-miR-204.(7) MMP1 was selected to be validated by experiments. Before gene silence, under the X-ray dose of 0.1 GY,0.5 GY,1 GY,3 GY and 6 GY, mRNA levels of MMP1 in SW620 were all decreased compared with the control (0 GY). Besides, within 0.5 GY, mRNA level of MMP1 was significantly reduced with the increase of X-ray dose.(8) SW620 cell proliferation and metastasis ability before MMP1 silence was significantly higher than that after MMP1 silence (p< 0.5). Furthermore, within 6 GY, SW620 cell proliferation and metastasis ability was gradually weakened with the increase of X-ray dose.Conclusion(1) Some DEGs associated with response to metal ion (e.g. SLC6A3, SLC30A4, RYR2 and NEDD4L), calcium signaling pathway and neuroactive ligand-receptor interaction pathway (e.g. CYSLTR2 and CHRM1), regulation of cell proliferation and complement and coagulation cascades (e.g. PLAU, FOSL1 and SERPINE1), collagen metabolism (e.g. MMP1 and MMP3), and ECM-receptor interaction (e.g. COL1A2, COL1A1 and COL4A1) were differentially expressed after radiotherapy.(2) Some miRNAs (e.g. hsa-miR-29c, hsa-miR-224, hsa-miR-204 and hsa-miR-222) and several TFs (e.g. PLAU and FOSL1) might be key in the tumorigenesis process of rectal cancer.(3) Different dose X-ray could reduce mRNA level of MMP1 in SW620, and within 0.5 GY, mRNA level of MMP1 was significantly reduced with the increase of X-ray dose.(4) MMP1 had a key role in the process of SW620 cell proliferation and metastasis.This study contributes to reveal the molecular mechanisms of preoperative radiotherapy for rectal cancer, and firstly validated the effect of different dose X-ray on mRNA level of MMP1 in rectal cancer. Additionally, the role of MMP1 in the process of SW620 cell proliferation and metastasis was validated by experiments. These findings may provide theoretical and experimental bases for measurement of tumor sensitivity during clinical radiotherapy of rectal cancer. |
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