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Biowire Winter 2011 — Knockout Rats — More Effective Pre-Clinical Models

Table of Contents

 


microRNA Target Validation: MISSION 3'UTR Lenti GoClone and Human microRNA Mimics

Abstract
microRNAs (miRNAs) may regulate hundreds of genes to control a cell’s response to developmental and environmental signals. The validation of potential target genes is essential in determining a miRNA’s role and function in these pathways. Here we use MISSION 3'UTR Lenti GoClone™ and Human microRNA Mimics to demonstrate validation of known and conserved miRNA targets. Known targets of hsa-miR-29b (MCL1), hsamiR- 124 (MAPK14), and hsa-miR-373 (LATS 2), and targets of conserved miRNA s hsa-miR-373 (RBL2) and hsa-miR-10a (HOXD10) were down-regulated by their respective mimic. These results demonstrate that the use of MISSION 3'UTR Lenti GoClone with Human microRNA Mimics is a viable option for miRNA target validation.

Introduction
microRNAs (miRNAs) are a class of naturally occurring small non-coding RNA molecules (21-25 bases in length) that regulate a variety of developmental and physiological processes1. The majority of miRNAs function by repressing their target mRNA’s translation, by promoting mRNA transcript cleavage and degradation, or both. As a consequence of their small size, miRNAs are predicted to have several hundred mRNA targets, meaning a single miRNA has the potential to impact the expression of many proteins2.

Many computational and experimental approaches have been used to identify biologically relevant miRNA gene targets3. Subsequently, validation of these putative targets is required, and reporter assays afford researchers an inexpensive and technically straightforward method for this process. For instance, plasmids harboring miRNA targets (target sequence or 3'UTR sequence; UTR: untranslated region) fused to a reporter (e.g., luciferase) are co-transfected into cells along with the corresponding miRNA mimic or miRNA inhibitor. The result is a reduction or an increase in reporter activity, respectively, that is simple to measure. Importantly, this readout provides substantive proof that the target can be regulated by the miRNA.

Here we report the use of MISSION 3'UTR Lenti GoClone and Human microRNA Mimics to demonstrate validation of known and conserved mRNA targets of human microRNAs.

Materials and Methods
MISSION 3'UTR Lenti GoClones (Table 1) were generated by SwitchGear Genomics and the MISSION operations group at Sigma-Aldrich. Two hundred thousand MCF7 cells (ATCC catalog No. HTB-22; see ATCC for medium composition) were transduced with MISSION 3'UTR Lenti GoClones at an MOI (Multiplicity of Infection) of 5. Five days post-transduction, medium was supplemented with 250 ng/ ml puromycin for selection of transduced cells. Puromycin-resistant cells were pooled and expanded for two weeks. Cells were then seeded at 10,000 cells per well in a 96- well culture plate and incubated overnight. Cells were then transfected with MISSION Human microRNA Mimics and MISSION miRNA Negative Control 1 (Product No. HMC0002) at a final concentration of 50 nM via Lipofectamine™ 2000 (Invitrogen). The microRNA mimic corresponds to its target MISSION 3'UTR Lenti GoClones (Table 1). Twenty-four hours post-transfection, cells were assayed for Renilla luciferase activity using the LightSwitch Assay System (SwitchGear Genomics) on a SpectraMax® L Microplate Reader (Molecular Devices).

Results and Discussion
Transduction of MCF Cells with MISSION 3'UTR Lenti GoClones
Historically, miRNA target validation has been achieved by co-transfecting cells with plasmids containing a reporter gene fused to a miRNA target along with miRNA mimics or inhibitors. The MISSION 3'UTR Lenti GoClone collection is a genome-wide collection of human 3'UTRs cloned into a lentiviral vector. Each viral vector encodes an optimized luciferase reporter gene (RenSP) fused with a 3'UTR sequence. The features of the GoClone lentiviral plasmid vector, pLSG UTR RenSP, are listed in Figure 1. MCF7 cells were transduced with the Lenti GoClones listed in Table 1. After selection and expansion of transduced MCF7 cells, luciferase assays were performed to assess expression of the Renilla reporter. As seen in Figure 2, the five cell lines transduced with the Lenti GoClones express Renilla luciferase above background. The variation in luciferase activity between the cell lines suggests that endogenous miRNAs or other factors affect the reporter via interactions with the fused 3'UTR.


Table 1.
MISSION 3'UTR Lenti GoClone and corresponding MISSION Human microRNA Mimics

GoClone Gene Symbol microRNA Mimic Reference
MCL1 hsa-miR-29b 4
MAPK14 hsa-miR-124 5, 6
HOXD10 hsa-miR-10a hsa-miR10b; ref. 7
LATS2 hsa-miR-373 8
RBL2 hsa-miR-373 mmu-miR-294; ref. 9


microRNA Target Validation Figure 1
Figure 1.
Features of Lentiviral Plasmid Vector pLSG UTR RenSP

 

Name Description
RPL10prom Constitutive human RPL10 promoter
RenSP Optimized Renilla luciferase gene
Xbal, Nhel, Avrll, Xhol, Fsel Multiple Cloning Site for human 3'UTR insertion
PGKprom Human phosphoglycerate kinase eukaryotic promoter
Puro Puromycin resistance gene for mammalian selection
LTRs Long terminal repeats
Ampr Ampicillin resistance gene for bacterial selection
ori Origin of replication
RRE Rev response element
 


microRNA Target Validation Figure 2
Figure 2.
Renilla Luciferase Reporter Activity Detected in Stable MCF7 Cells Transduced with Lenti GoClones. Puromycin-resistant MCF7 cells were assayed for Renilla luciferase activity using the LightSwitch Assay System on a SpectraMax L Microplate Reader. This was done with four replicates and luciferase output plotted as a value of relative luciferase units (RLU).
 

