No conflicts of interest are declared.

1. miR-143 and miR-506 transfection
CAUTION: Use latex gloves, protective eyeglasses, and a laboratory coat while performing the described experiments. When required, use the biosafety cabinet with the cabinet fan on, without blocking the airways or disturbing the laminar airflow. Always set the protecting glass window to the appropriate height, as described by the manufacturer.
<ol>
	<li>Seed NSCLC A549 cells in a T25 cm2 flask/6/96 well plate in DMEM/F12K media supplemented with 10% FBS and 1% penicillin-streptomycin (culture media) in a tissue culture hood and incubate overnight at 37 &#176;C with 5% CO2 in a tissue culture incubator.</li>
	<li>Suspend miR-143 and/or miR-506 mimics, or scramble siRNA with transfecting agent (2.4 &#181;g of miR were mixed with 14 &#181;L of transfecting agent; see Table of Materials) in 500 &#181;L of transfection media and 1.5 mL of serum and antibiotic-free DMEM/F12K media at a final miRNA concentration of 100 nM. miRNA amount may require optimization on different cells and concentrations. Appropriate approaches include the transfection of cells with increasing concentrations of miRNA (i.e., 50-200 nM) and evaluation of expression downregulation of the genes of interest.</li>
	<li>Remove culture media from flask/plate and wash once with 1x PBS.</li>
	<li>Add miRNA/scramble-transfecting agent complexes and incubate at 37 &#176;C with 5% CO2 for 6 h (flask size defines the added incubation volume).</li>
	<li>Replace the media with 4 mL of culture media and incubate cells for 24 h and/or 48 h.</li>
	<li>Harvest transfected cells by trypsinization, by adding 1 mL of trypsin-EDTA in each T25 cm2 flask, incubate for 5-10 min at 37 &#176;C, and add 3 mL of culture media to harvest the cells. Place the contents of each flask into a separate, marked 15 mL tube. Work in a tissue culture hood.</li>
	<li>Centrifuge at 751 x g for 5 min and remove the supernatant. 
	NOTE: Caution is required during supernatant removal, as agitation of the tube may cause loss of cells.</li>
	<li>Add 2 mL of 1x PBS and centrifuge for 5 min at 751 x g.</li>
	<li>Repeat steps 7 and 8 once to remove any traces of media and supernatant. 
	NOTE: At this stage, sample tubes can be stored at -80 &#176;C or can be used immediately.</li>
</ol>
2. RNA extraction
<ol>
	<li>Clean the work area by spraying with 70% isopropyl alcohol and RNAse decontamination solution.</li>
	<li>RNA extraction should be performed using an appropriate RNA kit (see Table of Materials).</li>
	<li>Remove the tubes from -80 &#176;C and allow them to thaw. Add 300 &#181;L of lysis buffer and pipette up and down to break the cell membrane.</li>
	<li>Add an equal volume of 100% ultra-pure ethanol.</li>
	<li>Mix well and place in separating column.</li>
	<li>Centrifuge between 11 and 16 x g for 30 s and remove the flow-through.</li>
	<li>Add 400 &#181;L of washing buffer and centrifuge to remove the buffer.</li>
	<li>Add 5 &#181;L of DNAse I with 75 &#181;L of DNA digestion buffer in each sample and incubate for 15 min.</li>
	<li>Wash the sample with 400 &#181;L of RNA prep buffer.</li>
	<li>Wash 2x with RNA wash buffer.</li>
	<li>Add nuclease-free water to the column, centrifuge, then collect the RNA.</li>
	<li>Measure total RNA concentration with a UV spectrophotometer.</li>
</ol>
3. RT-qPCR
<ol>
	<li>Prior to RT-qPCR, synthesize the cDNA. Subsequent to RNA concentration quantification, place 1 &#181;g of RNA in a 20 &#181;L final volume of reaction to prepare cDNA in a PCR tube. Always work on a clean bench.</li>
	<li>All other necessary ingredients are included in Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ingredients</td>
			<td>Quantity (&#181;L)/sample (20 &#181;L)</td>
		</tr>
		<tr>
			<td>5X cDNA Master mix</td>
			<td>4</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Random hexamers</td>
			<td>1</td>
		</tr>
		<tr>
			<td>RT enhancer</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Verso enzyme mix</td>
			<td>1</td>
		</tr>
		<tr>
			<td>DNase and RNase free water</td>
			<td>Required qty after adding RNA to make 20 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Materials for cDNA synthesis from RNA samples.&#160;Required quantities of respective ingredients to prepare a master mix for one sample for cDNA synthesis.
<ol start="3">
	<li>Incubate in a thermal cycler with the following temperature conditions: 42 &#176;C for 30 min; 95 &#176;C for 2 min; 4 &#176;C until collection of samples.</li>
	<li>Use immediately in regular PCR or qPCR, or store cDNA at -20 &#176;C.</li>
	<li>Prepare forward and reverse primer solutions with DNase/RNase-free water at a concentration of 10 &#181;M from stock primer solution.</li>
	<li>Prepare separate master mixes for each gene to be detected, according to the number of samples. For each cDNA sample (qPCR well), the reaction quantity is prepared according to Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ingredients</td>
			<td>Quantity (&#181;L)/sample (20 &#181;L)</td>
		</tr>
		<tr>
			<td>SYBR master mix</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Forward Primer - 10 &#956;M</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Reverse Primer - 10 &#956;M</td>
			<td>2</td>
		</tr>
		<tr>
			<td>DNase and RNase free water</td>
			<td>3</td>
		</tr>
		<tr>
			<td>cDNA sample</td>
			<td>3</td>
		</tr>
	</tbody>
</table>
Table 2: Materials for quantitative real-time PCR from cDNA samples.&#160;Required quantity of ingredients to prepare a master mix for one sample for qPCR.
<ol start="7">
	<li>Place each sample in the respective wells.</li>
	<li>Design the sample layout for the 96 well qPCR plate. For each sample and analyzed gene, perform the reaction in triplicates, or at least in duplicates.</li>
	<li>Seal the 96 well plate with an optically clear adhesive cover.</li>
	<li>Quick-spin the plate to allow the reaction mixture to reach the bottom of each well.</li>
	<li>Run RT-qPCR according to the following thermal gradient: 
