A signature of 12 microRNAs is robustly associated with growth rate in a variety of CHO cell lines

Abstract

As Chinese Hamster Ovary (CHO) cells are the cell line of choice for the production of human-like recombinant proteins, there is interest in genetic optimization of host cell lines to overcome certain limitations in their growth rate and protein secretion. At the same time, a detailed understanding of these processes could be used to advantage by identification of marker transcripts that characterize states of performance. In this context, microRNAs (miRNAs) that exhibit a robust correlation to the growth rate of CHO cells were determined by analyzing miRNA expression profiles in a comprehensive collection of 46 samples including CHO-K1, CHO-S and CHO-DUKXB11, which were adapted to various culture conditions, and analyzed in different growth stages using microarrays. By applying Spearman or Pearson correlation coefficient criteria of>|0.6|, miRNAs with high correlation to the overall growth, or growth rates observed in exponential, serum-free, and serum-free exponential phase were identified. An overlap of twelve miRNAs common for all sample sets was revealed, with nine positively and three negatively correlating miRNAs. The here identified panel of miRNAs can help to understand growth regulation in CHO cells and contains putative engineering targets as well as biomarkers for cell lines with advantageous growth characteristics.

Regulation of miR-29b-1/a transcription and identification of target mRNAs in CHO-K1 cells

“…Ectopic expression of miR-29b-1 and miR-29a decreased CHO-K1 cell proliferation, colony formation and cell invasion. These data agree with a report showing that miR-29b and miR-29a negatively correlated with CHO-K1 cell growth (Klanert et al, 2016) and corroborates miR-29's repressive functional role in cells (Garzon et al, 2009; Kwon et al, 2014; Mott et al, 2007; Xiong et al, 2010). Earlier we reported that 4-OHT increased miR-29b-1/a expression in LY2 endocrine-resistant breast cancer cells based on microarray analysis of miRNA expression (Manavalan et al, 2011) and recently confirmed this observation by qPCR (Muluhngwi et al, 2017).…”

“…miR-29b-1 and miR-29a were downregulated in growing CHO-K1 cells (Klanert et al, 2016). We observed 4-OHT stimulated the expression of miR-29b-1 and miR-29a in CHO-K1 cells (Fig.…”

“…miR-29b and miR-29a negatively correlate with CHO-K1 cell growth (Klanert et al, 2016) and tamoxifen (TAM) has previously been associated with cell cycle arrest (Lykkesfeldt et al, 1984). Since 4-OHT inhibits CHO-K1 cell growth depending on the culture medium used (Thomas et al, 2003), and because 4-OHT increased miR-29b-1 and miR-29a in CHO-K1 cells (Fig.…”

“…This has led to a variety of approaches for evaluating transcriptomics within CHO cell lines (Le et al, 2015), including de novo assembly of transcripts (Vishwanathan et al, 2015). For our analysis, like others (Klanert et al, 2016), we used previously identified sequence homology to mouse Ensembl transcripts.…”

“…Similarly, miR-29a was increased in the 6 d compared to the 3 d time period in a suspension-adapted CHO-K1 cell assay (Gammell et al, 2007). On the other hand, another study reported that miR-29b-3p, miR-29a-3p, and miR-29c-3p were negatively correlated with growth of CHO-K1 cells (Klanert et al, 2016). Thus, it is uncertain whether miR-29 promotes or represses CHO-K1 cell growth.…”