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Next Generation RNA Sequencing Analysis of AMPK Wild Type, AMPKα-KO and AMPKα1-2A U2OS cell Transcriptomes

ABSTRACT: Purpose: The goals of this study are to compare NGS-derived U2OS transcriptome profiling (RNA-seq) To get insight into the downstream signaling of AMPK and to evaluate protocols for optimal high-throughput data analysis Methods: RNA was extracted by using the Direct-zol™ RNA Miniprep Plus Kit (Zymo Research) and evaluated the purity and concentration of the RNA by ultraviolet spectroscopy (NanoDrop). RNA integrity numbers (RIN) were evaluated using the Agilent 2100 Bioanalyzer. RNA sequencing libraries were constructed using 1000 ng of total RNA from each sample and the TruSeqV2 kit from Illumina following manufacturer’s protocol. Illumina NextSeq sequencing and NGS data acquisition were conducted at the UNMC Genomics Core Facility. The libraries were subjected to 75 bp paired-end high-output sequencing using a NextSeq500 sequencer to generate approximately 33.3 to 41.6 million reads per sample. Fastq files were generated using the bc12fastq software, version 1.8.4 and provided to the UNMC Bioinformatics Core facility for further analysis. The original fastq format reads were trimmed and filtered using the fqtirm tool (https://ccb.jhu.edu/software/fqtrim) to remove adapters, terminal unknown bases (Ns) and low quality 3’ regions (Phred score < 30). The trimmed fastq files were processed by our facility’s newly developed standard pipeline utilizing STAR as the aligner and RSEM as the tool for annotation and quantification at both gene and isoform levels. TPM values were used for comparison results (student’s t-test) for all the available genes. The Benjamini-Hochberg (BH) adjusted p values were also provided to adjust for multiple testing-caused false discovery rate (FDR) with significant level of adjusted p value of ≤ 0.05. Comparative analysis of parental U2OS (control) vs. AMPKα-KO and control vs. AMPKα1-2A was performed using Ingenuity Pathway Analysis (IPA) to examine canonical pathways similarly altered in each to designate effects due to dysregulation of CDK1 phosphosites. Results: Comparative analysis of parental U2OS (control) vs. AMPKα-KO and control vs. AMPKα1-2A was performed using Ingenuity Pathway Analysis (IPA) to examine canonical pathways similarly altered in each to designate effects due to dysregulation of CDK1 phosphosites. Canonical pathways influencing actin dynamics such as actin cytoskeleton signaling, ILK signaling, and regulation of actin-based motility by Rho were predicted to be significantly activated. The expression of a multitude of genes involved in promoting cellular movement and migration were increased and several inhibitors of cell movement were diminished in AMPKα-KO and α1-2A cells compared to controls. By examining alterations of downstream gene expression, upstream analysis pinpointed numerous possible upstream regulators. Most of the highestscoring upstream effectors were analogously modulated between AMPKα-KO and α1-2A compared to controls, indicating that loss of phosphorylation of AMPK by CDK1 is comparable to AMPK knockout for alterations seen in these particular pathways. We previously detected stark reduction of p-Histone H3 (S10) in both the AMPKα-KO and α1-2A cells, which led to speculate that this was possibly due to either phosphatase dysregulation leading to hyperdephosphorylation, or perturbation of kinase signaling leading to inadequate phosphorylation of Histone H3. Interestingly, there were eight significantly up- or down-regulated phosphatases found to be changed mutually between the AMPKα-KO and α1-2A cells compared to controls. Furthermore, between the two treatments, the expression levels of ten kinases and two mitosis-associated kinases significantly changed compared to controls. Conclusions: Our study represents the first detailed analysis of AMPK Wild Type, AMPKα-KO and AMPKα1-2A U2OS cell Transcriptomes, with biologic replicates, generated by RNA-seq technology. Our results show that that CDK1 regulates AMPK control of mitotic entry and progression. It remains to be determined which signaling networks or processes CDK1-phosphorylated AMPK utilizes to drive these events.

ORGANISM(S): Homo sapiens

PROVIDER: GSE139293 | GEO | 2019/10/24

REPOSITORIES: GEO

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Next Generation RNA Sequencing Analysis of AMPK Wild Type, AMPKα-KO and AMPKα1-2A U2OS cell Transcriptomes

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