	1) 50 &#176;C for 2 min 
	2)&#160;95 &#176;C for 2 min 
	3)&#160;95 &#176;C for 15 s 
	4)&#160;Take reading 
	5)&#160;60 &#176;C for 1.5 min 
	6)&#160;Repeat step 3 for 39 times</li>
	<li>Run the following thermal gradient in continuation of the above to determine melting curve which indicates single product amplification. 
	7) 65 &#176;C for 0.31 min 
	8)&#160;+0.5 &#176;C/cycle 
	9)&#160;Plate read 
	10)&#160;Repeat step 8 for 60 times until reaching 95 &#176;C 
	11)&#160;72 &#176;C for 2 min</li>
</ol>
4. Agarose gel electrophoresis to confirm single gene amplification
<ol>
	<li>Prepare 1% agarose gel in 1x TBE buffer.</li>
	<li>Add ethidium bromide (EtBr) in warm (~50 &#176;C) gel until achieving a final concentration of approximately 0.2-0.5 &#181;g/mL. EtBR binds with DNA and allows it to be visualized under UV light in a gel imager.</li>
	<li>Pour warm gel in a gel tray supplied with an electrophoresis gel box. Attach the provided comb tightly for uniform wells.</li>
	<li>Allow the gel to rest and cool to room temperature (RT) for ~30 min.</li>
	<li>When the gel is solidified, place the gel and tray in the gel box.</li>
	<li>Fill the gel box with 1x TBE buffer until the gel is completely covered.</li>
	<li>Measure the concentration of the DNA from PCR amplification process using a UV spectrometer.</li>
	<li>Take ~15 ng of DNA in a small PCR tube, add 5 &#181;L of dye, and add the required amount of nuclease-free water to achieve a 15 &#181;L total volume.</li>
	<li>Load the DNA ladder and samples into the wells.</li>
	<li>Run the gel at 100 V until the dye line is approximately 75%-80% down the gel. 
	NOTE: Make sure the gel runs from a negative to positive charge.</li>
	<li>Remove the gel and place it in the gel imager to visualize DNA.</li>
</ol>
5. Cell cycle analysis
<ol>
	<li>Seed 5 x 105 cells for each sample in a T25 cm2 flask and perform a transfection according to the protocol described in section 1, steps 1-5.</li>
	<li>After 24 h and 48 h, then harvest the cells by trypsinization.</li>
	<li>Transfer the cell suspensions to 15 mL sterile tubes and label them properly.</li>
	<li>Centrifuge samples at 751 x g for 5 min and discard the supernatant.</li>
	<li>Add 2 mL of ice-cold 1x PBS, vortex, and centrifuge at 751 x g for 5 min. Discard the supernatant.</li>
	<li>Repeat the washing step with 1x PBS to remove residual media.</li>
	<li>Resuspend and break the pellet by adding 200 &#181;L of ice-cold 1x PBS by pipetting.</li>
	<li>Fix the cells by adding 2 mL of 70% ice-cold ethanol dropwise to the tube while vortexing gently.</li>
	<li>Incubate tubes for 30 min at RT and place the tubes at 4 &#176;C for 1 h.</li>
	<li>Remove tubes from 4 &#176;C and centrifuge at 751 x g for 5 min.</li>
	<li>Add 2 mL of ice-cold 1x PBS, vortex, and centrifuge at 751 x g for 5 min. Discard the supernatant.</li>
	<li>Add 500 &#181;L of 1x PBS with propidium iodide (50 &#181;g/mL) and ribonuclease A (200 &#181;g/mL)</li>
	<li>Incubate for 30 min at RT while protecting the samples from light.</li>
	<li>Acquire data on flow cytometer. Use forward vs. side scatter (FSC vs. SSC) to select the main population of cells, excluding debris at the bottom left corner of the FSC vs. SSC density plot and cell clusters at the top to top-right side of the FSC vs. SSC density plot.</li>
	<li>Analyze data for identification of cell populations per cell cycle stage with appropriate software.</li>
</ol>
6. Apoptosis assay
<ol>
	<li>Seed 5 x 105 cells for each sample in a T25 cm2 flask and perform a transfection according to the protocol described in section 1, steps 1-5.</li>
	<li>After 24 h and 48 h, harvest the cells by trypsinization.</li>
	<li>Transfer the cell suspensions to 15 mL sterile tubes and label them properly.</li>
	<li>Centrifuge at 751 x g for 5 min and discard the supernatant.</li>
	<li>Add 2 mL of ice-cold 1x PBS, vortex, and centrifuge at 751 x g for 5 min. Discard the supernatant.</li>
	<li>Repeat the washing step with 1x PBS to remove residual media.</li>
	<li>Dilute 10x Annexin V binding buffer to 1x with ice-cold dH2O.</li>
	<li>Add 1 mL of 1x Annexin V binding buffer to each sample tube and resuspend gently.</li>
	<li>Place 96 &#181;L of cell suspension in a 1.5 mL microcentrifuge tube.</li>
	<li>Add 1 &#181;L of Annexin V-FITC conjugate and 12.5 &#181;L of propidium iodide (PI) to the tube containing the cell suspension.</li>
	<li>Incubate the cell suspension for 10 min on ice in the dark.</li>
	<li>Add 250 &#181;L ice-cold 1x Annexin V binding buffer to each sample tube to dilute.</li>
	<li>Analyze samples with flow cytometer immediately.</li>
</ol>
7. Protein expression by antibody cell cycle microarray
<ol>
	<li>Seed 5 x 105 cells for each sample in T25 cm2 flasks and perform a transfection according to the protocol described in section 1, steps 1-5.</li>
	<li>After 24 h and 48 h, harvest cells by trypsinization.</li>
	<li>Transfer cell suspensions to 15 mL tubes and label them accordingly.</li>
	<li>Centrifuge at 751 x g for 5 min and discard the supernatant.</li>
	<li>Add 2 mL of ice-cold 1x PBS, vortex and centrifuge at 751 x g for 5 min. Discard the supernatant.</li>
	<li>Repeat the washing step with 1x PBS.</li>
	<li>Add 150 &#181;L of lysis buffer supplemented with protease inhibitors. Pipet up and down gently to disrupt the cell membranes.</li>
	<li>To prevent any lysis buffer interference, perform a buffer exchange to replace the lysis buffer with labeling buffer, using the manufacturer&#39;s solvent exchanging columns.</li>
	<li>Quantify the total protein with a BCA assay.</li>
	<li>Take 70 &#181;g of protein sample and add labeling buffer to achieve a final volume of 75 &#181;L.</li>
	<li>Add 100 &#181;L of dimethylformamide (DMF) to 1 mg of biotin reagent (biotin/DMF).</li>
	<li>Add 3 &#181;L of the biotin/DMF to each protein sample (biotinylated protein sample) and incubate for 2 h at RT.</li>
	<li>Add 35 &#181;L of stop reagent and mix by vortexing.</li>
