MicroRNAs (miRNAs) are an endogenous class of RNA molecules comprising 19-25 nucleotides that regulate the suitability or transflational efficiency of mRNAs. The mature miRNAs are processed from longer hairpin transcripts by the sequential action of the RNases Drosha1 and Dicer2,3. The occurance of miRNAs includes eukaryotic organisms from plants and animals4.
Based on complementary analyses of miRNAs to mRNA sequences in data bases, it has been estimated that each vertebrate miRNA targets ~200 mRNA molecules in average, suggesting that ~30% of the human genes may be post-translationally controlled by miRNAs5,6.
Figure 1. Proliferation rates of HeLa cells transfected with antisense oligonucleotides targeted against different miRNAs as measured with the Cell Proliferation Reagent WST-1.
Figure 2. Caspase levels of HeLa cells transfected with antisense oligonucleotides targeted against different miRNAs as measured with the Homogeneous Caspase Assay.
An increasing number of publications describe the participation of miRNAs in a variety of different regulation mechanisms such as cell growth, development, differentiation, and apoptosis7. They are involved in cancerogenic processes: miRNAs are frequently located in cancer-associated genomic regions, and altered miRNA expression patterns have been described in many human cancers8-10.
In order to measure the expression level of miRNAs and to influence their functional activity, different kinds of oligonucteotides have been used. Locked nucleic acids (LNAs) enhanced oligonucleotides are the latest generation of antisense moleculaes and offer an unprecedented affinity to their target miRNAs11. The high specificity to bind LNA-modified oligonucleotides enabled characterization of the temporal and special expression patterns of 115 different miRNAs in zebrafish embryos in in situ hybridization experiments12. Recently, the potential involvement of certain human miRNAs in cell proliferation and apoptotic events was observed using 2’-O-methyl-modified oligonucleotides13. Besides having a high affinity and specificity. LNA-modified oligonucleotides are highly stable in serum. Therefore, we decided to use LNA-modified antisense oligonucleotides in miRNA antisense studies.
Antisense oligonucleotides containing LNA monomers (miRCURY Knockdown probes) against h-mir 7, h-mir 21, h-mir 204, h-mir 214, h-mir 218 were a gift from Exiquon. The sequences of these oligonucleotides are the exact antisense copy of the mature miRNA sequences, published in the miRNA registry14.
Transfection of HeLa cells with X-tremeGENE™ siRNA transfection Reagent
HeLa cells (ECACC) were grown in MEM containing 2mM glutamine, 1x non-essential amino acids and 10% fetal calf serum. One day prior to transfection, 8x103 cells/well were plated into a 96-well plate. The cells were grown to 30% density when the transfection complex was added directly to the medium. The transfection complex was prepared as follows: 0.3ul X-tremeGENE™ siRNA Transfection Reagent and 5pmol of each antisense LNA oligonucleotides were individiually prediluted in 15ul OptiMEM, mixed, and incubated for 15 minutes at room temperature, 30ul of transfection complex were then added to the cells. After 24 hours of incubation, fresh medium was added and the cells were grown for additional 48 hours.
In a second experiemtn, a higher concentrated transfection complex consisting of 0.8ul X-tremeGENE™ siRNA Transfection Reagent and 11 pmoles of antisense LNA oligonucleotide against h-mir 204 was tested.
The occurenc of activated cysteinyl-aspartic-acid proteass (caspases) marks an early step of apoptosis. The Homogeneous Caspase Assay contains a fluorogenic peptide substrate that releases a fluorescent group depending on caspase activity in the samples. The caspasr assay was performed 72 hours after transfection, and the total caspase was measured according to the supplier’s instructions. The cells were trypsinized and washed in PBS buffer before they were lysed and the protease substrate was added.
Cell Proliferation Assay
Cell proliferation was measured 3 days fter transfection using the Cell Proliferation Reagent WST-1. WST-1 is a tetrazolium salt that is converted to a strongly colored compound by metabolically active cells. The extent of color formation directly correlates with metabolic activity. An amount of WST-1 equaling 10% of the volume in the well was added and incubated for 1 hour. Absorbance was then measured at 437nm against medium (reference wavelength 690nm). The cells in the same wells were then counted under the microscope.
