SIRT1 expression effect on migration and invasion of gastric cancer cells
ABSTRACT: Analysis of BGC-823 gastric cancer cells with SIRT1 overexpression or knockdown. SIRT1, a NAD+-dependent protein deacetylase, exerts inhibitory effects on migration and invasion of gastric cancer. Results provide insight into the role of SIRT1 in the metastasis of gastric cancer. Overall design: Four groups: Group 1, Stable lentivirus infected BGC-823 cells with the lentiviral expressing vector. Group 2, Stable lentivirus infected BGC-823 cells with the lentiviral expressing vector containing SIRT1 cDNA sequence. Group 3, Stable lentivirus infected BGC-823 cells with the lentiviral vector containing interference control sequence. Group 4, Stable lentivirus infected BGC-823 cells with the lentiviral vector containing interference sequence targeting SIRT1.
INSTRUMENT(S): Agilent-026652 Whole Human Genome Microarray 4x44K v2 (Probe Name version)
Project description:Cisplatin is the first-line agent utilized for the clinical treatment of a wide variety of solid tumors including gastric cancer. However, the intrinsic or acquired cisplatin resistance is often occurred in patients with gastric cancer and resulted in failure of cisplatin therapy. In order to investigate if miRNA involves in cisplatin resistance of human gastric cancer, we first screened and compared the expression of miRNAs between cisplatin resistant gastric cancer cell lines SGC-7901/DDP and BGC-823/DDP and their sensitive parental cells by miRNAs microarray. Overall design: Total miRNAs expression was examined in SGC-7901,SGC7901/DDP,BGC-823,BGC-823/DDP four cell lines.
Project description:Metastasis associated 1 family, member 2 (MTA2) gene is classified to metastasis associated gene family. We have previously reported that MTA2 gene was overexpressed in gastric cancer tissues, correlating with tumor invasion, lymph node metastasis, and advanced TNM stage. MTA2 knockdown significantly inhibited gastric cancer cell invasion and metastasis. Yet, its molecular mechanisms are still unclear. The aim of this study is to investigate the molecular mechanisms of MTA2 in regulating malignant behaviors of gastric cancer. This experiment captures the expression data between BGC-823/NC and BGC-823/MTA2, SGC-7901/NC and SGC-7901/shMTA2 cells using Whole human genome microarray 4×44K (Design ID: 014850, Agilent technologies).
Project description:We did the microarray to further compare the changes of gene expression between gastric cancer stem cells with CD44 knockdown by lentivirus and gastric cancer stem cells by scamble short hairpin RNA. Gastric cancer stem cells (Lentivirus) were infected with lentivirus that expressed human CD44-speciﬁc short hairpin RNA (shRNA). Control group of gastric cancer stem cells (Vector group) were only infected with scramble shRNA.
Project description:We did the microarray to further compare the changes of gene expression between gastric cancer stem cells with CD44 knockdown by lentivirus and gastric cancer stem cells by scamble short hairpin RNA. Overall design: Gastric cancer stem cells (Lentivirus) were infected with lentivirus that expressed human CD44-speciﬁc short hairpin RNA (shRNA). Control group of gastric cancer stem cells (Vector group) were only infected with scramble shRNA.
Project description:To elucidate whether GAS1 plays a role in gastric cancer tumorigenesis, a RNA-seq analysis was performed to compare the gene expression profiles of GAS1 shRNA and control shRNA transfectants. Overall design: RNA-seq was performed in BGC 823 gastric cancer cells after GAS1 shRNA and control shRNA virus infection
Project description:Pak1 as a serine/threonine kinase, has been implicated in cytoskeletal remodelling, cell motility, apoptosis and transformation. Pak1 plays important roles in multiple signal pathways. Pak1 protects cells from apoptosis through at least three different pathways including forkhead box O1 (FOXO1), B-cell CLL/lymphoma 2 (Bcl-2) and DLC1. Pak1 also regulates activity of Raf and Aurora kinases to affect cellular proliferation. Overexpression of Pak1 is involved in the regulation of actin assembly and disassembly through phosphorylations of LIM Kinase and cytoskeletal associated proteins such as Filamin A, Paxillin, Caldesmon, Cortactin and Arp2/3. Pak1 also regulates microtubule dynamics via activation of tubulin cofactor B (TCoB) and DLC1, and inhibition of stathmin. In spite of a large body of work about the mechanism of Pak1 action in cancer, it remains unknown whether Pak1 signaling could potentially regulate the biology of regulatory miRNAs. This is particularly relevant for gastric cancer because Pak1 can activate many regulators of miRNAs expression in gastric cancer cells including NF-kappaB and ERK, and Pak1 signaling has profound phenotypic effects on the biology of gastric cancer cells. We constructed Pak1 knockdown stable cell lines. The stable Pak1 knockdown gastric cancer BGC823 cells and control cells were performed miRNA chip analysis by CapitalBio company. Gastric cancer BGC823 cells with stable Pak1 knockdown and BGC-823 gastric cancer cells transfected with U6 were used in this experiment. Total RNA was extracted by trizol,Here we use a Capitalbio mammal microRNA V3.0(CapitalBio, Beijing, China) containing 509 well-characterized human, mouse and rat miRNAs and various controls to profile the expression levels of miRNA in 16 and conU6 group.three chip were test in each group, and the procedure was repeated twice.
