Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis
Summary
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.
Abstract
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.
Introduction
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'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.
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
No conflicts of interest are declared.
Materials
| -80 °C Freezer | VWR | VWR40086A | |
| 96 well plate | CELLTREAT Scientific | 50-607-511 | |
| 96-well Microwell Plates | Thermo Scientific | 12-556-008 | |
| A549 Non Small Cell Lung Cancer Cells | ATCC | ATCC CCL-185 | |
| Agarose | VWR | 0710-25G | |
| Agilent 2100 Bioanalyzer | Agilent Technologies | G2938c | |
| Ambion Silencer Negative Control No. 1 siRNA | Ambion | AM4611 | |
| Antibiotic-Antimycotic Solution (100x) | Gibco | 15240-062 | |
| Antibody Array Assay Kit, 2 Reactions | Full Moon Bio | KAS02 | |
| Bright field microscope | Microscoptics | IV-900 | |
| Bright field microscope | New Star Environment LLC | ||
| Cell Cycle Antibody Array, 2 Slides | Full Moon Bio | ACC058 | |
| Cell Logic+ Biosafety Cabinate | Labconco | 342391100 | |
| Cellquest Pro | BD bioscience | Steps 5.14; 6.13: Used for calculating the population distrubution according to the cell cycle phase and for calculating the population distribution for the analysis of apoptosis | |
| CFX96 Real Time System | BioRad | CFX96 Optics Module | |
| Chemidoc Touch Imaging System | BioRad | Chemidoc Touch Imaging System | |
| CO2 Incubator | Thermo Scientific | HERAcell 150i | |
| Cultrex Reduced Growth Factor Basement Membrane Matrix | Trevigen | 3433-010-01 | |
| Digital Camera | AmScope | FMA050 | |
| DMEM 4.5 g/L Glucose, w/out Sodium Pyruvate, w/ L-Glutamine | VWR | VWRL0100-0500 | |
| DNAse I | Zymo Research | E1010 | |
| Endothelial Cell Growth Supplement (ECGS) | BD Biosciences | 356006 | |
| Eppendorf Pipette Pick-A-Pack Sets | Eppendrof | 05-403-152 | |
| Ethanol, Absolute (200 Proof), Molecular Biology Grade, | Fisher BioReagents | BP2818500 | |
| Ethidium bromide | Alfa acar | L07462 | |
| F-12K Nutrient Mixture (Kaighn's Mod.) with L-glutamine, Corning | Corning | 45000-354 | |
| FACS Calibur Flowcytometer | Becton Dickinson | ||
| Fetal Bovine Serum – Premium | Antlanta Biologicals | S11150 | |
| Fetal Bovine Serum (FBS) | Fisher Scientific | 10438026 | |
| Fisherbrand Basix Microcentrifuge Tubes with Standard Snap Caps | Fisherbrand Basix | 02-682-002 | |
| Forma Series II water Jacket CO2 incubator | Thermo Scientific | ||
| Heparin Solution (5000 U/mL) | Hospira | NDC#63739-920-11 | |
| Horixontal Electrophoresis system | Benchtop lab system | BT102 | |
| hsa-miR-143-3p miRNA Mimic | ABM | MCH01315 | |
| hsa-miR-506-3p miRNA Mimic | ABM | MCH02824 | |
| Human Recombinant Vascular Endothelial Growth Factor (VEGF) | Thermo Scientific | PHC9394 | |
| Human Umbilical Vein Endothelial Cells (HUVEC) | Individual donors | IRB# A15-3891 | |
| HyClone Phosphate Buffered Saline (PBS) | Fisher Scientific | SH30256FS | |
| Ingenuity Pathway Analysis | Qiagen | Results: Used for bioinformatics pathway analysis | |
| Invitrogen UltraPure DNase/RNase-Free Distilled Water | Invitrogen | 10-977-015 | |
| Lipofectamine 2000 | Invitrogen | 11-668-027 | |
| Loading dye 10X | ward's science+ | 470024-814 | |
| Medium M199 (with Earle′s salts, L-glutamine and sodium bicarbonate) | Sigma Aldrich | M4530 | |
| Microscope Digital Camera | AmScope | MU130 | |
| Modfit LT | Verity Software | Step 5.15: Alternative software for analysis of cell cycle population distributions | |
| Nanodrop | Thermo Scientific | NanoDrop one C | |
| Opti-MEM | Gibco by life technologies | 31985-070 | |
| Penicillin-streptomycin 10/10 | Antlanta Biologicals | B21210 | |
| Power UP sybr green master mix | Applied Biosystems | A25780 | |
| Propidium Iodide | MP Biochemicals LLC | IC19545825 | |
| Proscanarray HT Microarray scanner | Perkin elmer | ASCNPHRG. We used excitation laser wavelength at 543 nm. | |
| q PCR optical adhesive cover | Applied Biosystems | 4360954 | |
| Quick-RNA Kits | Zymo Research | R1055 | |
| Ribonuclease A from Bovine pancreas | Sigma | R6513-50MG | |
| ScanArray Express | PerkinElmer | Step 7.33: Microarray analysis software | |
| Shaker | Thermo Scientific | 2314 | |
| SimpliAmp Thermal Cycler | Applied Biosystems | ||
| SpectraTube Centrifuge Tubes 15ml | VWR | 470224-998 | |
| SpectraTube Centrifuge Tubes 50ml | VWR | 470225-004 | |
| TBS Buffer, 20x liquid | VWR | 10791-796 | |
| Temperature controlled centrifuge matchine | Thermo Scientific | ST16R | |
| Temperature controlled micro centrifuge matchine | Eppendrof | 5415R | |
| Thermo Scientific BioLite Cell Culture Treated Flasks | Thermo Scientific | 12-556-009 | |
| Thermo Scientific Pierce BCA Protein Assay | Thermo Scientific | PI23225 | |
| Thermo Scientific Pierce RIPA Buffer | Thermo Scientific | PI89900 | |
| Thermo Scientific Thermo-Fast 96-Well Full-Skirted Plates | Thermo Scientific | AB0800WL | |
| Thermo Scientific Verso cDNA synthesis Kit (100 runs) | Thermo Scientific | AB1453B | |
| Ultra Low Range DNA Ladder | Invitrogen | 10597012 | |
| VWR standard solid door laboratory refrigerator | VWR |
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