Membrane changes resulting from late apoptotic events were determined 72 hours after transfection using the Annexin-V-FLUOS Staining Kit, following the instruction manual. With the Annexin-V-FLUOS Staining Kit, apoptotic cells exposing phosphatidylserine on their surface are stained and subsequently detected by FACS analysis.
Figure 3. Proliferation rates of HeLa cells transfected with different amounts of antisense oligonucleotides against miRNAs 204 as measured with the Cell Proliferation Reagent WST-1.
Silencing of Human miRNAs in HeLa Cells
A large panel of 2’-O-methyl modified antisense inhibitors against human miRNAs has been tested for growth inhibition in different cell lines13. From this panel, we selected five antisense oligonucleotides that showed either effects on cell proliferation, or on caspase activation levels and ordered those as LNA-modified versions. We used these LNA-modified oligonucleotides (miRCURY Knockdown probes) to transfect HeLa cells. After 72 hours, the cells number, proliferation rate, caspase activity, and membrane reorganization level were determined. As shown in Figure 1, the antisense molecules against h-mir 7 and h-mir 204 display a decreased proliferation rate, whereas the antisense molecules against h-mir 21 increase the proliferation rate to some degree. The antisense molecules against h-mir 214 and h-mir 218 do not effect on the proliferation rate when compared with the scrambled oligonucleotide or the non-transfected control. The observations for the LNA-modified oligonucleotides against h-mir 7, h-mir 204, and h-mir 21 corresponded qualitatively to the results with the 2’-O-methyl modified antisense inhibitors13, whereas the LNA-modified oligonucleotides against h-mir 214 and h-mir 218 did not. This might be due to the slightly different behavior exhibited by our HeLa cell line, as we were not able to influence the proliferation rate with the 2’-O-methyl modified antisense inhibitors against h-mir 214 and h-mir 218 (data not shown).
Figure 4. Caspase activity of HeLa cells transfected with different amounts of antisense oligonucleotides against miRNAs 204 as measured with the Homogeneous Caspase Assay.
Figure 5. Annexin V Staining of HeLa cells transfected with antisense oligonucleotides targeted against different miRNAs using the Annexin-V-FLUOS Staining Kit.
Caspase activity of the transfected cells was tested using the Homogeneous Caspase Assay. The obtained values were normalized to the WST-1 values to compensate for the difference in cell numbers The results can be seen in Figure 2, While the miRCURY Knockdown probes against h-mir 214, h-mir 218 and h-mir 21 do not influence the caspase levels, the caspase activity is increased after treatment with antisense inhibitors against h-mir 7 and h-mir 204. This indicates that these miRNA species prevent the HeLa cells from entering apoptosis, and a knockdown of these miRNAs starts apoptotic events like the activation of caspases.
To further analyze this effect, we repeated the experiment with the miRCURY Knockdown probes against h-mir 204 under two different concentrations of transfection complex. Again, we observed a strong effect on the cell proliferation rate and the total cell number (figure 3). The cell number correlated well with the proliferation rate as measured with the WST-1 assay. Under the higher concentration, the total cell number was less than half that of the controls (untransfected cell or cells transfected with scrambled oligonucleotides). Again, a high increase of caspase activity was seen with these knockdown probes reaching more than threefold elevated levels at the highest concentration (figure 4).
We also analyzed the transfectants for exposure of phosphatidylserine on the cell surface, a later apoptotic step. This was measured with a fluorescein-labeled Annexin V, a protein binding to the phosphatidylserine residues. Up to 35% of the cells displayed membrane changes (figure 5). This finding supports the view that h-mimr 204 is involved in regulating proliferation in HeLa cells and preventing the cell line from entering into apoptosis.
The recently discovered miRNAs have added a new level of known regulatory competents and thereby changed our view of gene regulation. It is of general interest to manipulate the levels of miRNAs in different cell types in order to elucidate their biological function. We have demonstrated that the X-tremeGENE™ siRNA Transfection Reagent is an excellent tool for transpoprting antisense molecules into the cell lines. The LNA-enhanced miRCURY Knockdown probes were actcive at very low concentrations and did not exhibit significant toxic side effects, indicating a very specific target recognition. By combining the technologies mentioned above, we have been able to demonstrate the role of different miRNAs in the proliferation of HeLa cells.
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