Project description:Transcriptional profiling of RIP products of human gastric cancer cells SGC7901-NM comparing control with SGC7901-NM infected with has-miR-625 lentivirus Stable transfected cell lines, SGC7901-NM-has-miR-625 vs. SGC7901-NM-NC, after RNA-binding protein immunoprecipitation with Ago2 antibody, the experimental group (Ago2) vs. the control group (input) per array.
Project description:P21-activated kinase 1 (Pak1) is a key oncogenic kinase and a lot of work about the mechanism of Pak1 action in cancer have been reported, while it remains unknown whether Pak1 could potentially regulate the biology of regulatory miRNAs by new interacting substrate. Here, we identified that Pak1 modulated the miR-132 expression in gastric cancer cells. Pak1 interacted with and phosphorylated activating transcription factor-2 (ATF2) on Serine 62 (Ser62), which blocked ATF2 translocation into cell nucleus. We further demonstrated that ATF2 induced miR-132 transcription via binding to the miR-132 promoter in the -30 to -39 region. Moreover, overexpression of miR-132 in gastric cancer cells significantly reduced cell adhesion, migration and invasion in vitro and hematogenous metastasis in vivo. MiR-132 targeted CD44 and fibronectin (FN) and promoted lymphocytes to gather around gastric cancer cells and kill them. More importantly, downregulation of miR-132 in gastric cancer was specifically associated with hematogenous metastasis, instead of lymph node or implantation metastasis. Taken together, miR-132 is a key negative regulator in the hematogenous metastasis of gastric cancer. A novel cell signaling pathway Pak1-ATF2-miR-132-CD44/FN is established and may be a new therapeutic target for hematogenous metastasis of gastric caner. Overall design: Gastric cancer BGC823 cells with stable Pak1 knockdown and BGC-823 gastric cancer cells transfected with U6 were used in this experiment. Total RNA was extracted by trizol,Here we use a Capitalbio mammal microRNA V3.0(CapitalBio, Beijing, China) containing 509 well-characterized human, mouse and rat miRNAs and various controls to profile the expression levels of miRNA in 16 and conU6 group.three chip were test in each group, and the procedure was repeated twice.
Project description:To explore the mechanism of Kindlin-2 regulating invasion and metastasis of human Hepatocellular carcinoma, we performed gene expression microarray analysis on Kindlin-2 knockdown LM3 cells and the control cells to compare the gene expression levels between the two groups. Overall design: Short hairpin RNAs (shRNA) targeting Kindlin-2 and negative control (NC) were synthesized and cloned into the lentiviral vector. Lentivirus expressing shRNA was generated and used to infect LM3 cells. After 72h of infection, the infected cell lines were cultured in the medium containing 2ug/ml puromycin to select Kindlin-2 stable knockdown cell line.
Project description:Aberrant DNA hypermethylation of CpG island (CGI) promoters are associated with transcriptional repression of many tumor suppressor genes and lead to tumor progression in many cancers. Most recently, one research group observed that aberrantly hypermethylated genes in multiple cancers are already repressed, but their promoters are maintained in a hypomethylated state in pre-cancerous tissues.Their studies didn't provide a clue to explain by what mechanisms those genes were repressed in pre-cancerous tissues. Another research group found that many genes with de novo promoter hypermethylation in colon cancer were among the subset of genes "bivalently" marked in embryonic stem cells and adult stem/progenitor cells by repressive Polycomb group proteins (PcG), which are known for maintaining low, but poised, transcription.These observations provide a clue that CGI promoter hypermethylation in cancers is associated with PcG target genes in pre-cancerous tissues.we took advantage of ChIP-BS-seq technology and applied it to examine H3K27me3 and H3K4me3 profiles for one normal lymphoblastoid cell line (YH) and three cancer cell lines including one cervical cancer cell line (Hela) and two gastric cancer (GC) cell lines (BGC-823 and AGS). We aplied ChIP-BS technology to examine H3K27me3 marks, which are catalyzed by the SET domain histone methyltransferase EZH2 and have a repressive function with 50bp pair-end sequencing. found H3K27me3 marks were enriched preferentially at CpG islands, (+/-500) transcription start sites (TSSs) and exons in two GC cell lines (BGC-823 and AGS). In YH cells, H3K27me3 marks were only preferentially enriched at CpG islands. In contrast, Hela cells presented a reverse pattern with highest H3K27me3 enrichment in intergenic regions. To confirm this result in Hela cells, we performed two independent replicates of ChIP-Seq and ChIP-BS-seq. Cause of useful was relative small. we still sequenced one 100bp pe reads replicate for H3K4me3 and two replicate for H3K27me3 ChIP-BS-seq.