Next Generation RNA Sequencing Analysis of AMPK Wild Type, AMPKα-KO and AMPKα1-2A U2OS cell Transcriptomes
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ABSTRACT:
PROVIDER: PRJNA579161 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10336239_1.fastq.gz | Fastqsanger.gz | |||
| SRR10336239_2.fastq.gz | Fastqsanger.gz | |||
| SRR10336240_1.fastq.gz | Fastqsanger.gz | |||
| SRR10336240_2.fastq.gz | Fastqsanger.gz | |||
| SRR10336241_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:Background Worldwide, gastric cancer is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. Objective To determine oncogenic mechanisms and novel therapeutic targets specific for gastric cancer by identifying commonly dys-regulated genes from the tumors of both Asian-Pacific and Caucasian patients. Design We generated transcriptomic profiles of 22 Caucasian gastric cancer tumors and their matched non-cancerous samples, and performed an integrative analysis across different gastric cancer gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signaling pathways by RNAi and/or pharmacologic inhibition. Results We found that HNF4α upregulation was a key signaling event in gastric tumors from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumor cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signaling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest, and tumor growth inhibition. HNF4α also regulated WNT signaling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumors. Conclusions Our results indicate that HNF4α is a targetable oncoprotein in gastric cancer, is regulated by AMPK signaling through AMPKα, and resides upstream of WNT signaling. HNF4α may regulate “metabolic switch” characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signaling cascade represents a potentially targetable pathway for drug development. Integrative analysis of Caucasian and Asian-Pacific gastric tumor expression datasets (including newly generated transcriptomic profiling of 22 tumors in this study) revealed a relatively small common sets of highly overexpressed genes.
2014-11-15 | E-GEOD-63288 | biostudies-arrayexpress
Project description:BHLHE40 is a basic helix-loop-helix transcription factor that is involved in multiple cell activities including differentiation, cell cycle, and epithelial-to-mesenchymal transition. While there is growing evidence to support the functions of BHLHE40 in energy metabolism, little is known about the mechanism. In this study, we found that BHLHE40 expression was downregulated in cases of endometrial cancer of higher grade and advanced disease. Knockdown of BHLHE40 in endometrial cancer cells resulted in suppressed oxygen consumption and enhanced extracellular acidification. Suppressed pyruvate dehydrogenase (PDH) activity and enhanced lactated dehydrogenase (LDH) activity was observed in the knockdown cells. Knockdown of BHLHE40 also led to dephosphorylation of AMPKα Thr172 and enhanced phosphorylation of PDHA1 Ser293 and LDHA Tyr10. These results suggested that BHLHE40 modulates PDH and LDH activity by regulating the phosphorylation status of PDHA1 and LDHA. We found that BHLHE40 enhanced AMPKα phosphorylation by directly suppressing the transcription of an AMPKα-specific phosphatase, PPM1F. Our immunohistochemical study showed that the expression of BHLHE40, PPM1F, and phosphorylated AMPKα correlated with the prognosis of endometrial cancer patients. Because AMPK is a central regulator of energy metabolism in cancer cells, targeting the BHLHE40‒PPM1F‒AMPK axis may represent a strategy to control cancer development.
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Project description:Background Worldwide, gastric cancer is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. Objective To determine oncogenic mechanisms and novel therapeutic targets specific for gastric cancer by identifying commonly dys-regulated genes from the tumors of both Asian-Pacific and Caucasian patients. Design We generated transcriptomic profiles of 22 Caucasian gastric cancer tumors and their matched non-cancerous samples, and performed an integrative analysis across different gastric cancer gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signaling pathways by RNAi and/or pharmacologic inhibition. Results We found that HNF4α upregulation was a key signaling event in gastric tumors from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumor cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signaling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest, and tumor growth inhibition. HNF4α also regulated WNT signaling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumors. Conclusions Our results indicate that HNF4α is a targetable oncoprotein in gastric cancer, is regulated by AMPK signaling through AMPKα, and resides upstream of WNT signaling. HNF4α may regulate “metabolic switch” characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signaling cascade represents a potentially targetable pathway for drug development.
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