	<li>Incubate samples at RT for 30 min.</li>
	<li>Remove the microarray slides from the refrigerator so that they warm to RT for 1 h before use.</li>
	<li>Perform blocking for non-specific binding by incubating the slides with 3% dry milk solution (in blocking reagent provided by manufacturer) in a Petri dish with continuous shaking for 45 min at RT.</li>
	<li>Wash the slides with ddH2O water (unless otherwise specified, washing takes place with ddH2O).</li>
	<li>Repeat the washing step ~10x to completely remove the blocking solution from the slide surfaces. This is important to achieve a uniform and low background.</li>
	<li>Remove excessive water from the slide surfaces and proceed to the next step without letting the slides dry.</li>
	<li>Prepare coupling solution by dissolving 3% dry milk in a coupling reagent.</li>
	<li>Add 6 mL of the coupling solution and to the full quantity of the previously prepared biotinylated protein sample from step 7.12.</li>
	<li>Place one slide in one well of the coupling chamber provided by the vendor and add ~6 mL of protein coupling mix to it. 
	NOTE: Ensure that the slide is completely submerged in protein coupling mix solution.</li>
	<li>Cover the coupling chamber and incubate for 2 h at RT with continuous agitation in an orbital shaker.</li>
	<li>Transfer the slides to a Petri dish and add 30 mL of 1x washing buffer. Place the Petri dish in orbital shaker, shake for 10 min, and discard the solution.</li>
	<li>Repeat step 7.24 2x.</li>
	<li>Rinse the slides with ddH2O water extensively as described in steps 7.17 and 7.18 and proceed to next step immediately to avoid drying.</li>
	<li>Add 30 &#181;L of Cy3-streptavidin (0.5 mg/mL) in 30 mL of detection buffer.</li>
	<li>Place the slide in a Petri dish and add 30 mL of detection buffer containing Cy3-streptavidin.</li>
	<li>Incubate in an orbital shaker for 20 min with continuous shaking protected from light. 
	NOTE: Cy-3 is a fluorescent dye. Cover with aluminum foil or operate under dark conditions to maintain fluorescence intensity.</li>
	<li>Perform steps 7.24-7.26 and allow the slide to dry using a gentle stream of air or placing the slide in a 50 mL conical tube and centrifuge at 1300 x g for 5-10 min.</li>
	<li>Place the slide in slide holder and cover with aluminum foil.</li>
	<li>Scan the slide in a microarray scanner with the appropriate excitation and emission wavelengths. In the case of Cy3, the excitation wavelength peak is at ~550 nm and emission peak at ~570 nm.</li>
	<li>Analyze the data (see Table of Materials) for software used).</li>
</ol>
8. RNA sequencing
<ol>
	<li>Seed 5 x 105 cells for each sample in T25 cm2 flasks and perform a transfection according to the protocol described in section 1, steps 1-5.</li>
	<li>After 24 h and 48 h, harvest the cells by trypsinization.</li>
	<li>Transfer the cell suspensions to 15 mL tubes and label them accordingly.</li>
	<li>Extract RNA according to section 2, steps 1-6.</li>
	<li>Check RNA quality as well as concentration with a bioanalyzer. An RNA integrity score (RIN) above eight and appropriate histograms are necessary to confirm RNA quality.</li>
	<li>From the total RNA, use ~2 &#181;g of the sample for RNA sequencing (messenger RNA from total RNA)</li>
	<li>Sequence using a next-generation sequencer11.</li>
	<li>Run quality trimming and map with a reference genome from FASTQ files generated from the RNA sequencing machine11.</li>
	<li>Upload FASTQ files and raw read the count data to Genebank, following the instructions of the &#60;https://www.ncbi.nlm.nih.gov/&#62; website. 
	NOTE: See accession number SRP133420 for previous results.</li>
</ol>
9. Tube formation assay
<ol>
	<li>Assess the angiogenic potential of miRNA-transfected human umbilical vein endothelial cells (HUVECs) 36 h post-transfection, as described in section 1, steps 1-5, using HUVEC cells instead of A549 cells.</li>
	<li>Starve HUVECs with M199 starvation media for 4 h at 37 &#176;C with 5% CO2.</li>
	<li>Remove reduced growth factor basement membrane matrix from -80 &#176;C and store at 4 &#176;C overnight, allowing gradual thawing to avoid bubble formation and polymerization.</li>
	<li>Carefully and slowly coat wells of a 96 well plate with 0.04 mL of reduced growth factor basement membrane matrix, avoiding bubble formation. Perform the whole procedure under a laminar flow hood.</li>
	<li>Fill adjacent wells of the 96 well plate with 0.1 mL of PBS to maintain humidity and preserve temperature.</li>
	<li>Incubate the 96 well plate at 37 &#176;C for at least 20 min so that polymerization of basement membrane extract is achieved. Do not incubate for more than 1 h.</li>
	<li>Trypsinize transfected HUVECs from each group and resuspend in medium M199 at a concentration of 1 x 105 cells/mL.</li>
	<li>Add 0.1 mL of each cell suspension to the wells containing polymerized basement membrane matrix in the 96 well plate.</li>
	<li>Incubate the 96 well plate at 37 &#176;C with 5% CO2 while preparation of the growth factors takes place. Preparation of growth factors also takes place under the laminar flow hood.</li>
	<li>Reconstitute growth factors (VEGF) in 2x the final desired concentration (4 ng/mL concentration for 2 ng/mL final concentration) and add 0.1 mL of growth factor-containing M199 starvation medium on top of the 0.1 mL of M199 starvation medium with the cells. For non-VEGF-treated wells, add 0.1 mL of M199 starvation medium on top of the 0.1 mL of M199 starvation medium with the cells.</li>
	<li>Incubate the 96 well plate for 6 h at 37 &#176;C with 5% CO2.</li>
	<li>At the end of the incubation period, obtain images of each well with 4x magnification, using a brightfield microscope connected with a digital camera.</li>
	<li>Process images with software equipped with an &#34;angiogenesis analyzer&#34; plug-in19. Use three parameters, the number of nodes, number of junctions, and total sprout length, to compare the effects of miRNA treatment on angiogenesis.</li>
</ol>

