From our characterization of mutation-specific patterns of miRNA expression, we define 12 modules of miRNA expression across our five mutant genotypes (with samples size greater than 1) and WT controls. As an example of pathway specific effects on miRNA expression, we highlight miRNAs that exhibit altered expression in response to a Smad4 mutation. We focus on miR-375-3p, a candidate tumor suppressor in CRC, and show that its expression is positively correlated with Tgf-B signaling. Therefore, we suggest that miR-375-3p mimics may serve as effective therapeutics for CRC patients with mutations that alter Tgf-B/Smad4 signaling.
As an example of mutation-independent effects on miRNA expression, we highlight that miR-24-3p is elevated across a spectrum of genetic contexts of CRC. Reduction of miR-24-3p in HCT116 cells and WT mouse enteroids results in a decrease in cell number. From TUNEL and RNA-seq analysis in miR-24-3p inhibited HCT116 cells, this decrease in cell number is, at least in part, due to an increase in apoptosis. By integrating RNA-seq and leChRO-seq analysis, we identify PRSS8 and HMOX1 as post-transcriptionally regulated gene targets that may contribute to the function of miR-24-3p. Our results support a model in which miR-24-3p elevation in CRC tumors with various genotypes downregulates multiple candidate tumor suppressor genes, including HMOX1 and PRSS8. Furthermore, we propose that downregulation of these tumor suppressor genes results in CRC resistance to apoptosis.
Overall, our results provide a novel perspective on the mutation-specific effects on oncogenic and tumor suppressive miRNAs in CRC. Understanding these patterns of expression are critical for the incorporation of miRNA-based therapeutics for precision medicine in CRC.
Experimental procedures
Generation of genetically modified mouse enteroids
The proximal half of the small intestine was isolated from 5–6 week-old C57BL/6 mice for crypt isolation. Cells were plated in Matrigel and grown for 3–4 weeks. Enteroids were then dissociated and transfected using the necessary Cre/CRISPR gene editors.
A, B, AKP, BKP: Cells with ApcQ883* mutation (A) and Ctnnb1S33F (B) were generated using CRISPR base editing as described in Schatoff et al. (2019) [29]. Selection for ApcQ883* and Ctnnb1S33F mutants was performed by culturing cells in the absence of RSPO1. KrasG12D (K) and Trp53−/− (P) mutations were generated using enteroids from the conditional LSL-Kras/p53fl/fl mouse model, as described in Dow et al., (2015) [30]. Cre was introduced to enteroids by transfection. Cells with Trp53−/− mutation were selected for by treating cells with 10 µM Nutlin3. To ensure KrasG12D mutation, cells were then cultured in the absence of EGF.
KRP, KRS, KRPS: KrasG12D mutations (K) were generated by using enteroids derived from the KrasLSL−G12D conditional model as described in Jackson et al. (2001)85. Cre was introduced to enteroids by transfection. Cells with the KrasLSL−G12D allele were selected for by adding 1 µM gefitinib to the culturing media. Cells with Ptprk-Rspo3 fusion (R) were generated via CRISPR/Cas9 chromosome rearrangement as described in Han et al., (2017)31. Selection for Ptprk-Rspo3 mutants was done by culturing cells in the absence of RSPO1. Trp53−/− (P)and Smad4KO (S) mutations were generated using CRISPR/Cas9 and single guide RNAs (sgRNA) as described in Han et al., (2020) [12]. Selection for Trp53−/− cells was completed by adding 5 µmol/L Nutlin-3 to the culturing media. Selection for Smad4KO cells was completed by adding 5 ng/mL TGFB1 to the culturing media.
