Project description:Background: Although TP53 gain-of-function (GOF) mutations promote cancer survival, its effect on EGFR-TKI efficacy remains unclear. We established EGFR-mutant lung cancer cell lines expressing various TP53 genotypes using CRISPR-Cas9 technology and found that TP53-GOF mutant cells develop an early resistance to EGFR-TKI osimertinib.The goal of this study is to elucidate the mechanisms underlying resistance to osimertinib treatment in TP53 GOF mutations through comprehensive gene analysis using RNA-seq with next-generation sequencing (NGS). Methods: Total RNA was isolated from PC-9 cells overexpressing TP53 R248Q mutation (PC9/p53R248Q: TP53 GOF mutation) and PC-9 cells overexpressing empty vector plasmid (PC9/p53EV: TP53 null) treated with DMSO or osimertinib for 24hours, using the RNeasy Micro Kit , in accordance with the manufacturer’s instructions. RNA samples were quantified by NanoDrop-2000 spectrophotometer, and the quality was confirmed with a 2200 TapeStation. rRNA was removed using MGI Easy rRNA Depletion Kit according to manufacturer's instructions followed by library construction using MGIEasy RNA Directional Library Prep Set (MGI). MGI DNBseq-G400 FAST was used to perform the amplicons deep sequencing following the standard operation protocol. The sequence format was 150bp pair read for all samples. All sequencing reads were trimmed low-quality bases and adapters with Trimmomatic (v.0.38) , and RNA sequencing reads were mapped to hg38 using HISAT2 software . Raw counts for each gene were estimated in each sample using RSEM version 1.3.0 and Bowtie 2. Calculation of the log fold-change (log FC) and p-value were performed using edgeR. Results: We explored the functions of specifically upregulated genes in TP53 GOF mutation after osimertinib treatment by KEGG pathway-enrichment analysis and found that the cytokine-cytokine receptor interaction was the most significantly altered pathway. Hallmark pathway analysis identified the TNF-α/NF-κB pathway was significantly enriched. Furthermore, TRRUST analysis showed enhanced activity of transcription factors especially RELA (p65) and NF-kB1. Conclusions: TP53 GOF mutaion induces osimertinib resistance by activating TNF-α/NF-κB pathway.

Project description:Objective:mRNA sequencing of sorted naive CD4+ T cells from the spleens of PIK3CD GOF mice and WT littermate mice. Methods:Naive CD4+ T cells were negatively isolated ex vivo(Stemcell Technologies).Total RNA from isolated naive CD4+ T cells was extracted using RNeasy Plus Mini Kit (Qiagen, GmBH, Germany) according to the manufacture’s instruction and checked for RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, US). Qualified total RNA was further purified by RNeasy micro kit (QIAGEN) and RNase-Free DNase Set (QIAGEN). Paired-end libraries were constructed using the TruSeq® RNA Sample Preparation Kit (Illumina, USA) according to the manufacturer’s instructions. The products are purified, enriched, quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer to confirm insert size and calculate the mole concentration. Cluster was generated using cBot with the library diluted to 10 pM and then were sequenced on the Illumina HiSeq 2500 (Illumina, USA). The library construction and sequencing was performed at Shanghai Biotechnology Corporation (Shanghai, China). Results:Compared to the expression profiles for the WT cells, those for the mutant TNaive showed 2,307 genes with differential expression, of which 1,749 were upregulated and 558 were downregulated. Gene ontology analysis showed that PIK3CD GOF TNaive upregulated the expression of genes encoding molecules involved in the cell cycle and mitosis, including E2f1, E2f2, cyclin A2, cyclin B2, Cdk1, Hells and Nuf2, all of which might synergistically promote entry into the cycling phase. In addition, the genes encoding T cell activation, cytokines and cytokines receptors, chemokines and chemokine receptors, transcription factors, and metabolic regulators were differentially regulated in PIK3CD GOF mice compared to WT mice. Kyoto Encyclopedia of Genes and Genomes analysis showed that the altered genes in TNaive from PIK3CD GOF mice displayed significant enrichment of several sets associated with infection, inflammation and autoimmune disease Conlusion:PIK3CD GOF results in a loss of quiescence-associated gene expression patterns in naive T cells by collectively coupling the cell cycle, nutrient metabolism, cell trafficking and signal transduction.

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Hoxb1 GoF RNA-seq

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