miR‐92a enhances recombinant protein productivity in CHO cells by increasing intracellular cholesterol levels

Abstract

BackgroundMicroRNAs (miRNAs) have emerged as promising targets for engineering of CHO cell factories to enhance recombinant protein productivity. Manipulation of miRNA levels in CHO cells have been shown to improve product yield by their effects on cellular processes such as increasing proliferation, resisting apoptosis and enhancing specific productivity (qP). We previously demonstrated increases in qP and titer of CHO-IgG cells by over-expressing miR-92a [1]. Stably-transfected pools showed 27% increase in qP and 21% increase in titer compared to parental clone, without significant alteration in proliferation rates. The highest producing clone isolated from the miR-92a pool demonstrated 114% increase in qP and 88% increase in titer. However, the mechanisms by which miR-92a enhances qP in CHO cells are still uninvestigated. Materials and methodsTo understand possible pathways via which miR-92a enhances productivity in CHO cells, we carried out transcriptomics study on 2 high-producing miR-92a clones (~33pg/cell-day) and blank-transfected pool (16pg/cell-day) using microarray. Genes identified to be differentially expressed in both clones relative to the blank-transfected pool were analyzed for pathway/gene ontology enrichment. Differential expression levels of Insig1 were confirmed using qPCR. Validation of insig1 as a direct target of miR-92a was done using a vector construct comprising of the 3' UTR sequence of insig1 downstream of luciferase reporter gene. Cholesterol assays were carried out on the cellular lipid extracts and the supernatant harvested from the miR-92a clones and blank-transfected pool. The clones were also stained with wheat germ agglutinin (WGA) conjugate for the visualization of the Golgi apparatus by confocal microscopy. The relative surface area of Golgi was calculated by normalizing surface area of Golgi compartment to that of the cell ResultsGenes involved in cholesterol synthesis and metabolism were found to be enriched among the differentially expressed genes identified by microarray. Using qPCR, decreased expression levels of insig1 were found in transient pools and stable clones over-expressing miR-92a. Co-transfection of miR-92a mimic with the (luciferaseinsig1 3'UTR) vector reduced luciferase expression bỹ 19% whereas miR-92a inhibitor significantly increased luciferase expression by 120%. INSIG1 inhibits cholesterol biosynthesis by preventing the activation of the SREBP transcription factor. The intracellular cholesterol concentration of both miR-92a clones were significantly increased by~30% compared to the blank-transfected pool. The extracellular cholesterol concentrations for the miR-92a clones were also~2-3-fold higher than that of the blank-transfected pool. Knock-out of miR-92a has been reported to reduce systemic cholesterol levels in mice [2], concurring with our observations that miR-92a overexpression increases cholesterol biosynthesis/secretion. Cholesterol is known to be essential for vesicular transport from the ER [3] and Golgi [4], and constitutes about 20% of th...

Exosomal miR-92a Concentration in the Serum of Shift Workers

“…Several miRNAs have proven to play a major role in BAT, such as controlling brown adipocytes differentiation [36]. Among these miRNAs, miR-92a has been involved in several metabolic processes, such as glucose homeostasis [24], regulation of intracellular cholesterol levels [37], and essential hypertension [38]. Furthermore, Chen and collaborators demonstrated that serum levels of exosomal miR-92a were inversely correlated with human BAT activity, confirming the data by FDG-PET/CT [39].…”

Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells

“…Identifying mRNA targets of identified microRNAs enables researchers to understand the pathways and mechanisms that are involved in improving recombinant protein expression [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 39 , 40 , 42 , 43 , 45 , 49 , 50 , 52 , 53 , 56 ]. In addition to detecting the mRNA-microRNAs interactions, investigators performed additional bioinformatics research to identify biological processes, gene ontology, and significant pathways that are affected by the targeted microRNAs.…”

“…In addition to detecting the mRNA-microRNAs interactions, investigators performed additional bioinformatics research to identify biological processes, gene ontology, and significant pathways that are affected by the targeted microRNAs. The researchers also aligned gene sequences between species, especially in the case of CHO cells where knowledge of genome is less evolved than that of the human genome [ 39 , 45 , 56 ]. These bioinformatics tools, also summarized in Table 2 , help to provide a comprehensive analysis, ensuring a robust approach to improving recombinant protein production.…”

“…The highest clones showed 130–140% increase in specific productivity and titer and that mir-17, mir-19b, and mir-92a were correlated with increased protein expression. The study was followed later by bioinformatics and reporter assays to identify insig1 as the gene target of mir-92a in CHO cells [ 56 ].…”

Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis.

“…Moreover, cholesterol regulation may also play a role on protein secretion reduction as it is an essential building block of secretion vesicles (Wang et al, 2000). Recently, it has been shown that the increase of cholesterol synthesis with the up-regulation of a micro RNA can increase the productivity of CHO cell lines by increasing their secretion capacity (Loh et al, 2017). It is then possible that the increase of productivity and glutathione content observed over time during the process are also link to cholesterol regulation.…”