Trizol LS RNA isolation
Cells were treated with 250 µL of cold 1X NUN Lysis Buffer (20 mM HEPES, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M Urea, 1% NP-40, 1 mM DTT, and 50 units/mL SUPERase In RNase Inhibitor (ThermoFisher Scientific, Waltham, MA), and 1X 50X Protease Inhibitor Cocktail (Roche, Branchburg, NJ)). Lysate was vortexed vigorously for 1 min to physically lyse cells. Samples were incubated for 30 min in Thermomixer C at 12 °C at 1500 rpm. Chromatin was pelleted out by centrifuging samples for 12,500 xg for 30 min at 4 °C. Supernatant containing RNA was removed from the tube and added to clean 1.5 mL centrifuge tube along with 750 µL Trizol LS (Life Technologies, 10,296–010). Samples were vortexed and stored at -80 °C until RNA isolation. Samples were thawed and allowed to incubate for 5 min. 200 µL of chloroform was added to each tube and vortexed for 20 s. Following a three-minute incubation, samples were centrifuged at 17,000 xg, 4 °C for 5 min. Aqueous layer was transferred to clean 1.5 mL centrifuge tube containing 2.5 µL of GlycoBlue. 1 mL of ice cold, 100% ethanol was added to aqueous phase and samples were then vortexed. Samples were then centrifuged at 17,000 xg at 4 °C for 15 min. Supernatant was removed and pellet was washed with 75% ice cold ethanol. Samples were vortexed and RNA was pelleted by centrifuging at 17,000 xg at 4 °C for 5 min. Supernatant was removed and RNA pellets were allowed to dry for 10 min at room temperature. RNA was resuspended in 30 µL of RNase-free water.
Small RNA library preparation and sequencing
Total RNA was isolated using the Total RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) according to manufacturer’s instructions or Trizol LS method described above. RNA purity and concentration was determined using the Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA). RNA integrity was quantified using the 4200 Tapestation (Agilent Technologies, Santa Clara, CA) or Fragment Analyzer Automated CE System (Advanced Analytical Technologies, Ankeny, IA). Libraries were prepared at the Genome Sequencing Facility of the Greehey Children’s Cancer Research Institute (University of Texas Health Science Center, San Antonio, TX) using the CleanTag Small RNA Library Prep kit (TriLink Biotechnologies, San Diego, CA). Libraries were then sequenced on the HiSeq2000 platform (Illumina, San Diego, CA).
RNA library preparation and sequencing
Total RNA was isolated using the Total RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s instructions or using the Trizol LS method described above. RNA purity and concentration was determined using the Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA). RNA integrity was quantified using the 4200 Tapestation (Agilent Technologies, Santa Clara, CA) or Fragment Analyzer Automated CE System (Advanced Analytical Technologies, Ankeny, IA). Libraries were prepared using the NEBNext Ultra II Directional Library Prep Kit following Ribosomal Depletion (mouse enteroid RNA) or PolyA enrichment (HCT116 RNA) at the Cornell Transcriptional Regulation and Expression Facility (Cornell University, Ithaca, NY). Libraries were then sequenced using the NextSeq500 platform (Illumina, San Diego, CA).
Small RNA-seq analysis
Read quality was assessed using FastQC. Trimming, mapping and quantification was performed using miRquant 2.0 as described in Kanke et al., (2016) [32]. In short, reads were trimmed using Cutadapt, aligned to the genome using Bowtie and SHRiMP, and aligned reads were quantified and normalized using DESeq2 [50]. We accounted for sequencing batch, RIN, and genotype in our model. Defining groups of miRNAs with similar patterns of expression across genotypes: Raw miRNA count matrices produced by miRquant were analyzed using a likelihood ratio-test from DESeq2. miRNA annotations in the 5000 s are degradation products and removed from the analysis, and miRNAs with an adjusted p-value greater than 0.05 and baseMean expression less than 500 were discarded. An rlog transformation was applied to the raw counts and batch effects were removed using the limma function removeBatchEffects. Clusters of miRNAs with similar expression patterns were identified using the DEGreport (v1.26.0) function degPatterns (minc = 5). Only clusters containing greater than five miRNAs were considered. Fold change heatmaps: Transformation and batch correction of miRNA expression and grouping of miRNAs is described above. Normalized expression of miRNAs for each mutant enteroid sample was subtracted from average WT expression and heatmaps were made using the R package pheatmap (v1.0.12).
