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>

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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. 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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.

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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Quick-RNA Kits</td>
			<td>Zymo Research</td>
			<td>R1055</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ribonuclease A from Bovine pancreas</td>
			<td style="width:210px;">Sigma</td>
			<td>R6513-50MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">ScanArray Express</td>
			<td>PerkinElmer</td>
			<td style="width:1167px;">Step 7.33: Microarray analysis software</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Shaker</td>
			<td style="width:210px;">Thermo Scientific</td>
			<td style="width:1167px;">2314</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">SimpliAmp Thermal Cycler</td>
			<td style="width:210px;">Applied Biosystems</td>
			<td style="width:1167px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">SpectraTube Centrifuge Tubes 15ml</td>
			<td style="width:210px;">VWR</td>
			<td style="width:1167px;">470224-998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">SpectraTube Centrifuge Tubes 50ml</td>
			<td style="width:210px;">VWR</td>
			<td style="width:1167px;">470225-004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TBS Buffer, 20x liquid</td>
			<td style="width:210px;">VWR</td>
			<td>10791-796</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Temperature controlled&#160; centrifuge matchine</td>
			<td style="width:210px;">Thermo Scientific</td>
			<td style="width:1167px;">ST16R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Temperature controlled micro centrifuge matchine</td>
			<td style="width:210px;">Eppendrof</td>
			<td style="width:1167px;">5415R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermo Scientific BioLite Cell Culture Treated Flasks</td>
			<td>Thermo Scientific</td>
			<td>12-556-009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Thermo Scientific Pierce BCA Protein Assay</td>
			<td style="width:210px;">Thermo Scientific</td>
			<td style="width:1167px;">PI23225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermo Scientific Pierce RIPA Buffer</td>
			<td style="width:210px;">Thermo Scientific</td>
			<td>PI89900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermo Scientific Thermo-Fast 96-Well Full-Skirted Plates</td>
			<td style="width:210px;">Thermo Scientific</td>
			<td style="width:1167px;">AB0800WL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermo Scientific Verso cDNA synthesis Kit (100 runs)</td>
			<td style="width:210px;">Thermo Scientific</td>
			<td>AB1453B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Ultra Low Range DNA Ladder</td>
			<td style="width:210px;">Invitrogen</td>
			<td style="width:1167px;">10597012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">VWR standard solid door laboratory refrigerator</td>
			<td style="width:210px;">VWR</td>
			<td style="width:1167px;"></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. 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.

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

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.

Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems.

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...