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GRNA-seq from Ishikawa after medroxyprogesterone treatment

ABSTRACT: Hormone therapy serves as a primary choice for fertility preservation and is also considered for advanced and recurrent cases with endometrial cancer. While a number of patients fail to respond favorably to hormone treatments. In order to explore the mechanisms underpinning hormone resistance, Here we constructed a whole-genome library for EAC cell lines using the CRISPR-Cas9 system and performed high-throughput sequencing after medroxyprogesterone treatment in this study. For CRISPR screening, ishikawa cells were infected pooled GeCkov2 lentiviral library with functional MOI of 0.3. Genomic DNA from each group was isolated and amplified by PCR. The PCR products were purified and subjected to NGS by using the Novaseq 6000-PE150 platform. After 48h of puromycin selection, the cells were divided into three groups, the baseline group cells were collected and frozen at -80 degree centigrade, the control group cells were treated with DMSO for 10 days while the MPA group cells were treated with 15 μM MPA for 10 days.

ORGANISM(S): Homo sapiens

PROVIDER: GSE241070 | GEO | 2024/02/12

REPOSITORIES: GEO

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Project description:Context: Progesterone is important physiologically and therapeutically to maintain uterine quiescence during pregnancy. It acts in part through controlling myometrial gene expression. Objective: To use expression microarray and qRT-PCR validation to determine the changes in gene expression induced by prolonged exposure to a synthetic progestogen. Design: Myometrial explants, obtained at elective Caesarean section (n=9 patients), were maintained in culture, under 0.6g tension, for 3 days in the presence or absence of medroxyprogesterone acetate (MPA, 100nM). Expression array was performed using Illumina human-8 v3 beadchip arrays. Approximately 30% of differentially expressed transcripts were validated in biological replicates (n=10) using qRT-PCR. Results: Transcripts from 114 genes were significantly differentially expressed. These were significantly enriched in inflammatory response (P=0.00001), growth factor activity (P=0.0004) and cytokine activity genes (P=0.008). 34 transcripts were validated using qRT-PCR using an additional independent sample set obtained from 10 further women. There was very close agreement in the fold changes obtained by array and qRT-PCR analysis (r2=0.9, P<0.0001). We confirmed significant down-regulation of a number of genes which have been well characterized as progesterone sensitive, such as IL1B, IL6, PTGS2 and GJA1. However, the top and 6th most down-regulated genes encoded two cytokines, IL11 and IL24, respectively, not previously implicated in mediating the effects of progesterone in myometrium. Both were validated by qRT-PCR (4.3-fold and 2.2-fold down-regulated, both P<0.001). Conclusions: Progestogens control expression of multiple genes in myometrium, including many with no previously recognised role, including IL11 and IL24. It is plausible that proteins encoded by some of these genes may have important, but as yet uncharacterized effects in controlling human parturition. 9 paired samples of human myometrium treated with MPA (10-7M) or vehicle

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GRNA-seq from Ishikawa after medroxyprogesterone treatment

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