<ol>
	<li>Schafer, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+cycle:+a+review.">The cell cycle: a review.</a> Veternary Pathology. 35 (6), 461-478 (1998).</li><li>Barnum, K. J., O'Connell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+regulation+by+checkpoints.">Cell cycle regulation by checkpoints.</a> Methods in Molecular Biology. 1170, 29-40 (2014).</li><li>Malumbres, M., Barbacid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle,+CDKs+and+cancer:+a+changing+paradigm.">Cell cycle, CDKs and cancer: a changing paradigm.</a> Nature Reviews Cancer. 9 (3), 153-166 (2009).</li><li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+CDK+inhibitor+dinaciclib+suppresses+neuroblastoma+growth+via+inhibiting+CDK2+and+CDK9+activity.">Multiple CDK inhibitor dinaciclib suppresses neuroblastoma growth via inhibiting CDK2 and CDK9 activity.</a> Science Repository. 6, 29090 (2016).</li><li>Brown, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CDK1+structures+reveal+conserved+and+unique+features+of+the+essential+cell+cycle+CDK.">CDK1 structures reveal conserved and unique features of the essential cell cycle CDK.</a> Nature Communications. 6, 6769 (2015).</li><li>Sanchez-Martinez, C., Gelbert, L. M., Lallena, M. J., de Dios, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+dependent+kinase+(CDK)+inhibitors+as+anticancer+drugs.">Cyclin dependent kinase (CDK) inhibitors as anticancer drugs.</a> Bioorganic &amp; Medicinal Chemistry Letters. 25 (17), 3420-3435 (2015).</li><li>Shah, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+Approval:+Ribociclib+for+the+Treatment+of+Postmenopausal+Women+with+Hormone+Receptor-Positive,+HER2-Negative+Advanced+or+Metastatic+Breast+Cancer.">FDA Approval: Ribociclib for the Treatment of Postmenopausal Women with Hormone Receptor-Positive, HER2-Negative Advanced or Metastatic Breast Cancer.</a> Clinical Cancer Research. , (2018).</li><li>Asghar, U., Witkiewicz, A. K., Turner, N. C., Knudsen, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+and+future+of+targeting+cyclin-dependent+kinases+in+cancer+therapy.">The history and future of targeting cyclin-dependent kinases in cancer therapy.</a> Nature Reviews Drug Discovery. 14 (2), 130-146 (2015).</li><li>Mullard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+approves+Novartis's+CDK4/6+inhibitor.">FDA approves Novartis's CDK4/6 inhibitor.</a> Nature Reviews Drug Discovery. 16 (4), 229 (2017).</li><li>Walker, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+Approval+of+Palbociclib+in+Combination+with+Fulvestrant+for+the+Treatment+of+Hormone+Receptor-Positive,+HER2-Negative+Metastatic+Breast+Cancer.">FDA Approval of Palbociclib in Combination with Fulvestrant for the Treatment of Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer.</a> Clinical Cancer Research. 22 (20), 4968-4972 (2016).</li><li>Hossian, A., Sajib, M. S., Tullar, P. E., Mikelis, C. M., Mattheolabakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipronged+activity+of+combinatorial+miR-143+and+miR-506+inhibits+Lung+Cancer+cell+cycle+progression+and+angiogenesis+in+vitro.">Multipronged activity of combinatorial miR-143 and miR-506 inhibits Lung Cancer cell cycle progression and angiogenesis in vitro.</a> Science Repository. 8 (1), 10495 (2018).</li><li>Inamura, K., Ishikawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+In+Lung+Cancer:+Novel+Biomarkers+and+Potential+Tools+for+Treatment.">MicroRNA In Lung Cancer: Novel Biomarkers and Potential Tools for Treatment.</a> Journal of Clinical Medicine. 5 (3), (2016).</li><li>Mizuno, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+expression+signature+of+small+cell+lung+cancer:+tumor+suppressors+of+miR-27a-5p+and+miR-34b-3p+and+their+targeted+oncogenes.">The microRNA expression signature of small cell lung cancer: tumor suppressors of miR-27a-5p and miR-34b-3p and their targeted oncogenes.</a> Journal of Human Genetics. 62 (7), 671-678 (2017).</li><li>Zhang, B., Pan, X., Cobb, G. P., Anderson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNAs+as+oncogenes+and+tumor+suppressors.">microRNAs as oncogenes and tumor suppressors.</a> Developmental Biology. 302 (1), 1-12 (2007).</li><li>Peng, Y., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+MicroRNAs+in+human+cancer.">The role of MicroRNAs in human cancer.</a> Signal Transduction and Targeted Therapy. 1, 15004 (2016).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+recurrence+in+early+stage+non-small+cell+lung+cancer+using+computer+extracted+nuclear+features+from+digital+H&amp;E+images.">Prediction of recurrence in early stage non-small cell lung cancer using computer extracted nuclear features from digital H&amp;E images.</a> Science Repository. 7 (1), 13543 (2017).</li><li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cancer Statistics, 2017. CA: A Cancer Journal for Clinicians. 67 (1), 7-30 (2017).</li><li>Saxon, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p52+expression+enhances+lung+cancer+progression.">p52 expression enhances lung cancer progression.</a> Science Repository. 8 (1), 6078 (2018).</li><li>Carpentier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+Analyzer+for+ImageJ.">Angiogenesis Analyzer for ImageJ.</a> ImageJ User and Developer Conference. , (2012).</li><li>Robinson, M. D., McCarthy, D. J., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edgeR:+a+Bioconductor+package+for+differential+expression+analysis+of+digital+gene+expression+data.">edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.</a> Bioinformatics. 26 (1), 139-140 (2010).</li><li>Robinson, M. D., Oshlack, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scaling+normalization+method+for+differential+expression+analysis+of+RNA-seq+data.">A scaling normalization method for differential expression analysis of RNA-seq data.</a> Genome Biology. 11 (3), R25 (2010).</li><li>DeCicco-Skinner, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+tube+formation+assay+for+the+in+vitro+study+of+angiogenesis.">Endothelial cell tube formation assay for the in vitro study of angiogenesis.</a> Journal of Visualized Experiments. (91), e51312 (2014).</li><li>Kong, D. H., Kim, M. R., Jang, J. H., Na, H. J., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+Anti-Angiogenic+Targets+for+Monoclonal+Antibody+Cancer+Therapy.">A Review of Anti-Angiogenic Targets for Monoclonal Antibody Cancer Therapy.</a> International Journal of Molecular Science. 18 (8), (2017).</li><li>Wong, P. P., Bodrug, N., Hodivala-Dilke, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+Novel+Methods+for+Modulating+Tumor+Blood+Vessels+in+Cancer+Treatment.">Exploring Novel Methods for Modulating Tumor Blood Vessels in Cancer Treatment.</a> Current Biology. 26 (21), R1161-R1166 (2016).</li><li>Evan, G. I., Brown, L., Whyte, M., Harrington, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+and+the+cell+cycle.">Apoptosis and the cell cycle.</a> Current Opinion in Cell Biology. 7 (6), 825-834 (1995).</li><li>Haab, B. B., Dunham, M. J., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+microarrays+for+highly+parallel+detection+and+quantitation+of+specific+proteins+and+antibodies+in+complex+solutions.">Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.</a> Genome Biology. 2 (2), RESEARCH0004 (2001).</li><li>Sutandy, F. X., Qian, J., Chen, C. S., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+protein+microarrays.">Overview of protein microarrays.</a> Currrent Protocols in Protein Science. 27, 21 (2013).</li><li>St-Pierre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+sequencing+of+neonatal+thymic+epithelial+cells.">Transcriptome sequencing of neonatal thymic epithelial cells.</a> Science Repository. 3, 1860 (2013).</li></ol>

