Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of a Medicago truncatula P450 monooxygenase mutant

ABSTRACT: The aim of the experiment was to characterise a P450 monooxygenase mutant involved in the biosynthetic pathway of haemolytic saponins. Saponins are a group of glycosidic compounds with an antimicrobial function. The gene CYP716A12 is responsible for an early step in saponin biosythesis. Transcriptome analysis of a CYP716A12 mutant was performed to investigate its function. 6 samples were used in the experiment: 3 biological replicates of CYP716A12 mutant plants and 3 biological replicates of wild-type plants.

ORGANISM(S): Medicago truncatula

SUBMITTER: Ornella Calderini

PROVIDER: E-GEOD-22835 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background/Aim: Our previous in vitro high throughput (HTP) botanical anticancer cytotoxic screenings indicated that the wild yam extract (Dioscorea villosa (WYE)) has very potent anticancer effects in terms of espousing cell death with a low IC50 across diverse cancer cell lines including glioblastoma, melanoma, colon, neuroblastoma, and breast. However, unlike other botanicals we have focused on, WYE contains a high content of detergent steroidal saponins. As surfactants, they act by direct contact to rapidly reduce oil-water interfacial tensions causing disintegration of bio-lipid membranes. This study examined the WYE at sub-lethal concentrations where the saponin content does not create an interfering variable of lytic cell death in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Methods: A method validation study was first set up to cross-confirm the inverse relationship between saponin content with lytic cell death, where saponins were associated with loss of plasma and mitochondrial membrane potential and cytotoxic effects in both 2D and 3D cultures. Once appropriate sub-lethal concentrations were established, we then monitored changes in whole transcriptomic (WT) mRNA, miRNA, and long intergenic non-coding RNAs and cross- confirm this data by cell proliferation studies spot test antibody-protein arrays, and ELISA. Results: Upregulated DEGS reflected immune-stimulating effects centering on TNF signaling, COX2, cytokine release, and cholesterol/steroid biosynthesis. Downregulated DEGs show a very different transcriptome signature, revealing severe, extensive damage to cell cycle processes, including loss of transcripts for cell division cycle (CDC) genes (25A,45,6,7, A2, A7, A7L,8) cyclins (CCN.) (A2, B1, B2, E2, F) as well as cyclin-dependent kinases (CDKs), centromere proteins (CENP), kinesin family members (KIFs), and polo-like kinases (PLKs) with exception to a corresponding upregulated DEG for p21 gene CDKN1A, all which were corroborated by the cytostatic effects confirmed during a 6-day proliferation study. Conclusions: In conclusion, WYE appears to mediate a saponin lytic effect at high concentration on direct contact, however at lower concentrations and independent of cell death, there exist three distinct biological consequences as found in this breast cancer cell model, including pro-inflammatory, steroid biosynthetic, and collapse of the cell cycle.

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Characterization of a Medicago truncatula P450 monooxygenase mutant

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