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 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.
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.
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.
2. RNA extraction
3. RT-qPCR
Ingredients | Quantity (µL)/sample (20 µL) |
5X cDNA Master mix | 4 |
dNTPs | 2 |
Random hexamers | 1 |
RT enhancer | 1 |
Verso enzyme mix | 1 |
DNase and RNase free water | Required qty after adding RNA to make 20 µL |
Table 1: Materials for cDNA synthesis from RNA samples. Required quantities of respective ingredients to prepare a master mix for one sample for cDNA synthesis.
Ingredients | Quantity (µL)/sample (20 µL) |
SYBR master mix | 10 |
Forward Primer – 10 μM | 2 |
Reverse Primer – 10 μM | 2 |
DNase and RNase free water | 3 |
cDNA sample | 3 |
Table 2: Materials for quantitative real-time PCR from cDNA samples. Required quantity of ingredients to prepare a master mix for one sample for qPCR.
4. Agarose gel electrophoresis to confirm single gene amplification
5. Cell cycle analysis
6. Apoptosis assay
7. Protein expression by antibody cell cycle microarray
8. RNA sequencing
9. Tube formation assay
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 scramble siRNA with the transfecting agent permitted evaluation of any interference from the procedure on detected gene downregulation, which was minimal. The data were statistically analyzed using a two-tailed student's t-test, andp < 0.05 was considered statistically significant (Figure 1). Prior to qPCR, the primer sequences were evaluated using primer-BLAST <https://www.ncbi.nlm.nih.gov/tools/primer-blast/> for single gene amplification. This was also confirmed by analyzing the amplification products through gel electrophoresis. A single band of DNA products was detected for each analyzed gene (Figure 1D), confirming single gene amplification. CDK6 single amplification was confirmed (data not shown).
Figure 1: Relative expression of CDK1, CDK4, and CDK6 genes as detected by qPCR, and gel electrophoresis analysis of the DNA amplification products. miR-143 and miR-506 transfection of A549 cells induced downregulation of CDK1 (A), CDK4 (B), and CDK6 (C) downregulation at 24 h and 48 h post-transfection. DNA amplification products were evaluated by gel electrophoresis (D) to confirm single gene amplification. GAPDH was used as reference gene. Average ± SEM, *p < 0.05; **p < 0.01, two-tailed t-test. This figure has been modified from Hossian et al.11 Please click here to view a larger version of this figure.
Cell cycle distribution using flow cytometry
Propidium iodide staining of cellular nucleic acids is a standard method to visualize cell population in different stages of the cell cycle by quantitation of DNA content. The combinatorial treatment of miR-143 and miR-506 halted the cell cycle at two checkpoints, G0/G1 and G2/M, as indicated through flow cytometric analysis (Figure 2).
Figure 2: Cell cycle analysis of A549 cells transfected with miR-143 and miR-506 at 24 h and 48 h post-transfection. Cell populations percentages for each cell cycle were determined by flow cytometry and DNA-binding propidium iodide. Average ± SEM. This figure is reprinted with modifications from Hossian et al.11 Please click here to view a larger version of this figure.
Annexin V/PI apoptosis assay by flow cytometry
Following transfection of A549 cells with miR-143 and miR-506, an apoptosis assay was performed using Annexin V and PI staining and flow cytometry. It was identified that the combinatorial treatment induced significant apoptosis at 24 h and 48 h timepoints. Compared to the negative controls, the percent-change of apoptotic cells was determined as detected by the Annexin V positive cells, due to the miR treatment as presented in Figure 3.
Figure 3: Illustrative analysis of apoptotic cells. Transection with miR-143 and miR-506 increased the percent of Annexin V positive A549 cells. Average ± SEM. *p < 0.05, **p < 0.01, two-tailed t-test. This figure has been modified from Hossian et al.11 Please click here to view a larger version of this figure.
Cell cycle antibody microarray
Mechanistic responses to treatment can be identified through changes in protein expression. Differential expression was evaluated at a protein level of genes associated with the cell cycle pathway using a pathway-specific antibody microarray. Protein extracts were used for analysis from cells transfected with miR-143/506. The microarray analysis allowed for semi-quantitative analysis of ~60 cell cycle-associated proteins, with six replicates for each specific antibody. The approach allows a broader perspective of mechanistic behavior within a specific pathway, identification of molecular targets for further evaluation, and performing of analysis at the post-translational level. Due to the semi-quantitative principle of the method, any results on specific genes need to be confirmed through western blotting. Indicatively, in this analysis, a decreased expression of proteins associated with cell cycle progression was detected. This included the targeted CDK1 and CDK4 at both 24 h and 48 h post-transfection (Figure 4A), as detected by qPCR.
Figure 4: Gene dysregulation as detected by microarray and RNA sequencing analysis. (A) Heatmap of cell cycle pathway gene expressions as detected by microarray analysis in protein extracts from A549 cells transfected with miR-143 and miR-506, at 24 h and 48 h post-transfection. (B) Fold change of cell cycle pathway gene expressions from A549 cells transfected with miR-143 and miR-506 at 24 h post-transfection as detected by RNA sequencing. (C) Pathway activity as analyzed by pathway analysis software from data obtained from RNA sequencing. This figure has been modified from Hossian et al.11 Please click here to view a larger version of this figure.
