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...

<ol>
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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;">Heparin Solution (5000 U/mL)</td>
			<td style="width:210px;">Hospira</td>
			<td style="width:1167px;">NDC#63739-920-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Horixontal Electrophoresis system</td>
			<td style="width:210px;">Benchtop lab system</td>
			<td style="width:1167px;">BT102</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-143-3p miRNA Mimic</td>
			<td style="width:210px;">ABM</td>
			<td>MCH01315</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-506-3p miRNA Mimic</td>
			<td style="width:210px;">ABM</td>
			<td>MCH02824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Human Recombinant Vascular Endothelial Growth Factor (VEGF)</td>
			<td style="width:210px;">Thermo Scientific</td>
			<td style="width:1167px;">PHC9394&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:210px;">Individual donors</td>
			<td style="width:1167px;">IRB# A15-3891</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">HyClone Phosphate Buffered Saline (PBS)</td>
			<td style="width:210px;">Fisher Scientific</td>
			<td style="width:1167px;">SH30256FS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Ingenuity Pathway Analysis</td>
			<td style="width:210px;">Qiagen</td>
			<td style="width:1167px;">Results: Used for bioinformatics pathway analysis</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Invitrogen UltraPure DNase/RNase-Free Distilled Water</td>
			<td style="width:210px;">Invitrogen</td>
			<td>10-977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Lipofectamine 2000&#160;</td>
			<td style="width:210px;">Invitrogen</td>
			<td style="width:1167px;">11-668-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Loading dye 10X</td>
			<td style="width:210px;">ward&#39;s science+</td>
			<td style="width:1167px;">470024-814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Medium M199 (with Earle&#8242;s salts, L-glutamine and sodium bicarbonate)</td>
			<td style="width:210px;">Sigma Aldrich</td>
			<td style="width:1167px;">M4530</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Microscope Digital Camera</td>
			<td style="width:210px;">AmScope&#160;</td>
			<td style="width:1167px;">MU130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Modfit LT</td>
			<td style="width:210px;">Verity Software</td>
			<td style="width:1167px;">Step 5.15: Alternative software for analysis of cell cycle population distributions</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Nanodrop</td>
			<td style="width:210px;">Thermo Scientific</td>
			<td style="width:1167px;">NanoDrop one C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM</td>
			<td style="width:210px;">Gibco by life technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin 10/10</td>
			<td>Antlanta Biologicals</td>
			<td>B21210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Power UP sybr green master mix</td>
			<td style="width:210px;">Applied Biosystems</td>
			<td>A25780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium Iodide</td>
			<td style="width:210px;">MP Biochemicals LLC</td>
			<td>IC19545825</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:519px;">Proscanarray HT Microarray scanner</td>
			<td style="width:210px;">Perkin elmer</td>
			<td style="width:1167px;">ASCNPHRG. We used excitation laser wavelength at 543 nm.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">q PCR optical adhesive cover</td>
			<td style="width:210px;">Applied Biosystems</td>
			<td style="width:1167px;">4360954</td>
			<td></td>
		</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 ...

