ObjectiveTo investigate the pathogenesis of atherosclerosis (AS) by detecting different expression genes between normal Wistar rats and rats with atherosclerosis through the technology of gene chip. MethodsThe rats were treated with standard diet with saline injection (control group) or high-cholesterol diet with vitamin D3 injection and balloon injury (model group). Total cholesterol (TC) and triglyeride (TG) in serum were detected 90 days later to ensure the success of establishment of the atherosclerosis model. Abdominal aorta was isolated and stained with HE. Total RNA was isolated from the aorta for gene chip analysis to explore the differential gene expression. ResultsCompared with the control group, the TC and TG level in the model group were highly advanced (P<0.05). AS model was confirmed by pathological observation. Gene chip detection showed that 511 genes were up-regulated and 1 219 ones were down-regulated which were interrelated with lipid metabolism, inflammatory reaction, oxidative stress and apoptosis as well. ConclusionThe expression change with multiple gene in AS suggests that the nosogenesis of AS is adjusted and controlled complicatedly. Intensive study of some important genes will contribute to the prevention and improvement of prognosis of AS.
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.
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 investigate the microRNA (miRNA) expression profile during chondrogenic differentiation of human adipose-derived stem cells (hADSCs), and assess the roles of involved miRNAs during chondrogenesis. MethodshADSCs were harvested and cultured from donors who underwent elective liposuction or other abdominal surgery. When the cells were passaged to P3, chondrogenic induction medium was used for chondrogenic differentiation. The morphology of the cells was observed by inverted phase contrast microscopy. Alcian blue staining was carried out at 21 days after induction to access the chondrogenic status. The expressions of chondrogenic proteins were detected by ELISA at 0, 7, 14, and 21 days. The miRNA expression profiles at pre- and post-chondrogenic induction were obtained by microarray assay, and differentially expressed miRNAs were verified by real-time quantitative PCR (qRT-PCR). The targets of the miRNAs were predicted by online software programs. ResultshADSCs were cultured successfully and induced with chondrogenic medium. At 21 days after chondrogenic induction, the cells were stained positively for alcian blue staining. At 7, 14, and 21 days after chondrogenic induction, the levels of collogen type Ⅱ, Col2a1, aggrecan, Col10a1, and chondroitin sulfate in induced hADSCs were significantly higher than those in noninduced hADSCs (P<0.05). Eleven differentially expressed miRNAs were found, including seven up-regulated and four down-regulated. Predicted target genes of the differentially expressed miRNAs were based on the overlap from three public prediction algorithms, with the known functions of regulating chondrogenic differentiation of stem cells, selfrenewal, signal transduction, intracellular signaling cascade, and cell cycle control. ConclusionA group of miRNAs and their target genes are identified, which may play important roles in regulating chondrogenic differentiation of hADSCs. These results will facilitate the initial understanding of the molecular mechanism of chondrogenic differentiation in hADSCs and subsequently control hADSCs differentiation, and provide high performance seed cells for cartilage tissue engineering.
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 determine the effect of insulin-like growth factor-1 (IGF-1) on angiogenesis in mouse breast cancer model of lower and normal serum IGF-1 levels after using angiogenesis inhibitor ginsenoside Rg3 （GS Rg3）. Methods The breast cancer models were established in control mice and liver specific IGF-1 deficient (LID) mice by feeding DMBA and were treated with GS Rg3. Vascular endothelial growth factor (VEGF) and F8-RAg were detected by immunohistochemical method in breast cancer tissues. IGF-1 gene and angiogenesis relating genes were detected by gene chip in breast cancer and normal breast tissue. Results The incidence rate of breast cancer in LID mice was lower than that in control mice (P＜0.05). VEGF expression and microvessel density of LID mice were lower than those in control mice (P＜0.05). Compared to the control mice, IGF-1, FGF-1, TGF-β1 and HGF genes were increased, and FGFR-2, PDGF-A and PDGF-B genes were decreased in breast cancer of LID mice. After GS Rg3 treatment, VEGFa, EGF, EGFR, PDGF-A and FGFR-2 genes were increased, IGF-1 and TGF-β1 genes were decreased in breast cancer of LID mice compared with the control mice. Conclusion IGF-1 may be involved in mouse breast cancer progression and associated with the growth of blood vessels. Angiogenesis inhibitor may play an antitumor role by IGF-1 and TGF-β1.
Objective To investigate the change of immunologic gene expression in cases of colorectal cancer with liver metastasis. Methods The total RNAs were extracted from tumor tissues of original lesions in 16 patients with colorectal cancer， DNA microarray was used to examine the change of immunologic gene expression in colorectal cancer patients with or without liver metastasis. Results Compared with samples without liver metastases， the expressions of 11 immunologic genes obviously down-regulated in the tumor tissues of colorectal cancer patients with liver metastasis， including：carboxypeptidase D；Fc fragment of IgE， high affinityⅠreceptor for gamma polypeptide；Fc fragment of IgG， low affinityⅢa receptor （CD16a）；free fatty acid receptor 2；interleukin 2 receptor gamma；protein tyrosine phosphatase receptor type C；complement factor B；major histocompatibility complex， classⅡ， DM alpha；major histocompatibility complex， classⅡ， DM beta；major histocompatibility complex， classⅡ， DQ alpha 1；granzyme B. The functions involved the growth and activation of immunologic cell， signal transduction， cell apoptotic， cell factors， receptors， complement， apoptotic， and immunogenicity of tumor cell. Conclusions Down-regulation of a various of immunologic gene expression in colorectal cancer patients with liver metastasis inhibits the function of immunology， and tumor cells escaped the destruction of immunology system results in metastasis.
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.