| As a common digestive cancer occurred in rectum or colon, colorectal cancer(CRC) is also named as rectal cancer or colon cancer. CRC is the second common cancer in female and the third common cancer in men, and its mortality ranks the second in cancers. According to the statistics of the world health organization’s international agency for research on cancer, CRC had 1.2 million newly diagnosed cases worldwide in 2008 and was responsible for 8% of all cancer-induced deaths.In recent years, along with the improvement of economic income and quality of life in our country, morbidity and mortality of CRC in the Chinese population have increased. The incidence of CRC is the third and its mortality is the fifth in malignant tumors happened in China. CRC is increasingly becoming a threat to people’s health and life. According to the three elements of distant organ metastases(M), regional lymph node involvement(N) and tumor infiltration situation(T),scholars put forward the TNM staging of CRC. When cancer cells only exist in intestinal wall of CRC patients(phase I and II), there has the hope of cure. If left untreated, the cancer cells will spread to the lymph nodes(phase III), and then develop into the distant metastases(IV). CRC patients in stage I and II can be cured by surgical removal of the lesions, about 73% of the CRC patients in stage III can be cured with surgery and chemotherapy. Although chemotherapy after surgical resection can improve the survival rates of CRC patients, CRC patients in stage IV often cannot be cured due to metastasized cancer cells. We screened the differentially expressed genes(DEGs) between CRC tissues and normal adjacent tissues, as well as searched for CRC genes(that is seed genes). Then, the protein-protein interaction network for the seed genes and the DEGs were built.Afterwards, random walk with restart analysis was performed to identify the top 50 nodes with higher affinity scores in the network, and enrichment analysis was conducted for the DEGs among the top 50 nodes. Subsequently, possible interactions between interested gene and antineoplastic drugs were searched. This study is aimed to find out the key genes involved in the development of CRC. Then,expression levels of the key genes were detected in CRC tissues and cell lines to verify the results of bioinformatics analysis. In order to reveal the action mechanisms of the key genes, the influences of the key genes on the cell proliferation, cell cycle and cell apoptosis of CRC cells were also detected.GSE32323 was downloaded from Gene Expression Omnibus, including 17(2in stage I, 7 in stage II, 5 in stage III, and 3 in stage IV) pairs cancer-normal matched colorectal tissue samples. The DEGs were screened using limma package and CRC genes(also denoted as seed genes) were searched from Online Mendelian Inheritance in Man database. Then, protein-protein interaction(PPI) pairs were searched from STRING database and sub-network(also known as CRC.PPI) of DEGs and seed genes were visualized by Cytoscape software. Besides, the top 50 nodes with higher affinity scores in the CRC.PPI were identified using random walk with restart analysis. And potential functions of the DEGs among the top 50 nodes were predicted by DAVID online tool. Using DGldb database, drug-gene interaction analysis was performed to search antineoplastic drugs interacted with genes. Using quantitative real-time polymerase chain reaction(q RT-PCR) experiments, the expression of key genes in CRC tissues and cell lines were detected. Then, the effects of key genes on cell proliferation of SW480 cells were determined by MTT and CCK-8 experiments. Finally, the impacts of key genes on cell cycle and cell apoptosis of SW480 cells were detected using flow cytometry.Compared with normal samples, there were 1640 DEGs screened in the CRC samples, including 850 up-regulated and 790 down-regulated genes. Pathway enrichment showed that up-regulated pituitary tumor-transforming gene(PTTG),proliferating cell nuclear antigen(PCNA), cyclin-dependent kinase 1(CDK1),minichromosome maintenance(MCM) and histone deacetylase(HDAC) were significantly enriched in cell cycle(p = 7.86E-13). The CRC genes or seed genes searched from OMIM database included cyclin D1(CCND1), aurora kinase A(AURKA) and so on. After the CRC.PPI of DEGs and seed genes were obtained, the top 50 nodes with higher affinity scores(e.g. DEP domain containing MTOR-interacting protein, DEPTOR; breast carcinoma amplified sequence-1,BCAS1; neural precursor cell expressed developmentally down-regulated 9, NEDD9;mitogen-activated protein kinase kinase 2, MAP2K2) in the CRC.PPI were identified by random walk with restart analysis. The PPI network of the top 50 nodes(including 14 up-regulated genes, 17 down-regulated genes and 19 non-DEGs) had224 interactions(e.g. DEPTOR-CCND1, AURKA-BCAS1, CCND1-BCAS1,CCND1-NEDD9 and CCND1-MAP2K2). There were 31 DEGs(including 5 seed genes and 26 non-seed genes) included in the top 50 nodes. The 31 DEGs in the PPI network of the top 50 nodes mainly were enriched in the functions of cell cycle phase(p= 0.005651, which involved CCND1, NEDD9 and AURKA), cell cycle(p = 0.045692,which involved CCND1, NEDD9 and AURKA) and intracellular signaling cascade(p =0.01978, which involved CCND1, MAP2K2 and AURKA). In addition, the enriched pathway for the 31 DEGs included cytokine-cytokine receptor interaction(p = 0.04602).The interactions between antineoplastic drugs and the 31 DEGs in the CRC.PPI network were screened. There were only three DEGs(CCND1, AURKA and DEPTOR) had interactions with their corresponding antineoplastic drugs.Considering that the differential expressed genes including CCND1, NEDD9 and AURKA were enriched in cell cycle, we next investigated their expression and functions in colorectal cancer by experimental methods. Using semiquantitative Reverse Transcription- Polymerase Chain Reaction(semiquantitative RT-PCR)and/or quantitative real time- Polymerase Chain Reaction(q RT-PCR) experiments,the expression of CCND1,AURKA and NEDD9 in CRC tissues and cell lines were detected. The results showed that CCND1, AURKA and NEDD9 were significantly up-regulated in CRC tissues and SW480 cells. The change in AURKA was mostsignificantly, followed by CCND1. The result of MTT experiment indicated that cell proliferation capacity of SW480 cells with AURKA silencing was significantly decreased. Flow cytometry analysis showed that the G1-S phase progression of SW480 cells with AURKA silencing were significantly inhibited. Moreover,apoptosis of SW480 cells with AURKA silencing were increased. In addition, the result of CCK-8 test indicated that cell proliferation capacity of SW480 cells with CCND1 silencing was markedly decreased. Flow cytometry analysis showed that the G1-S phase progression of SW480 cells with CCND1 silencing were significantly suppressed. Moreover, apoptosis of SW480 cells with CCND1 silencing were increased.In conclusion, we carried out a comprehensive bioinformatics analysis of genes which may be involved in CRC. We screened 1640 DEGs in the CRC samples compared with normal samples. Besides, PTTG, PCNA, CDK1, MCM, HDAC,DEPTOR, AURKA, CCND1, BCAS1, NEDD9 and MAP2K2 might have a correlation with CRC. Moreover, antineoplastic drugs which might interact with CCND1, AURKA and DEPTOR were obtained. However, further verifications are still necessary to unravel their action mechanisms in CRC. CCND1 and AURKA were significantly up-regulated in CRC tissues and SW480 cells, which was consistent with the previous results of bioinformatics analysis. Besides, cell viability and proliferation capacity of SW480 cells with AURKA or CCND1 silencing were significantly decreased. The G1-S phase progression of SW480 cells with AURKA or CCND1 silencng were significantly inhibited. Moreover, apoptosis of SW480 cells with AURKA or CCND1 silencing were increased. These declared that AURKA and CCND1 could promote proliferation and cell cycle progression, as well as inhibit apoptosis of SW480 cells. |
PDF Full Text Request