Effects of Inhibiting Two Cell Cycle Modulating microRNAs in Recombinant Human IgG Producing Chinese Hamster Ovary Cells

By: Nan Lin1, Ken Heuermann,2, Jessica Schlueter,2, Carol Kreader, Scott Knight,2 and Kevin Kayser1, 1. Cell Engineering, SAFC, Sigma-Aldrich, 2909 Laclede Ave., Saint Louis, MO 63103, U.S.A. 2. Research Biotechnology, Sigma-Aldrich, 2909 Laclede Ave., Saint Louis, MO 63103, U.S.A.

Project Overview

background-experimental-design

Figure 1. Background and Experimental Design

Results

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Figure 2. Differential Expression of miR-221 and miR-222 in IgG-Producing DG44 Lines by qRT-PCR

Differential expression of miR-221 (left panel), miR-222 (right panel) in four IgG-producing DG44-derived cell lines (500 nM MTX cultures). Relative expression levels were normalized to miR-16.
*p < 0.05, ANOVA and Dunnett’s Multiple Comparison Test.

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Figure 3. Post-Amplification Expression of miR-221 and miR-222 in the MTX Responsive and Non-Responsive Cell Lines

Differential expression of miR-221 (left panel), miR-222 (right panel) in cell line C and A (Fig.2) Amplified by sequential increase of MTX concentration. Cell line A demonstrated increased IgG productivity but not cell line C (data not shown here) Relative expression levels were normalized to miR-16.
*p < 0.05, ANOVA and Dunnett’s Multiple Comparison Test.

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Figure 4. Modulating miR Expression to Control Cell Cycle for Increased IgG Secretion

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Figure 5. Relative Expression of miR-221/222 Target - Cdkn1b (p27Kip1)2 by qRT-PCR

Differential expression of Cdkn1b in IgG producing cell lines A – D (left panel), and amplified cell line C and A (right panel). Relative expression levels were normalized to ActR5 (multiplexed qRT-PCR).
*p < 0.05, ANOVA and Dunnett’s Multiple Comparison Test.

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Figure 6. Relative Expression of miR-17 Target - Cdkn1a (p21WAF1)3 by qRT-PCR

Differential expression of Cdkn1a in IgG producing cell lines A – D (left panel), and amplified cell line C and A (right panel). Relative expression levels were normalized to ActR5 (multiplexed qRT-PCR).
*p < 0.05, ANOVA and Dunnett’s Multiple Comparison Test.

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Figure 7. Transient Knockdown of miR-221/222 Using LNA miR Inhibitors

Cells were collected 96 hours after electroporation of LNA miR Inhibitors for miR-221 and/or miR-222 in cell lines A and C. miR-221 (left panel) and miR-222 (right panel) relative expression levels were normalized to ActR5 (multiplexed qRT-PCR).

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Figure 8. IgG Secretion 96-Hour Post Transient Knockdown of miR-221/222

Upper panels: Cells were plated 24 hours after LNA electroporation for the in situ IgG Secretion Assay4. Detection was performed 48 hrs after electroporation. High IgG secretion cutoff is 90th percentile of the control population. LNA inhibition of miR-221 and miR-222 led to mild increase in high IgG secreting population in Cell Line C but not Cell Line A. ANOVA, p < 0.05. Dunnett’s Multiple Comparison Test, LNA non-targeting as control, *p <0.05

Lower panels: Cells were plated in 6-well static cultures 24 hours after LNA electroporation, and harvested 5 days after plating for viable cell density and IgG productivity using ForteBio Octet system.

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Figure 9. Stable Inhibition of miR-221 in Two IgG Producing DG44 Lines

Panel A: Lentiviral expression vector for proprietary miR inhibitors. Puromycin selection was performed after transduction to generate stable pools for miR-221 knockdown. Growth and productivity assays in Erlenmeyer flask cultures were performed using the miR-221 KD lines and the wildtype lines.
Panel B: Relative expression of miR-221 by qRT-PCR. Expression of miR-222 showed no significant change (data not shown).
Panel C: Relative expression of Cdkn1b on Day 3 and Day 7 by qRT-PCR. Numeric increase in Cell Line A-miR-221 KD on day 3 and in Cell Line C-miR-221 on day 7 was observed.
Panel D: Volumetric IgG productivity of the miR-221 KD and wildtype cell lines.

Conclusions

  • In the IgG producing CHO DG44 cell lines, miR-221 and miR-222 are differentially expressed compared to the parental CHO DG44 cell line. In an MTX-amplified cell line, miR-221 is significantly down-regulated at increased MTX concentrations albeit without significant dose-response
  • Cdkn1b and Cdkn1a, two of the validated target genes for cell cycle modulating miRs (miR-221, miR-222 and miR-17) are significantly up-regulated in the IgG-producing DG44 lines
  • LNA miR Inhibitors effectively inhibited miR-221 and miR-222 with high specificity. Transient inhibition of both miR-221 and miR-222 expression led to mild increase in IgG secretion per cell in one of the two IgG producing lines (Line C)
  • Stable miR inhibition was achieved by Lentiviral expression of a proprietary miR inhibitor with high specificity for miR-221. However, neither cell line A nor C demonstrated significant increase in IgG productivity after transduction. Both cell lines showed numerical increase in miR-221 target Cdkn1b expression
  • Based on the transient inhibition data, ongoing work includes simultaneous stable inhibition of miR-221 and miR-222 in both cell lines A and C. Expression of miR-17 mimics will also be explored.

Acknowledgements

Kevin Forbes (Research Biotechnology, Sigma-Aldrich); Henry George (SAFC, Sigma-Aldrich)

Materials

     

References

  1. Dzau (2002) Nature Medicine 8, 1249 - 1256. 2. Galardi et al. (2007) J Biol Chem 282(32):23716-24. 3. Cloonan et al. (2008) Genome Biol 9(8):R127. 4. Lin et al. (2008) Curr Protoc Cytom. Chapter 2:Unit 2.14.

 

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