ObjectiveTo study the mechanism of the effect on invasion and metastasis of colorectal cancer by down-regulating c-Met gene.Methodsc-Met genes were knocked down in SW480 cells, differential genes were screened by gene chip, functional cluster analysis of differential genes was carried out, and IPA was used to analyze the interaction network of cell signal pathway and related differential genes, as well as the ralationship between related genes and upstream regulatory molecules. The related genes in the suppressed signal pathway were selected for qPCR verification.ResultsAfter knockdown of c-Met, the number of up-regulated genes and down-regulated genes in SW480 cells was 399 and 286, respectively. Cluster analysis showed that c-Met knockdown had a great effect on the gene expression level of SW480 cells, IPA pathway analysis showed that HGF signaling pathway was suppressed, and after c-Met knockdown, IPA interaction network suggested that AKT2, PIK3CA, and MAP2K4 in HGF pathway were down-regulated, and qPCR verified that the above genes were also down-regulated, which was consistent with the results of microarray.Conclusionc-Met may affect the invasion and metastasis of colorectal cancer through the regulation of AKT2, PIK3CA, and MAP2K4 in HGF pathway.
Objective To identify genes associated with hepatocellular carcinoma (HCC) as candidate diagnostic markers in a genome-wide scale. Methods The gene expression profiles of 40 pairs of HCC tumor tissue and peripheral non-tumorous liver tissue were analyzed by using gene chip technology.The gene chips were fabricated at the National Cancer Institute (NCI). Each gene chip contained 9 180 genes. The fluorescent targets were prepared by a direct labeling approach using two kinds of fluorescences as following: 100 μg of total RNA from non-cancerous liver tissue was labeled with Cy3-dUTP and 200 μg of total RNA from HCC was labeled with Cy5-dUTP. The targets were mixed together and hybridized with genes on the gene chips. Unsupervised hierarchical clustering analysis was done by CLUSTER and TREEVIEW software using median centered correlation and complete linkage. Results A total of 10 genes were found up-regulated in over 80% of primary tumors comparing with that of their corresponding non-tumorous liver tissues at a two-fold filter with an unsupervised hierarchical clustering algorithm, including protocadherin-alpha 9, ESTs, Homo sapiens cDNA FLJ, KPNA2, RPS20, SNRPE, CDKN2A, UBD, MDK and ANXA2.Conclusion These genes are supposed to be candidates for the diagnosis of HCC. Further investigation of these genes in a large scale of patients with HCC and patients with non-malignant hepatic diseases will be needed to disclose whether they could be used clinically as novel diagnostic tumor markers for HCC.
Objective To explore the pathogenesis of the level of gene and therapeutic target genes associated with intestinal obstruction by analyzing the differential expression gene. Methods The gene expression data that came from public database gene expression omnibus （GEO） which provided adhesion formation’ gene expression data on 1， 3， 7，and 14 days after operation （n=8） and normal intestinal tissues’ gene expression data （n=2） of mouse were collected. The gene function and differential expression of genes were analyzed by using gene ontology （GO） and significance analysis of microarray （SAM）. Results There were a lot of response stimulated up-regulation of gene expression when occurrence of adhesion， and the products of these genes were distributed on cell membrane. The analysis results of gene expression at different time point after operation showed that expression up-regulated of Hmgcs 2 gene occurred on 3-14 days ofter operation and expression up-regulated of Stxbp 5 gene occurred on 14 days ofter operation. Conclusions The adhesion formation may be closely associated with the genes of response to stimulus and the gene product in membrane. The Hmgcs 2 and Stxbp 5 genes may be closely associated with the occurrence of other diseases which induced by adhesion formation.This provides a basis for the discovery of potential therapeutic targets.
ObjectiveTo screen for the differentially expressed genes in steroid-induced osteonecrosis of the femoral head (ONFH) by gene microarray. MethodsThe femoral head tissue of ONFH was harvested from 3 patients with steroid-induced ONFH, aged 25, 31, and 38 years, respectively. Normal tissue was harvested from a 26-year-old male remains contributor. HE staining of the specimens was performed for observing the histology manifestation; the total RNA was extracted for measuring the purity; cDNA probe was synthesized by reverse transcription, and then were hybridized as the cDNA microarray for scanning of fluorescent signals and differentially expressed genes in the tissues. ResultsHE staining of normal tissue showed complete unit composed of lamellar bone, continuous and complete lamellar bone with a concentric arrangement around blood vessels, and normal bone cells in the trabecular bone lacuna. In ONFH tissue, adipose tissue increased in the medullary cavity, with increased fat cells filling in the medullary cavity and extruding capillary, and with decreased bone cells in the bone trabecula, which had deeply-stained nuclear chromatin, pyknotic or cracking nucleus, and even bone cells disappeared in the part of the bone lacuna, and trabecular bone became thin, sparse, interrupt, reduced area in visual field/unit. Total RNA extraction electrophoretogram displayed clear bands of 28S and 18S, and the brightness ratio of the 28S:18S was 2:1, indicating good total RNA quality. And 44 genes were differentially expressed, and there were 28 up-regulated genes and 16 down-regulated genes, including cell/organism defense genes, cell structure/motility genes, cell division genes, cell signaling/cell communication genes, cell metabolism genes, gene/protein expression genes, and unclassified genes. ConclusionThe analysis of the gene expression profile of steroid-induced ONFH can provide evidence for the pathogenesis of ONFH.