The authors would like to acknowledge John Caskey at Louisiana State University for his assistance on the pathway analysis of the RNA sequencing data, and the University of Texas Southwestern Medical Center, McDermott Center Next Generation Sequencing Core for performing the RNA sequencing and data analysis. This research was supported by the College of Pharmacy, University of Louisiana Monroe start-up funding, and the National Institutes of Health (NIH) through the National Institute of General Medical Science Grants 5 P20 GM103424-15, 3 P20 GM103424-15S1.

miRNAs can operate as targeted therapies for cancer treatment, recognizing the dysregulation of expression levels in diseased vs. normal tissues. This study aimed to determine miRNAs that potentially halt cell cycle progression during multiple stages. It was identified that miR-143 and miR-506 halt the cell cycle of cancer cells, and the presented protocols aimed to comprehend the activity of this combinatorial miRNA treatment.
The described methodologies provide an overarching understanding o...

An erratum was issued for:&#160;<a href="https://www.jove.com/video/59460/analysis-combinatorial-mirna-treatments-to-regulate-cell-cycle">Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis</a>.&#160; An author name was updated.
One of the authors&#39; names was corrected from:
George Matthaiolampakis
to:
George Mattheolabakis

An erratum was issued for: <a href="https://www.jove.com/video/59460/analysis-combinatorial-mirna-treatments-to-regulate-cell-cycle">Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis</a>.&#160; An author name was updated.

Erratum: Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

The cell cycle is a combination of multiple regulatory events that allow duplication of DNA and cell proliferation through the mitotic process1. Cyclin-dependent kinases (CDKs) regulate and promote the cell cycle2. Among them, the mitotic CDK (CDK1) and interphase CDKs (CDK2, CDK4, and CDK6) have a pivotal role in cell cycle progression3. Retinoblastoma protein (Rb) is phosphorylated by the CDK4/CDK6 complex to allow cell cycle progression4, and CDK1 activation is essential for successful cell division5. Numerous CDK inhibitors have been developed and evaluated in clinical trials over the last few decades, indicating the potential of targeting CDKs in cancer treatment. In fact, three CDK inhibitors have been approved for the treatment of breast cancer recently6,7,8,9,10. Thus, CDKs, and in particular, CDK1 and CDK4/6, are of great interest in regulating cancer cell progression.
miRNAs (miRs) are small, non-coding RNAs and post-transcriptional regulators of gene expression, regulating approximately 30% of all human genes11. Their activity is based on translational repression or degradation of messenger RNAs (mRNAs)12. Illustrative of their biological significance, more than 5,000 miRNAs have been identified and a single miRNA molecule can regulate multiple genes11,13. More importantly, miRNA expression has been associated with different diseases and disease statuses, including cancer13. In fact, miRNAs have been characterized as oncogenic or tumor suppressors, being capable to either promote or suppress tumor development and progression14,15. The relative expression of miRNAs in diseased tissues can regulate disease progression; thus, exogenous delivery of miRNAs has therapeutic potential.
Lung cancer is the leading cause of cancer-related deaths and greater than 60% of all lung malignancies are non-small cell lung cancers16,17, with a 5-year survival rate of less than 20%18. The use of miR-143-3p and miR-506-3p was recently evaluated for targeting the cell cycles in lung cancer cells11. miR-143 and miR-506 have sequences that are complementarity to CDK1 and CDK4/CDK6, and the effects of these two miRs on A549 cells were analyzed. The experimental details are presented and discussed in this paper. Gene expression, cell cycle progression, and apoptosis were evaluated using different experimental designs and timepoints following transfection. We used real-time quantitative PCR (RT-qPCR) methods along with microarray analysis to measure specific gene expression, and next-generation RNA sequencing was used to determine global gene dysregulation11. The latter method identifies the relative abundance of each gene&#39;s transcript with high sensitivity and reproducibility, while thousands of genes can be analyzed from a single experimental analysis. Additionally, apoptotic analysis due to miRNA treatment was performed and is described here. Bioinformatics supplemented the pathway analysis. Presented here are protocols used for analysis of the therapeutic potential of the combinatorial miR-143 and miR-506.
The main purpose of this protocol is to identify the effects of miRNAs in cells, with a focus on the cell cycle. The variety of techniques presented here span from gene expression analysis pre-translation (using qPCR) to elaborate and novel techniques for gene analysis at the protein level, such as microarray analysis. It is hoped that this report is helpful for researchers interested in working with miRNAs. Additionally, methodology for flow cytometric analysis of the cell cycle and apoptosis of cells is presented.

