Changes in the expression of microRNAs regulate podophyllotoxin accumulation in Linum album cells to phenylalanine

Podophyllotoxin (PTOX) is a significant lignan in Linum album that is widely utilized as a precursor to produce excellent anticancer derivatives. Our previous study illustrated that exogenous phenylalanine (Phe) had a beneficial impact on the production of PTOX in L. album cell culture. The current study was focused on investigating the molecular mechanisms through which Phe (1000 µM) regulates the biosynthesis of PTOX in L. album cells. In the current work, the results indicated that Phe increases hydrogen peroxide (H2O2), salicylic acid (SA), and nitric oxide (NO) formation in L. album cells during the treatment period. Also, RT-qPCR analysis implied that Phe upregulates four PTOX biosynthetic genes including PAL, CAD, CCR, and PLR. As expected, Phe accelerates the metabolic fluxes towards PTOX accumulation through a change in the rate of PAL and PLR action in the entry and terminal points of biosynthesis of PTOX in L. album cells. Moreover, a Poly(A)-tailed RT-qPCR analysis for microRNAs revealed that Phe feeding differentially modulates the expression levels of seven conserved miRNAs, namely miR156, miR159, miR171, miR172, miR393, miR396, and miR408. DSPC analysis revealed a strong relationship between the transcript levels of PTOX biosynthetic genes and the expression levels of these miRNAs. Overall, this work demonstrated that Phe feeding regulates the PTOX content, primarily by changing SA, H2O2, and NO generation and, secondly by regulating the expression levels of biosynthetic genes and miRNAs involved in PTOX in L. album cells. Phe can increase the production of PTOX in L. album through inducing SA, H2O2, NO generation as well as modulating the transcription of specific genes involved in lignan biosynthesis and conserved miRNAs.