RNA-seq analysis of A498 cell line treated with siSETD2 or si-NC
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE133521 | GEO | 2019/07/31
REPOSITORIES: GEO
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Project description:Finding the downstream miRNAs of SETD2 in the renal cell carcinoma cell line the coordinated expression of SETD2-miRNAs-MAPK/JNK may be predictive of poor prognostic in patients with RCC. Our findings also emphasize the therapeutic potential of MAP4K4 in RCC therapy and support the development of an effective therapeutic strategy to target MAP4K4 by molecularly targeted approaches
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Project description:Set domain-containing 2 (SETD2) is the most frequently mutated gene among all the histone methyltransferases (HMTs) in Clear cell renal cell carcinoma (ccRCC). Loss of function of SETD2 is significantly associated with poor prognosis in patients with ccRCC. A better understanding of the roles of SETD2 played in ccRCC can greatly improve the prognosis and quality of life of patients with kidney cancer. Clear cell renal carcinoma cell A498 were treated with si-SETD2 and si-NC, and the exosomes were extracted.
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Project description:Immune checkpoint blockade (ICB) demonstrates durable clinical benefit only in a minority of renal cell carcinoma (RCC) patients. Identifying molecular features that determine response and developing approaches to enhance the response remain an urgent clinical need. Here we found that, in multiple RCC cell lines, targeting the ATR-CHK1 axis with pharmacological inhibitors increased cytosolic DNA accumulation, activated the cGAS-IRF3-dependent cytosolic DNA sensing pathway, and resulted in the inflammatory cytokine expression. SETD2 mutated RCC cell lines or tumor samples were associated with preferential ATR-CHK1 activation over ATM-CHK2 activation. SETD2 knockdown promoted the cytosolic DNA sensing pathway and conferred greater sensitivity in response to ATR-CHK1 inhibition. In murine Renca tumors, Setd2 knockdown and ATR inhibitor VE822 synergistically promoted cytosolic DNA sensing pathway, immune cell infiltration, and immune checkpoint protein expression. Setd2 deficient Renca tumors demonstrated greater vulnerability to ICB monotherapy or in combination with VE822 than Setd2 proficient tumors. SETD2 mutations were associated with a higher response rate and prolonged overall survival in ICB-treated RCC patients, but not in non-ICB-treated RCC patients. This study provides a mechanism-based guidance to develop more personalized combination therapy regimens for RCC patients with SETD2 mutations.
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Project description:Purpose: SETD2 deficiency, a driving force of depleted levels of H3K36me3 due to impaired methyltransferase activity, plays an important role in diverse forms of cancer, most notably in aggressive and metastatic clear cell renal cell carcinomas (ccRCC). Developing novel treatment schema targeting SETD2-compromised cancer cells are urgently needed. Methods: Human kidney cancer cell lines with or without SETD2 deficiency were treated with a DNA Hypomethylating Agent (HMA), a poly(ADP-ribose) polymerase inhibitor (PARPi), and both (HMA + PARPi) in combination in both in vitro and in vivo settings. Cell inhibition, Comet, flow cytometry, western blot, and RNA-sequencing (RNA-seq) gene expression assays as well as bioinformatic analyses were performed to explore the underlying mechanism action. Results: By taking advantage of involvement of SETD2 in DNA methylation and DNA repair, we found the combination treatment of an HMA (5-Aza-CdR/DAC) and a PARPi (Talazoparib/BMN-673) generated increased cytotoxicity in in vitro SETD2-deficient RCC cell lines than in SETD2-proficient cell lines. Furthermore, our results also demonstrate synergetic anti-tumor effects in SETD2 deficiency RCC cell lines when treated with DAC and BMN-673 that include apoptotic induction, increased DNA damage, insufficient DNA damage repair, upregulation of immune responses by STING and transposable elements (TEs), as well as increased genomic instability. Finally, the combination treatment for SETD2-deficient RCCs was also validated using in vivo mouse models. Conclusion: SETD2 deficiency creates a vulnerable epigenetic status to generate efficacious anti-tumor effects via the combination treatment of DAC and BMN-673 in human and mouse RCC systems, making it a precise medicine-based approach for SETD2-compromised cancers.
