High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells

Abstract

CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs-miRNAs-is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells.

Keywords: CHO; hyperproductivity; miR-574-3p; miRNA screening; p300; p53; pri-miRNA processing.

Figure 1

A functional high-throughput miRNA mimic screening in CHO-EPO and CHO-ETN cells. (a) Overview of the normalized results of 2042 miRNA mimics on the recombinant CHO-EPO cells. EPO titer, cell specific EPO productivity and viable cell density are represented as fold-changes with respect to the negative control. The pie charts illustrate the percentages of miRNA mimics that induce at least a 1.2-fold increase to at least a 0.8-fold decrease. (b) The effect of selected 35 miRNA mimics transiently transfected in CHO-EPO and CHO-ETN cells. Normalized volumetric productivities are presented as fold-changes relative to the respective negative control. Data is presented as the mean of 3 independent experiments ± SEM. Statistical analysis was performed using two-way ANOVA with Fisher’s analysis (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001).

Figure 1

A functional high-throughput miRNA mimic screening in CHO-EPO and CHO-ETN cells. (a) Overview of the normalized results of 2042 miRNA mimics on the recombinant CHO-EPO cells. EPO titer, cell specific EPO productivity and viable cell density are represented as fold-changes with respect to the negative control. The pie charts illustrate the percentages of miRNA mimics that induce at least a 1.2-fold increase to at least a 0.8-fold decrease. (b) The effect of selected 35 miRNA mimics transiently transfected in CHO-EPO and CHO-ETN cells. Normalized volumetric productivities are presented as fold-changes relative to the respective negative control. Data is presented as the mean of 3 independent experiments ± SEM. Statistical analysis was performed using two-way ANOVA with Fisher’s analysis (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001).

Figure 1

A functional high-throughput miRNA mimic screening in CHO-EPO and CHO-ETN cells. (a) Overview of the normalized results of 2042 miRNA mimics on the recombinant CHO-EPO cells. EPO titer, cell specific EPO productivity and viable cell density are represented as fold-changes with respect to the negative control. The pie charts illustrate the percentages of miRNA mimics that induce at least a 1.2-fold increase to at least a 0.8-fold decrease. (b) The effect of selected 35 miRNA mimics transiently transfected in CHO-EPO and CHO-ETN cells. Normalized volumetric productivities are presented as fold-changes relative to the respective negative control. Data is presented as the mean of 3 independent experiments ± SEM. Statistical analysis was performed using two-way ANOVA with Fisher’s analysis (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001).

Figure 2

(a) Analysis of the relative level of mature miRNAs in stable CHO-ETN cells expressed from human pri-miRNAs. MiRNAs expression is shown as fold-change relative to the CHO-ETN control cells transduced with non-targeting miRNA and normalized to miR-191-5p. Data is presented as the mean of three independent experiments ± SEM (b) RT-PCR analyses of the total RNA extracted from CHO-ETN stable clones overexpressing hsa-miR-18b/3667/3939 confirmed the presence of the precursor miRNA transcripts in the CHO cells (c) Analysis of the relative level of mature miRNAs in stable CHO-ETN cells expressed from different pri-miRNA scaffolds. MiRNAs expression is shown as fold-change relative to the CHO-ETN control cells transduced with non-targeting miRNA and normalized to miR-191-5p (d) Effects of stable overexpression of miR-143 on ETN production. Data is presented as the mean of 3 independent experiments ± SEM. Statistical analysis was done using one-way ANOVA followed by Bonferroni’s post-hoc test (ns: not significant, * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001). (e) Predicted secondary structure of pri-miRNAs of interest shows a hairpin, double-stranded stem and three bulges located within the miRNA stem. The most probable secondary structure of pri-miRNAs transcripts was obtained using UnaFold software.

Figure 3

Effects of stable overexpression of seven miRNAs on EPO and ETN production. Relative mature miRNAs levels in (a) stable CHO-EPO expressing seven miRNAs and (d) stable CHO-ETN also expressing seven miRNAs shown as fold-change relative to the respective control cells and normalized to miR-191-5p. Normalized volumetric productivities of (b) EPO and (e) ETN are presented as a fold change to the respective negative control. Normalized specific productivities of (c) EPO and (f) ETN are presented as a fold change to the respective negative control. Data are presented as the means of three independent experiments ± SEM. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc comparisons tests (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001).

Figure 3

Effects of stable overexpression of seven miRNAs on EPO and ETN production. Relative mature miRNAs levels in (a) stable CHO-EPO expressing seven miRNAs and (d) stable CHO-ETN also expressing seven miRNAs shown as fold-change relative to the respective control cells and normalized to miR-191-5p. Normalized volumetric productivities of (b) EPO and (e) ETN are presented as a fold change to the respective negative control. Normalized specific productivities of (c) EPO and (f) ETN are presented as a fold change to the respective negative control. Data are presented as the means of three independent experiments ± SEM. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc comparisons tests (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001).