ObjectiveThrough the analysis of gene enrichment in gastric cancer samples, the changes of RNA alternative splicing and related molecular mechanisms were explored.MethodsThe pathological samples of three cases of gastric cancer patients and adjacent tissues were obtained clinically, and the data were obtained by cell culture, protein quantitative labeling, gene chip detection, high-throughput sequencing, etc. GO enrichment was performed on samples by DAVID and other network software, KEGG pathway analysis yielded relevant information for screening for variable splicing of differential genes.ResultsA total of 605 genes with individual ENSG IDs consistent with the gene identification of the ENSEMBL database were screened, and the gene levels of cancer tissues and adjacent tissues were compared. There were 411 non-differentiated genes, 119 differentially up-regulated genes, and 75 differentially down-regulated genes. A total of 69 differentially spliced genes were screened out. Functional annotation and pathway analysis revealed that the detection genes were mainly concentrated in molecular metabolic processes, cell migration, extracellular matrix tissue, blood coagulation, cell matrix adhesion, signal transduction, negative apoptosis regulation, angiogenesis, platelet activation, complement system, adipokines signaling pathway, peroxisome, cancer pathway, transforming growth factor (TGF) signaling pathway, axon guidance, cell cycle, etc.ConclusionThere are a large number of differentially spliced genes in gastric cancer tissue samples, and the difference in expression due to changes in splice sites may play an important role in the development of gastric cancer.
Objective To explore the microRNA （miRNA） expression changes and related miRNA characteristics of colorectal cancer （CRC） with hepatic metastasis by miRNA microarray. Methods The fresh specimens of primary CRC were collected in 10 patients during operation， which with hepatic metastasis or not. miRNA microarray analysis was performed to compare the miRNA expression levels in two groups. The different expression levels of miRNA were validated by quantitative real-time PCR analysis. Results A total of six dysregulated miRNAs were identified in the CRC patients with hepatic metastasis comparing with CRC patients without hepatic metastasis， including 3 up-regulated miRNAs （miR-224， miR-1236， and miR-622） and 3 down-regulated miRNAs （miR-155， miR-342-5p， and miR-363）， and the quantitative real-time PCR result of miR-224 consisted with the microarray finding. Conclusions miR-224 may be involved in the process of CRC with hepatic metastasis pathogenesis. miR-224 would be a research direction on a new biomarker or therapic method in CRC with hepatic metastasis.
ObjectiveTo analyze the expression profile changes of osteogenic-related genes during spontaneous calcification of rat bone marrow mesenchymal stem cells (BMSCs). MethodsBMSCs were isolated from 3-day-old healthy Sprague Dawley rats;cells at the 4th generation were used to establish the spontaneous calcification model in vitro. Spontaneous calcification process was recorded by inverted phase contrast microscope observation and alizarin red staining after 7 and 14 days of culture. For gene microarray analysis, cell samples were collected at 0, 7, and 14 days after culture; the differentially expressed genes were analyzed by bioinformatics methods and validated by real-time quantitative PCR (RT-qPCR) assay. ResultsRat BMSCs calcified spontaneously in vitro. When cultured for 7 days, the cells began to aggregate and were weakly positive for alizarin red staining. After 14 days of culture, obvious cellular aggregation and typical mineralized nodules were observed, the mineralized nodules were brightly positive for alizarin red staining. A total of 576 gene probe-sets expressed differentially during spontaneous calcification, corresponding 378 rat genes. Among them, 359 gene probe-sets expressed differentially between at 0 and 7 days, while only 13 gene probe-sets expressed differentially between at 7 and 14 days. The 378 differentially expressed genes were divided into 6 modes according to their expression profiles. Moreover, according to their biological functions, differentially expressed genes related to bone cell biology could be classified into 7 major groups:angiogenesis, apoptosis, bone-related genes, cell cycle, development, cell communication, and signal pathways related to osteogenic differentiation. In cell cycle group, 12 down-regulated genes were linked with each other functionally. Matrix metalloproteinase 13 (Mmp13), secreted phosphoprotein 1 (Spp1), Cxcl12, Mmp2, Mmp3, Apoe, and Itga7 had more functional connections with other genes. The results of genes Spp1, Mgp, Mmp13, Wnt inhibitory factor 1, Cxcl12, and cyclin A2 by RT-qPCR were consistent with that of gene microarray. ConclusionThe first 7 days after rat BMSCs were seeded are a key phase determining the fate of spontaneous calcification. Multiple genes related with cell communication, bone-related genes, cell cycle, transforming growth factor-β signaling pathway, mitogen-activated protein kinase signaling pathway, and Wnt signaling pathway are involved during spontaneous calcification.