<table><tbody><tr><td>-80 &amp;deg;C Freezer</td><td>VWR</td><td>VWR40086A</td><td></td></tr><tr><td>96 well plate</td><td>CELLTREAT Scientific&amp;nbsp;</td><td>50-607-511</td><td></td></tr><tr><td>96-well Microwell Plates&amp;nbsp;&amp;nbsp;</td><td>Thermo Scientific</td><td>12-556-008</td><td></td></tr><tr><td>A549 Non Small Cell Lung Cancer Cells</td><td>ATCC</td><td>ATCC&amp;nbsp;CCL-185</td><td></td></tr><tr><td>Agarose</td><td>VWR</td><td>0710-25G</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2938c</td><td></td></tr><tr><td>Ambion Silencer Negative Control No. 1 siRNA</td><td>Ambion</td><td>AM4611</td><td></td></tr><tr><td>Antibiotic-Antimycotic Solution (100x)</td><td>Gibco</td><td>15240-062&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Antibody Array Assay Kit, 2 Reactions</td><td>Full Moon Bio</td><td>KAS02</td><td></td></tr><tr><td>Bright field microscope&amp;nbsp;&amp;nbsp;</td><td>Microscoptics&amp;nbsp;</td><td>IV-900</td><td></td></tr><tr><td>Bright field microscope&amp;nbsp;&amp;nbsp;</td><td>New Star Environment LLC</td><td></td><td></td></tr><tr><td>Cell Cycle Antibody Array, 2 Slides</td><td>Full Moon Bio</td><td>ACC058</td><td></td></tr><tr><td>Cell Logic+ Biosafety Cabinate</td><td>Labconco</td><td>342391100</td><td></td></tr><tr><td>Cellquest Pro</td><td>BD bioscience</td><td>Steps 5.14; 6.13: Used for calculating the population distrubution according to the cell cycle&amp;nbsp; phase and for&amp;nbsp; calculating the population distribution for the analysis of apoptosis&amp;nbsp;</td><td></td></tr><tr><td>CFX96 Real Time System</td><td>BioRad</td><td>CFX96 Optics Module</td><td></td></tr><tr><td>Chemidoc Touch Imaging System</td><td>BioRad</td><td>Chemidoc Touch Imaging System</td><td></td></tr><tr><td>CO2 Incubator</td><td>Thermo Scientific</td><td>HERAcell 150i</td><td></td></tr><tr><td>Cultrex Reduced Growth Factor Basement Membrane Matrix</td><td>Trevigen</td><td>3433-010-01</td><td></td></tr><tr><td>Digital Camera</td><td>AmScope&amp;nbsp;</td><td>FMA050</td><td></td></tr><tr><td>DMEM 4.5 g/L Glucose, w/out Sodium Pyruvate, w/ L-Glutamine</td><td>VWR</td><td>VWRL0100-0500</td><td></td></tr><tr><td>DNAse I</td><td>Zymo Research</td><td>E1010</td><td></td></tr><tr><td>Endothelial Cell Growth Supplement (ECGS)</td><td>BD Biosciences</td><td>356006</td><td></td></tr><tr><td>Eppendorf Pipette Pick-A-Pack Sets</td><td>Eppendrof</td><td>05-403-152</td><td></td></tr><tr><td>Ethanol, Absolute (200 Proof), Molecular Biology Grade,&amp;nbsp;</td><td>Fisher BioReagents</td><td>BP2818500</td><td></td></tr><tr><td>Ethidium bromide</td><td>Alfa acar</td><td>L07462</td><td></td></tr><tr><td>F-12K Nutrient Mixture (Kaighn&#039;s Mod.) with L-glutamine, Corning</td><td>Corning</td><td>45000-354</td><td></td></tr><tr><td>FACS Calibur Flowcytometer</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Antlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Fisher Scientific</td><td>10438026</td><td></td></tr><tr><td>Fisherbrand Basix Microcentrifuge Tubes with Standard Snap Caps</td><td>Fisherbrand Basix</td><td>02-682-002</td><td></td></tr><tr><td>Forma Series II water Jacket CO2 incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Heparin Solution (5000 U/mL)</td><td>Hospira</td><td>NDC#63739-920-11</td><td></td></tr><tr><td>Horixontal Electrophoresis system</td><td>Benchtop lab system</td><td>BT102</td><td></td></tr><tr><td>hsa-miR-143-3p miRNA Mimic</td><td>ABM</td><td>MCH01315</td><td></td></tr><tr><td>hsa-miR-506-3p miRNA Mimic</td><td>ABM</td><td>MCH02824</td><td></td></tr><tr><td>Human Recombinant Vascular Endothelial Growth Factor (VEGF)</td><td>Thermo Scientific</td><td>PHC9394&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Human Umbilical Vein Endothelial Cells (HUVEC)</td><td>Individual donors</td><td>IRB# A15-3891</td><td></td></tr><tr><td>HyClone Phosphate Buffered Saline (PBS)</td><td>Fisher Scientific</td><td>SH30256FS</td><td></td></tr><tr><td>Ingenuity Pathway Analysis</td><td>Qiagen</td><td>Results: Used for bioinformatics pathway analysis</td><td></td></tr><tr><td>Invitrogen UltraPure DNase/RNase-Free Distilled Water</td><td>Invitrogen</td><td>10-977-015</td><td></td></tr><tr><td>Lipofectamine 2000&amp;nbsp;</td><td>Invitrogen</td><td>11-668-027</td><td></td></tr><tr><td>Loading dye 10X</td><td>ward&#039;s science+</td><td>470024-814</td><td></td></tr><tr><td>Medium M199 (with Earle&amp;prime;s salts, L-glutamine and sodium bicarbonate)</td><td>Sigma Aldrich</td><td>M4530</td><td></td></tr><tr><td>Microscope Digital Camera</td><td>AmScope&amp;nbsp;</td><td>MU130</td><td></td></tr><tr><td>Modfit LT</td><td>Verity Software</td><td>Step 5.15: Alternative software for analysis of cell cycle population distributions</td><td></td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td>NanoDrop one C</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco by life technologies</td><td>31985-070</td><td></td></tr><tr><td>Penicillin-streptomycin 10/10</td><td>Antlanta Biologicals</td><td>B21210</td><td></td></tr><tr><td>Power UP sybr green master mix</td><td>Applied Biosystems</td><td>A25780</td><td></td></tr><tr><td>Propidium Iodide</td><td>MP Biochemicals LLC</td><td>IC19545825</td><td></td></tr><tr><td>Proscanarray HT Microarray scanner</td><td>Perkin elmer</td><td>ASCNPHRG. We used excitation laser wavelength at 543 nm.</td><td></td></tr><tr><td>q PCR optical adhesive cover</td><td>Applied Biosystems</td><td>4360954</td><td></td></tr><tr><td>Quick-RNA Kits</td><td>Zymo Research</td><td>R1055</td><td></td></tr><tr><td>Ribonuclease A from Bovine pancreas</td><td>Sigma</td><td>R6513-50MG</td><td></td></tr><tr><td>ScanArray Express</td><td>PerkinElmer</td><td>Step 7.33: Microarray analysis software</td><td></td></tr><tr><td>Shaker</td><td>Thermo Scientific</td><td>2314</td><td></td></tr><tr><td>SimpliAmp Thermal Cycler</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>SpectraTube Centrifuge Tubes 15ml</td><td>VWR</td><td>470224-998</td><td></td></tr><tr><td>SpectraTube Centrifuge Tubes 50ml</td><td>VWR</td><td>470225-004</td><td></td></tr><tr><td>TBS Buffer, 20x liquid</td><td>VWR</td><td>10791-796</td><td></td></tr><tr><td>Temperature controlled&amp;nbsp; centrifuge matchine</td><td>Thermo Scientific</td><td>ST16R</td><td></td></tr><tr><td>Temperature controlled micro centrifuge matchine</td><td>Eppendrof</td><td>5415R</td><td></td></tr><tr><td>Thermo Scientific BioLite Cell Culture Treated Flasks</td><td>Thermo Scientific</td><td>12-556-009</td><td></td></tr><tr><td>Thermo Scientific Pierce BCA Protein Assay</td><td>Thermo Scientific</td><td>PI23225</td><td></td></tr><tr><td>Thermo Scientific Pierce RIPA Buffer</td><td>Thermo Scientific</td><td>PI89900</td><td></td></tr><tr><td>Thermo Scientific Thermo-Fast 96-Well Full-Skirted Plates</td><td>Thermo Scientific</td><td>AB0800WL</td><td></td></tr><tr><td>Thermo Scientific Verso cDNA synthesis Kit (100 runs)</td><td>Thermo Scientific</td><td>AB1453B</td><td></td></tr><tr><td>Ultra Low Range DNA Ladder</td><td>Invitrogen</td><td>10597012</td><td></td></tr><tr><td>VWR standard solid door laboratory refrigerator</td><td>VWR</td><td></td><td></td></tr></tbody></table>

analysis of combinatorial mirna treatments to regulate cell cycle and angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

Gene expression analysis using RT-qPCR and gel electrophoresis
Differential gene expression analysis using RT-qPCR demonstrated significant downregulation of the targeted genes CDK1, CDK4, and CDK6. CDK1 and CDK4/6 were shown to be instrumental for the G2/M and G1/S transitions, respectively. The performed analysis allowed direct comparison between individual miRs and combinatorial miR activity. The use of scram...

Watch this Scientific Journal Video about Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis at JoVE.com

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

analysis-combinatorial-mirna-treatments-to-regulate-cell-cycle

Research

JoVE Journal

Cancer Research

7.5K Views.  University of Louisiana Monroe. Deze methode zal helpen begrijpen en uitvoeren lab technieken die nodig zijn om de activiteit van twee microRNA in longkankercellen in vitro te identificeren. Longkanker is wereldwijd de belangrijkste oorzaak van kankergerelateerde sterfgevallen. Hier presenteren we technieken die zich uitstrekken van basislabmethoden tot complexere protocollen, zoals genexpressieanalyse en antilichaameiwitmicroRNA, specifiek gebruikt voor het identificeren van het effect van de acties van microRNA.De ...

Video: Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Genetics

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Medicine

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Biology

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

mirMachine: A One-Stop Shop for Plant miRNA Annotation

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of gene expression, miRNAs play key roles in various cellular pathways, including both development and response to a/biotic stress, such as drought and diseases. Having high-quality reference genome sequences enabled identification and annotation of miRNAs in several plant species, where miRNA sequences are highly conserved. As computational miRNA identification and annotation processes are mostly error-prone processes, homology-based predictions increase prediction accuracy. We developed and have improved the miRNA annotation pipeline, SUmir, in the last decade, which has been used for several plant genomes since then. 
This study presents a fully automated, new miRNA pipeline, mirMachine (miRNA Machine), by (i) adding an additional filtering step on the secondary structure predictions, (ii) making it fully automated, and (iii) introducing new options to predict either known miRNA based on homology or novel miRNAs based on small RNA sequencing reads using the previous pipeline. The new miRNA pipeline, mirMachine, was tested using The Arabidopsis Information Resource, TAIR10, release of the Arabidopsis genome and the International Wheat Genome Sequencing Consortium (IWGSC) wheat reference genome v2.

Herein, we present a new and fully automated miRNA pipeline, mirMachine that 1) can identify known and novel miRNAs more accurately and 2) is fully automated and freely available. Users can now execute a short submission script to run the fully automated mirMachine pipeline.

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of ...

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

Cell Cycle Analysis: An Approach to Study Cell Cycle Regulation of miRNA-transfected Lung Cancer Cells

Source: Hossian, A. K. M. N., et al. <a href="https://www.jove.com/v/59460/analysis-combinatorial-mirna-treatments-to-regulate-cell-cycle">Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis.</a>&#160;J. Vis. Exp.&#160;(2019).
miRNA or microRNA are key regulators of cell cycle progression. This video describes a flow cytometry-based cell cycle analysis of lung cancer cells transfected with miRNA, to study the effect of miRNA on cell cycle regulation. Inhibition of proliferation by halting cell cycle progression has shown potential to be a promising approach for cancer treatment.

Source: Hossian, A. K. M. N., et al. Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis.&#160;J. Vis. Exp.&#160;(2019).
...

JoVE Encyclopedia of Experiments

Lung Cancer

In vitro study

Antibody Microarray: A Technique to Study the Protein Expression of miRNA Treated Lung Cancer Cells

Source: Hossian, A. et. al. <a href="https://www.jove.com/v/59460/analysis-combinatorial-mirna-treatments-to-regulate-cell-cycle">Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis.</a> J. Vis. Exp. (2019).
This video describes the technique to study the protein expression in miRNA transfected lung cancer cells by using antibody microarray to measure specific gene expression. In the featured protocol, we perform antibody microarray for the identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Source: Hossian, A. et. al. Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis. J. Vis. Exp. (2019).
This video describes ...

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

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