Dataset Information

Glutaminase (GLS) was a target gene downstream of lithocholic acid inhibiting gallbladder cancer

ABSTRACT: Purpose:To further explore the molecular mechanisms of LCA treatment underlying its tumor suppressive functions. Methods:For the NOZ cells cultures, there were two different conditions performed in biological triplicates: (I) mock (DMSO), (II) 150 μM LCA for 24 h. A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Paired t-test was used to normalize and statistically analyze the data. The threshold we set to screen for upregulated and downregulated genes with a fold change >0 and a padj-value < 0.05. Hierarchical clustering was performed and a heat map was constructed with edgeR R package (3.18.1) to visualize differential mRNA expression. Gene set enrichment analysis was conducted with the local version of the GSEA analysis tool http://www.broadinstitute.org/gsea/index.jsp. Results: By means of RNA-seq and subsequent data analysis with volcano plot and hierarchical clustering, we found that LCA treatment led to a different gene expression profile with the upregulation of 3607 genes and the downregulation of 3501genes compared with those in cells treated with mock. Gene Set Enrichment Analysis (GSEA) suggested that alanine, aspartate, and glutamate metabolism pathways were top-listed in the inhibitory regulation of LCA, and the heatmap plots showed that the pathways involved a set of 35genes. In addition, qRT-PCR was used to confirm the expression of several metabolism genes including GLS, ASS1, CPS1, ASNS, ABAT, and ADSSL. We were particularly interested in the commonly downregulated GLS in both NOZ and EH-GB1 cells, which mediates glutamate and glutamine metabolism. As the result, protein levels of GLS were dramatically inhibited by LCA in NOZ and EH-GB1 cells in a time-dependent manner as compared with the untreated cancer cells, Conclusion: GLS was a downstream effector of LCA treatment in gallbladder cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE192577 | GEO | 2023/12/31

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Purpose: To understand the biological function of citratemt accumulation, we performed RNA-seq analysis. Methods:MLE12 cells were transfected simultaneously with shIdh3α lentivirus (MOI: 50) and shSlc25a1 lentivirus (MOI: 50) and negative control (shCtrl lentivirus, MOI: 50) in the presence of 1HitransG P (Lot: REVG005, GeneChem).Empty construct was used as control for all experiments. The cells were washed with fresh complete medium 16 h later and then cultivated for an additional 80 h.Total RNA was extracted from control and Slc25a1 plus Idh3α-deficient MLE12 cells. The transcriptome library for sequencing was generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® following the manufacturer’s recommendations. The index codes were added to attribute sequences to each sample. After the quality of sequencing libraries was confirmed on the Agilent 2100 system, clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, San Diego, CA, USA) according to the manufacturer’s instructions. The library preparations were then sequenced on an Illumina Hiseq 2500 platform (Illumina, San Diego, CA, USA), and paired-end reads were generated. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity with min_kmer_cov set to 2 by default and all other parameters set to default. Results: Using an optimized data analysis workflow,We found that 215 genes were upregulated in MLE12 cells with citratemt accumulation . To determine the functional properties of the top 30 genes upregulated by citratemt, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. KEGG pathway analysis indicated that the 30 genes were involved in regulating the TNF signaling pathway , one of the critical signaling pathways for triggering necroptosis Conclusions: These results further suggest that citratemt accumulation specifically drives necroptosis

Dataset Information

Glutaminase (GLS) was a target gene downstream of lithocholic acid inhibiting gallbladder cancer

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure