Computational protocol: Elevated free fatty acid uptake via CD36 promotes epithelial-mesenchymal transition in hepatocellular carcinoma

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Protocol publication

[…] De-identified, IRB-approved frozen samples were obtained from the “International Registry of Patients with or at Risk for Hepatobiliary Cancers, Including Hepatocellular Carcinoma, Cholangiocarcinoma, and Gallbladder Adenocarcinoma, and those Patients with Normal Risk Factors”. This Human Subjects Research falls under Exemption 4 because the research involves the study of existing data, records, and pathological or diagnostic specimens of de-identified IRB-exempt samples from Dr. Lewis Roberts. The research was approved by the Mayo Clinic Institutional Review Board (IRB) under protocol 14-005015 with waiver of the requirement to obtain informed consent and was carried out in accordance with the approved guidelines. All experimental protocols were approved by the Mayo Clinic IRB. Patient clinical information, including body mass index, was provided by Mayo Clinic. Total cellular proteins were extracted from the tumor samples using T-PER protein extraction reagent (Pierce, 78510) supplemented with protease inhibitor cocktail tablets (Roche, 04693159001) according to the manufacturer’s protocol.Liver cancer gene expression data (mRNA, normalized RNAseqV2 RSEM) were retrieved from the TCGA database using the cBioPortal for cancer genomics. Expression levels were log2 transformed and EMT scores were calculated using a previously published metric. EMT score = Sum of mesenchymal gene expression (CDH2, FN1, SNAI1, SNAI2, TWIST1, TWIST2, VIM, ZEB1, ZEB2) – Sum of epithelial gene expression (CDH1, CLDN4, CLDN7, MUC1, TJP3). Hierarchical clusters and heatmap visualizations were generated using the GENE-E matrix analysis platform (http://www.broadinstitute.org/cancer/software/GENE-E/index.html) with TCGA gene expression data (mRNA, RNAseq z-scores) retrieved using the cBioPortal. Clusters were obtained using the average linkage method with 1-Pearson’s correlation coefficient as the distance metric. Gene set enrichment analyses were performed using GSEA module available from the GenePattern genomics server (http://genepattern.broadinstitute.org/). [...] Total mRNA for qRT-PCR and PCR array studies was extracted from cells using RNeasy Mini Plus kit (Qiagen, 74134) according to the manufacturer’s protocol. cDNA was synthesized from 1μg of total mRNA using High-capacity cDNA reverse transcript kit (Applied Biosystems, 4368814) following the manufacturer’s protocol. qRT-PCR experiments were performed in a MyiQ thermal cycler (BioRad) with Sybr-Green mastermix (BioRad, 170–8882) and 200 ng of template/reaction using primers (Operon) designed using the Primer3/NCBI Primer-BLAST tool ().The following thermal cycling settings were used: 95 °C for 10 min. followed by 40 cycles at 95 °C for 30 sec., 62 °C for 1 min. and 72 °C for 1 min. The fold-change values were calculated as delta-delta Ct (ddCt) values from a minimum of three independent biological replicates.The PCR array study was performed with an 84-gene EMT pathway-specific array (SABiosciences, PAHS-090Z) according to the manufacturer’s protocol. The top 20 genes were ranked by fold change magnitude and analyzed for KEGG pathway enrichment using the DAVID bioinformatics tool. […]

Pipeline specifications

Software tools Primer3, Primer-BLAST, DAVID
Databases KEGG PATHWAY
Application qPCR
Organisms Homo sapiens
Diseases Carcinoma, Hepatocellular, Liver Neoplasms, Neoplasms