MISSION Human microRNA Mimics Knockdown 3'UTR Lenti GoClone Targets
After transduction with MISSION 3'UTR Lenti GoClones and selection for stable integration, miRNAs of interest may be introduced to determine whether or not they target the encoded 3'UTR. Published Human miRNA targets (GoClones in Table 1) for hsa-miR-29b (MCL1), hsa-miR-124 (MAPK14), hsa-miR-10a (HOXD10) and hsa-miR-373 (LATS 2; RBL2) were tested for knockdown of luciferase activity via their 3'UTR with their corresponding miRNA. The latter hsa-miR-373 target, RBL2, is a published target of Mouse mmu-miR-2949, but targeting by the corresponding human miRNA has not been reported. Mmu-miR-294 and hsa-miR-373 have the same seed sequence and are presumed to be from the same miRNA family (Figure 3A). The regulation of RBL2 by mmu-miR-294 in mice has a direct effect on DNA methyltransferases, in turn controlling DNA methylation and telomere recombination. Validation of human RBL2 3'UTR targeting by hsa-miR-373 suggests that hsa-miR-373 may have the same effect on the regulation of DNA methylation and telomere recombination in human cells. This phenomenon will need to be further explored. The GoClone target HOXD10 is a reported target of hsa-miR-10b7. Here we used hsamiR- 10a for targeting HOXD10, which is from the same miRNA family and only differs by one base when compared to hsa-miR-10b (Figure 3A). As seen in Figure 3B, all five targets were knocked down in stable MCF7 cells when their corresponding miRNA (mimic) was transfected into the cell.


microRNA Target Validation Figure 3
Figure 3.
microRNA Mimics Knockdown Reporter Activity of Stable MCF7 Cells Transduced with Lenti GoClones. (A). Alignment of conserved miRNAs hsa-miR10a and 10b and hsa-miR-373 and mmu-miR294. The blue highlighted regions are conserved bases and red highlighted regions are non-conserved bases. (B). Stable MCF7 cells transduced with Lenti GoClones were transfected with a final concentration of 50nM MISSION Human microRNA Mimics, in parentheses (red bars) and with MISSION miRNA Negative Control 1 (white bars). Twenty-four hours post-transfection, cells were assayed for Renilla luciferase activity using the LightSwitch Assay System on a SpectraMax L Microplate Reader. This was done with four replicates, and the values were plotted as a percent expression compared to GoClone plus negative control as 100 percent expression. In addition, all five microRNA mimics were also transfected individually into MCF7 cells transduced with Lenti GoClone-GAPDH, and no knockdown of reporter activity was observed (data not shown).
 

Conclusion
The results shown in Figure 3B demonstrate that delivery of MISSION 3'UTR Lenti GoClones along with MISSION Human miRNA Mimics into cells provide a viable option for miRNA target validation. The advantages of the Lenti GoClone format are the ability to permit infection and integration into cell lines recalcitrant to transfection, and the ability to isolate cells stably expressing the reporter. Furthermore, the pre-cloned 3'UTR sequences provide researchers a measurable savings in both time and money and facilitate the efficient acquisition of validation data.

In conclusion, MISSION 3'UTR Lenti GoClone and Human microRNA Mimics will aid microRNA researchers in target validation and allow for creation of stable cell lines with a luciferase reporter regulated by the users.

Acknowledgements
We would like to acknowledge the following members of Sigma-Aldrich Biotechnology R&D, in particular Drs. Carol Kreader, Scott Knight and Heather Holemon, for their assistance throughout development of this product. We would like to thank the Sigma-Aldrich Operations group, in particular Stacy Deeds and her group and our colleagues at SwitchGear Genomics, Drs. Patrick Collins and John Garcia for providing the Lenti GoClones. Last but not least, we would also like to acknowledge our Marketing team of Drs. Nikos Hontzeas and Steve Suchyta.

References

  1. Ghildiyal, M.; Zamore, P.D. Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10, 94-108 (2009).
  2. Filipowicz, W.; Bhattacharya, S.N.; Sonenberg, N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9, 102-114 (2008).
  3. Thomas, M.; Lieberman, J. Lal A. Desperately seeking microRNA targets. Nat. Struct. Mol. Biol. 17, 1169-1174 (2010).
  4. Mott, J.L. et al. mir-29 regulates Mcl-1 protein expression and apoptosis. Oncogene 26, 6133-6140 (2007).
  5. Krek, A. et al. Combinatorial microRNA target predictions. Nat. Genet. 37, 495-500 (2005).
  6. Lim, L.P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature. 433, 769-773 (2005).
  7. Ma, L.; Teruya-Feldstein, J.; Weinberg, R.A. Tumour invasion and metastasis initiated by microRNA 10b in breast cancer. Nature. 449, 682-688 (2007).
  8. Voorhoeve, P.M. et al. A Genetic Screen Implicates miRNA-372 and miRNA-373 as Oncogenes in Testicular Germ Cell Tumors. Cell. 124, 1169-1181 (2006).
  9. Benetti, R. et al. A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2- dependent regulation of DNA methyltransferases. Nat. Struct. Mol. Biol. 15, 268-279 (2008).

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