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Project description:Purpose: to find the potential down-stream target gene of circFAM120A Methods: mRNA profiles of decidualized human endometrial stromal cells (hESCs) after down-regulated FAM120A and control using siRNA were generated by NGS and compared by bioinformatics analysis Results: we sequenced 26414 mRNAs in hESCs between the si-NC and si-circFAM120A groups and identified 242 differentially expressed mRNAs, between which 161 were downregulated and 81 were up-regulated in si-circFAM120A group Conclusions: Our study represents the detailed analysis of decidualized hESCs transcriptomes between si-NC and si-circFAM120A groups.
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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the difference of mRNA between si-nc and si-SRSF11 in LOVO cell line Methods: LOVO mRNA profiles of si-nc and si-SRSF11 were generated by deep sequencing, in triplicate, using Illumina Novaseq . The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Sample collection and preparation RNA quantification and qualification 1RNA degradation and contamination was monitored on 1% agarose gels. 2RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA) . 3RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Library preparation for Transcriptome sequencing 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. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing (Genchem Experimental Department) The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) 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. Data Analysis Quality control Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Reads mapping to the reference genome Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools. Novel transcripts prediction The mapped reads of each sample were assembled by StringTie (v1.3.3b) (Mihaela Pertea.et al. 2015) in a reference-based approach. StringTie uses a novel network flow algorithm as well as an optional de novo assembly step to assemble and quantitate fulllength transcripts representing multiple splice variants for each gene locus. Quantification of gene expression level featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels. Differential expression analysis (For DESeq2 with biological replicates) Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed. (For edgeR without biological replicates) Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the edgeR R package (3.18.1). The P values were adjusted using the Benjamini & Hochberg method. Corrected P-value of 0.05 and absolute foldchange of 2 were set as the threshold for significantly differential expression. GO and KEGG enrichment analysis of differentially expressed genes Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias wascorrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways. SNP analysis GATK2 (v3.7) software was used to perform SNP calling. Raw vcf files were filtered with GATK standard filter method and other parameters (cluster:3; WindowSize:35; QD < 2.0 o; FS > 30.0; DP < 10 and SnpEff software was used to annotation for the Variablesite. AS analysis Alternative Splicing is an important mechanism for regulate the expression of genes and the variable of protein. rMATS(3.2.5) software was used to analysis the AS event. PPI analysis of differentially expressed genes PPI analysis of differentially expressed genes was based on the STRING database, which known and predicted Protein-Protein Interactions. Weighted correlation network analysis WGCNA (Weighted correlation network analysis) is a systematic biological method used to describe the gene association modes among different samples. it can be used to identify gene sets that are highly synergistic changed, and identify candidate biomarkers or therapeutic targets based on the coherence of gene sets and the correlation between gene sets and phenotypes. The R package WGCNA is a set of functions used to calculate various weighted association analysis, which can be used for network construction, gene screening, gene cluster identification, topological feature calculation, data simulation and visualization. WGCNA is suitable for multi- sample data. Generally, more than 15 samples are required. One input file is sample information, that is, a matrix describing the traits of the sample: the traits used for association analysis must be numeric; If it is a regional or categorical variable, it needs to be converted to a 0-1 matrix. The other is gene expression data. For transcriptome sequencing ,FPKM can be used as gene expression data.
2022-12-01 | GSE199094 | GEO
Project description:RNA-seq was used to analyze the differential expression of mRNAs between si-THAP7-AS1 and si-NC468 cells.
2021-10-31 | GSE160685 | GEO
Project description:To detect the gene profiles in si-NC and si-PR-SET7 KD hTSCs, hTSCs are collected and subjected to RNA-Seq. After aligned to mouse hg38 by HISAT2, RPKM value was calculated by Edger. Our results show that PR-SET7 as the key regulator for hTSCs. There are also some genes differentially expressed after PR-SET7 KD, some of the DEGs were further confirmed by qPCR, the DEGs were associated with viral mimic inflammatory activities, antiviral innate immune response, genomic instability, and programmed cell death, indicating the essential role of PR-SET7. This RNA-Seq data provides fundamental information for our further physiological study of PR-SET7.
2023-06-28 | GSE196223 | GEO