This method will help understand and perform lab techniques necessary to identify the activity of two microRNA in lung cancer cells in vitro. Lung cancer is the leading cause of cancer-related death worldwide. Here, we present techniques spanning from basic lab methodologies to more complex protocols, such as gene expression analysis and antibody protein microRNA, specifically used for identifying the effect of microRNA's actions.The following methods will help us answer key questions regarding the effect of two microRNAs, microRNA 143 and microRNA 506, on regulating the cell cycle in lung cancer cells. We present different lab techniques, like cell transfection, RNA extraction, quantitative real time PCR and microRNA analysis. We hope our presentation will contribute on successfully completing these analyses.First, seat the cultured cells in an appropriate flask sufficient for RNA and protein extraction, to perform gene expression using qPCR or micro-array analysis. Then incubate overnight with media supplemented with 10 percent FBS and 1 percent penicillin streptomycin. The next day, prepare the microRNA sample for transfection by diluting microRNA 143 and microRNA 506 in media at a concentration of 100 nanomolar each.Take out the flask from the incubator and remove the media. Wash with 1x PBS. All the untreated controls will contain only incomplete media with cells.Incubate the cells with transfection media for six hours in an incubator. After six hours, remove the media and replace with four milliliters of fresh, complete media. Harvest the cells after 24 hours and 48 hours by trypsinization.Next, wash twice with 1x PBS in each tube and remove supernatant. In order to perform RNA extraction at a later time, freeze the cells at minus 80 degrees Celsius. RNA extraction was performed using an RNA kit, following manufacturer's instructions.First, remove the tubes from minus 80 degrees Celsius and allow to thaw. Add 300 microliters of lysis buffer and pipette up and down to break cell membrane. Add equal volume of 100 percent ultra-pure ethanol.Then mix well and place in separated column. Centrifuge and remove the flow-through. Add 400 microliters of wash and buffer and centrifuge to remove the buffer.Add 5 microliters of DNAse 1 with 75 microliters VNA digestion buffer in each sample, and incubate for 15 minutes. Then wash the sample with 400 microliter RNA prep buffer. Next, wash twice with RNA wash buffer.Now, add nuclease-free water to the column, centrifuge and then collect the RNA. Measure the RNA concentration. In this stage, two micrograms of RNA sample can be sent for the RNA sequencing.Prepare the cDNA according to the protocol described in the text section, using thermocycler. Prepare forward and reverse primer solutions with DNAse RNAse-free water to a concentration of 10 micromolar. Prepare a master mix for each gene to be detected according to the number of samples.For each cDNA sample, reaction quantity is according to the table described in the text. Place each sample to their respective wells. For qPCR, it is always good to prepare an Excel document with 96 well plate layout, in order to keep track of the samples.For each sample and analyzed gene, perform their reaction in triplicates, or at least duplicates. Seal the plastic plate with transparent, optically clear plastic sealer. Avoid touching the thin, plastic layer with fingers, as this can produce false signal.Quickly spin the plate to mix all of the reagents to the bottom of their wells. Next, run the sample plate using a quantitative, real-time PCR machine. Get the CT values generated by the qPCR machine, and analyze for relative gene expression.Transfect the cells as described in the earlier section of this video. Harvest cells from each sample in separate, 15 milliliter sterile tubes for trypsinization. Next, wash the cells twice with 1x PBS, by centrifugation at 751G for five minutes, and then remove the supernatant.Re-suspend and break the palette by adding 200 microliter ice-cold 1x PBS through a pipette. Add two milliliters of 70 percent ice-cold ethanol, drop-wise to the tube, while vortexing the tube gently to fix the cells. Incubate tubes for 30 minutes at room temperature and place them in four degrees Celsius for one hour.Remove the tubes from four degrees Celsius and centrifuge. Add 2ml ice-cold PBS and vortex, and then remove the supernatant by centrifugation. Add 500 microliters of 1x PBS containing propidium iodide and ribonuclease A with concentration 50 and 200 micrograms per milliliter, respectively, in each sample, and incubate for 30 minutes at room temperature.Transfer samples into appropriate tubes, protecting from light, and run on flow cytometer. This experiment is to identify all the cell cycle pathway proteins'expression changed by the microRNA treatment. Collect protein samples according to the procedure described in the transfection section, and quantify with BCA assay.Remove the slides from minus four degrees Celsius one hour before experiment to bring to room temperature and to remove moisture. Take 70 microgram of protein samples and prepare sample slides according to the manufacturer's instructions, which is also described step-by-step in the text section. Here to note, proper washing of slides with ultra-pure deionized water is another important step, as this will help to reduce the background of the slides.Additionally, it is very critical in this experiment not to dry up the sample slides until the final step. Finally, scan the slide using micro-array machine and analyze the data. The qPCR analysis of the microRNA-treated samples took place to determine the gene expression of CDK1, CDK4 and CDK6.These genes regulate the cell cycle of normal and cancer cells, and have been targeted as a means for inhibiting tumor progression. The data demonstrated the downregulatory effect of these genes due to the treatment with microRNA 143 and 506. Cell cycle distribution data from flow cytometry demonstrated a population increase in the G0 and G1 phase, and a population decrease in the S phase due to the effect of combinatorial treatment of microRNA 143 and 506.The cell cycle's specific micro-array analysis detected protein expression changes of approximately 60 genes. The represented heat map indicated downregulation at the protein level of multiple genes necessary for the progression of the cell cycle and proliferation, such as CDK2, Helen 1 and 3, RE2F1. In contrast, proteins commonly recognized by their grown inhibitory effect were upregulated.Although the results are semi-quantitative and further analysis is required, the micro-array analysis provides a quick overview of the pathway's activity. We believe, after watching this video, you will be able to perform fundamental, as well complex, lab experimentation that will assist you on the identification of microRNA activity in cells.

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

Research

JoVE Journal

Cancer Research

7.5K visualizações.  University of Louisiana Monroe. Este método ajudará a entender e executar técnicas de laboratório necessárias para identificar a atividade de dois microRNA em células cancerígenas de pulmão in vitro. O câncer de pulmão é a principal causa de morte relacionada ao câncer em todo o mundo. Aqui, apresentamos técnicas que abrangem desde metodologias básicas de laboratório até protocolos mais complexos, como análise de expressão genética e microRNA de proteína de anticorpos, especificamente utilizados para ide...