RNA sequencing and pathway analysis using pathway analysis software
Next-generation sequencing accurately analyzes gene expression at the RNA level. The method allows for identification of multiple gene changes through a single analysis (in this protocol, the analysis detected the expression of >18,000 genes). Due to the large number of detected genes, bioinformatics analysis was used for efficient determination of pathway behavior (Figure 4B). Software was then used (see Table of Materials) to predict G1/S and G2/M phase arrests and the downregulation of S phase initiation (Figure 4C). Furthermore, the RNA sequencing results can be compared to qPCR data. In this study, the RNA sequencing confirmed the findings from the qPCR analysis, indicating a downregulation of CDK1 (48%, p < 0.001, FDR < 0.001), CDK4 (68%, p < 0.001, FDR < 0.001), and CDK6 (71%, < 0.001, FDR < 0.001) due to combinatorial miR-143 and miR-506 activity. Statistical analysis was performed by the EdgeR software used for calculation of the relative gene expression, calculating p values using Negative Binomial20,21. Bioinformatics analysis can be performed for the functional evaluation of miRNA activity and prediction of potential molecular targets, as illustrated in Figure 5.
Figure 5: Illustrative pathway and mechanistic analysis as presented by pathway analysis aoftware. RNA sequencing data was analyzed from A549 cells transfected with miR-143 and miR-506, 24 h post-transfection, using pathway analysis software and identified canonical pathways with the lowest (A) or highest (B) activation score. The software also provided predicted functions (C) and potential upstream regulators/targets (D). This figure is reprinted from Hossian et al.11 Please click here to view a larger version of this figure.
Endothelial tube formation assay
The in vitro endothelial tube formation assay is widely used to study angiogenesis and is reliable, automated, and quantifiable22. Vascular endothelial growth factor (VEGF) is a well-known angiogenic growth factor23,24 and endothelial tube formation promoter. In this study, it was identified that the combinatorial treatment of miR-143 and miR-506 abrogates VEGF-induced angiogenesis. Indicative images of tube formation and the effects of treatment are presented in Figure 6.
Figure 6: Representative images of endothelial sprouts of VEGF-treated vs. non-treated HUVECs transfected with scramble miRNA, miRNA-143, miRNA-506, or a combination thereof. Pictures were obtained under a brightfield microscope equipped with a digital camera under 4x magnification. Please click here to view a larger version of this figure.
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 on the function of miRNAs. The challenges of studying miRNAs are associated with their capacity to target multiple genes and thus affect multiple pathways. The described qPCR analysis allows identification of the expression of specific genes of interest, if specific targets are identified prior to treatment. For example, the main focus here was the expression of CDK1 and CDK4/6 and the cell cycle.
Thus, cell cycle analysis using propidium iodide and flow cytometry protocol is a reliable approach to detect alterations in the cell populations according to their stage in the cell cycle. The method relies on the proportionate increase of fluorescence signal by the PI, which binds to DNA, corresponding to the stage of the cell cycle. Briefly, cells in the S phase synthesize DNA, inducing higher signals than cells in the G0/G1 phase, and cells in the G2 phase have duplicated their DNA, producing the most intense signal.
Accumulating evidence indicates the connection of damage to the cell cycle and triggering of apoptosis25. The flow cytometry method using Annexin VI/PI has consistently been used for the identification of induced apoptosis in cells from chemotherapy treatment. Indicatively, a strong apoptotic response was identified due to the combinatorial miRNA therapy, which was more potent compared to the individual miRNAs.
The semi-quantitative protein antibody microarray is a sensitive and reliable method to identify protein expression alterations related to specific biological responses26,27. This protocol used a cell cycle pathway-specific antibody microarray, which detected expression changes in ~60 genes between treated and untreated cells. Caution is required during the washing steps to ensure that the process has been thoroughly performed and to minimize the background signal. Additionally, the slides should not become dry until completion of the experiment.
In contrast, RNA sequencing provides quantitative gene expressions analysis of multiple genes, at the post-transcription level. The significantly large number of analyzed genes (>18,000 for RNA-seq vs. 60 for microarray) allows for the simultaneous analysis of multiple pathways and molecular targets, and with increased accuracy. Such broad analysis is important, as a single miRNA can bind to and target different mRNAs. In contrast, the pathway analysis of large numbers of gene dysregulations is inherently challenging. For example, although the RNA sequencing confirmed our qPCR data regarding CDK1 and CDK4/6 downregulation due to the miRNA treatment, the analysis also provided data on thousands of genes that were also down-or up-regulated. To provide context to such numerous gene dysregulations, pathway analysis software was used to determine overall effects of the treatment on different pathway and cellular functions. Indicatively, the software provided scores representative to activation (positive z score) or inactivation (negative z score) of specific functions or pathways, as well as statistical significance of the analysis (Figure 5)28.
In conclusion, the study of miRNA activity is a challenging procedure. The inherent capacity of miRNAs to affect multiple genes requires the utilization of multiple elaborate and complicated analytical methods to identify potential activity. Not surprisingly, further work is required to fully comprehend the activities of miR-143 and miR-506 in lung cancer.
The authors have nothing to disclose.
No conflicts of interest are declared.
-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 |