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Next Generation Sequencing Facilitates Quantitative Analysis of Control and FoxM1-knockdown C2C12 Transcriptomes

ABSTRACT: Purpose:To identify FoxM1-dependent genes and the molecular pathways involved in the regulation of muscle stem cells function, we performed RNA-Sequence of C2C12 from shControl and shFoxM1 cells. Methods: C2C12 mRNA and lncRNA profiles of shControl and shFoxM1 cells (quiescent or activated states) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. Results:Using an optimized data analysis workflow, we mapped about 80 million sequence reads per sample to the mouse genome (build mm10) and identified 49,586 transcripts in the control C2C12 and FoxM1-knockdown C2C12 with BWA workflow. Conclusions: Our results represents the first detailed analysis of C2C12 transcriptomes and found that knockdown of FoxM1 perturbed multiple mRNA pathways, as well as for novel lncRNA pathways.

ORGANISM(S): Mus musculus

PROVIDER: GSE102685 | GEO | 2017/09/01

SECONDARY ACCESSION(S): PRJNA398379

REPOSITORIES: GEO

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Project description:Purpose: Small cell lung cancer (SCLC) continues to cause poor clinical outcomes due to limited advances in sustained treatments for rapid cancer cell proliferation and progression. The transcriptional factor Forkhead Box M1 (FOXM1) regulates cell proliferation, tumor initiation and progression in multiple cancer types. However, its biological function and clinical significance in SCLC remain to be established. Hence, the goals of this study are to decipher if FOXM1 is crucial to SCLC cells. Methods: NCI-H1688 stable cells, which contain shFOXM1 or shControl inducible expressing cassette based on pInducer20, plated as triplicates in 60-mm dishes for culturing overnight were induced by doxycycline for 48 hours. Total RNAs were collected by NucleoSpin RNA mini kit and sent for RNA-seq by Novogene. Bioanalyzer 2100 was used to analyze the RNA integrity and cDNA library was constructed with NEBNext UltraTM RNA Library Prep Kit for Illumina. Differential expression gene (DEG) analysis was performed using DESeq2 R package. DEGs were then subjected to ClusterProfiler R package for GO and KEGG enrichment analysis. Results: 1809 downregulated genes and 1781 upregulated genes were enriched in FOXM1-depleted NCI-H1688 cells. SCLC related KEGG pathway was found to be repressed, while multiple cell adhesion and junction associated GOs were induced in FOXM1-depleted NCI-H1688 cells. Furthermore, Foxm1 knockout inhibited SCLC formation in the Rb1fl/flTrp53fl/flMycLSL/LSL (RPM) mouse model associated with increased levels of neuroendocrine markers, Ascl1 and Cgrp, and decrease in Yap1. Consistently, FOXM1 depletion in NCI-H1688 SCLC cells reduced migration and enhanced apoptosis and sensitivity to cisplatin and etoposide. SCLC with high FOXM1 expression (N=30, 57.7%) was significantly correlated with advanced clinical stage, extrathoracic metastases and decrease in overall survival (OS), compared with the low-FOXM1 group (7.90 vs. 12.46 months). Moreover, the high-FOXM1 group showed shorter progression-free survival (PFS), compared with the low-FOXM1 group (3.90 vs. 8.69 months). Our collective findings support the utility of FOXM1 as a prognostic biomarker and potential molecular target for SCLC.

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Next Generation Sequencing Facilitates Quantitative Analysis of Control and FoxM1-knockdown C2C12 Transcriptomes

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