Figure 4

Comparison of relative mature miRNA-574-3p expression in CHO-ETN and CHO-EPO stable clones from pri-miR-574 and pri-miR-143 Chinese hamster flanking sequences. Quantitative RT-PCR analysis of the relative level of mature miRNAs shown as a fold-change relative to the respective control cells and normalized to miR-191-5p. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc comparisons tests (ns: not significant, *** p ≤ 0.001 and **** p ≤ 0.0001).

Figure 5

Relative ETN and EPO mRNA levels in stable overexpressing miR-574-3p CHO clones. Quantitative RT-PCR analysis of the relative levels of ETN and EPO mRNAs. MMADHC mRNA was used for normalization and results are expressed as the mean of fold change in expression in CHO-ETN/EPO-miR-574-3p cells compared to the control cells (SEM as error bars, n = 3). Statistical analysis is performed using t-test followed by Bonferroni post hoc test (* p ≤ 0.05, ** p ≤ 0.01).

Figure 6

Effect of stable overexpression of miR-574-3p on EPO production. (a) EPO production in serum-free media over 12-days. (b) Western blot analysis of EPO produced in serum-free media in stable overexpressing miR-574-3p and CHO control cells (anti-EPO antibody). (c) Isoelectric focusing pattern of differently glycosylated EPO isoforms purified from CHO-EPO-miR-574-3p cells and CHO-EPO control cells.

Figure 7

miR-574-3p overexpression induces less apoptosis in CHO-ETN cells. Cell-cycle analysis by flow cytometry after BrdU and propidium iodide staining of CHO-ETN cells stably expressing miR-574-3p, shows less subG0/G1 cells in comparison to the control cells after 4 days of cultivation. All data is expressed as the mean ± SEM. (ns: not significant, *** p ≤ 0.001, t-test).

Figure 8

(a) Relative mRNA levels of nine annotated miR-574-3p target genes in CHO-ETN cells overexpressing miR-574-3p shown as fold-change relative to the control cells and normalized to MMADHC mRNA. Data is presented as the mean of three independent experiments ± SEM. Statistical analysis was done using one-way ANOVA followed by Bonferroni’s post-hoc comparisons tests (ns: not significant, *** p ≤ 0.001 and **** p ≤ 0.0001) (b) Western blot analysis shows downregulation of p300, SMAD4 and RXRA in CHO-EN stable cells overexpressing miR-574-3p.

Figure 8

(a) Relative mRNA levels of nine annotated miR-574-3p target genes in CHO-ETN cells overexpressing miR-574-3p shown as fold-change relative to the control cells and normalized to MMADHC mRNA. Data is presented as the mean of three independent experiments ± SEM. Statistical analysis was done using one-way ANOVA followed by Bonferroni’s post-hoc comparisons tests (ns: not significant, *** p ≤ 0.001 and **** p ≤ 0.0001) (b) Western blot analysis shows downregulation of p300, SMAD4 and RXRA in CHO-EN stable cells overexpressing miR-574-3p.

Figure 9

Impact of p300 and SMAD4 knockdown on ETN transcription in CHO-ETN cells. (a) Quantitative real-time RT-PCR analysis of relative levels of p300 and SMAD4 mRNAs in siRNA transfected CHO-ETN cells. (b) Western blot analysis shows significant downregulation of p300 in CHO-ETN cells. The graph shows the mean ± SD from three independent experiments (unpaired t-test; ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001) (c) Quantitative real-time RT-PCR analysis of relative levels of ETN and SMAD4 in siRNA transfected CHO-ETN clones. MMADHC mRNA was used for normalisation and results are expressed as the mean of fold change in expression in CHO-ETN cells compared to the control cells (SEM as error bars, n = 3).

Figure 10

MiR-574-3p downregulates p53. (a) CHO cells stably expressing miR-574-3p were exposed to etoposide (ETO 250 μM) for 16 h. Western blot analysis shows significant downregulation of p53 in CHO-ETN cells. The graph shows the mean ± SEM from three independent experiments (unpaired t-test, *** p < 0.001) (b) Proposed miR-574-3p mode of action through p300 and p53 downregulation 1. In the wt cells, under different stresses, such as prolonged cell cultivation and nutrient deprivation, p300 is activated as well as p53. p300 is a major acetyltransferase for p53 that acetylates p53 and therefore stabilizes it. p53 suppresses viral promoter activity interfering with the basal transcription machinery. Upon miR-574-3p overexpression, p300 is downregulated resulting in a lower p53 acetylation, destabilization and degradation removing viral promoter repression and increasing heterologous gene transcription.