RNA-seq Analysis
Read quality was assessed using FastQC. RNA-seq reads were aligned to either the mm10 genome release for mouse enteroids or the hg38 genome release for the human HCT116 cells using STAR (v2.7.9a). Quantification was performed with Salmon (v1.4.0) using the GENCODE release 25 annotations. Normalization and differential expression analyses were performed utilizing DESeq2 (v1.30.1). We accounted for cell culture batch effects, RIN, and genotype in our model. Enrichr was used for KEGG pathway analysis as described in Chen et al. (2013) [52]. miRhub analysis was performed as described in Baran-Gale et al. (2013) [51]. In short, miRhub scans input gene lists for miRNA binding sites defined by TargetScan v5.2 [86]. For our analyses, we filtered for binding sites that are conserved in mice and at least one of the following species (cons1): human, rat, dog and/or chicken. miRNA-gene scores were generated based on seed sequence strength, conservation, and frequency of target sites in the 3'-UTR while controlling for 3' UTR length. These scores were added together for each miRNA to generate a cumulative value that represents the miRNA targeting score. A Monte Carlo simulation repeated this analysis 1000 × using lists of randomly selected genes. An empirical p-value was then calculated for each miRNA by comparing the targeting score from input gene lists to the targeting scores calculated calculated using the lists consisting of randomly selected genes.
Quantitative PCR
Total RNA from HCT116 cells was extracted using the Total RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) according to manufacturer’s instructions. Reverse-transcription for miRNA expression was performed using the Taqman MicroRNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA). Quantification of miRNA expression was done using the TaqMan Universal PCR Master Mix (ThermoFisher Scientific, Waltham, MA). miRNA expression was normalized to U6 (assay ID: 001,973). miRNA Taqman assays: miR-375-3p (assay ID: 000,564), miR-24-3p (assay ID: 000,402). Reverse-transcription for gene expression was performed using the High-Capacity RNA-to-cDNA kit (ThermoFisher Scientific, Waltham, MA). Quantification of gene expression was done using the TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Waltham, MA). Gene expression was normalized to RPS9 (assay ID: Hs02339424_g1). Gene Taqman assays: HMOX1 (assay ID: Hs01110250_m1), PRSS8 (assay ID: Hs00173606_m1), Rps9 (assay ID: Mm00850060_s1), Fn1 (assay ID: Mm01256744_m1), Col1a1 (assay ID: Mm00801666_g1). Measurements were taken using the BioRad CFX96 Touch Real Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA).
The Cancer Genome Atlas (TCGA) analysis
Data Download: RNA-seq High Throughput Sequencing (HTSeq) counts files for 382 primary colon tumor and 39 solid normal tissue samples was downloaded using the NIHGDC Data Transfer Tool. Normalization and differential expression were identified using DESeq2. For our miRhub analysis, we filtered for binding sites that are conserved in humans and at least two of the following species (cons2): mouse, rat, dog and/or chicken. miRNA quantification files, that used mirbase21, for 371 primary colon tumor and 8 solid normal tissue samples were also downloaded using NIHGDC Data Transfer Tool. Of the 371 colon tumor samples with miRNA data, 326 had simple somatic mutation (TCGA v32.0) and copy number variation (CNV; TCGA v31.0) information. Tumor samples were assigned APC (A), TP53 (P), and SMAD4 (S) mutations if they contained a non-synonymous mutation and/or CNV loss for a given gene. For A, P, and S designations, samples with a CNV gain and a non-synonymous mutation were not included. Mutations in CTNNB1 (B) and KRAS (K) were assigned to tumor samples with a non-synonymous mutation and/or CNV gain for a given gene. For B and K designations, samples with a CNV loss and a non-synonymous mutation were not included.
TCGA small RNA-seq across cancer types: Small RNA sequencing expression data was downloaded from TCGA for 23 tumor types using the R package TCGA-assembler. Expression was reported as the reads per million mapped to miRNAs (RPMMM). Log2 fold change was calculated by dividing the tumor expression by the expression in non-tumor tissue followed by log2 transformation.
Mouse enteroid culture
Crypts from the jejunum of 3–5 month old male B62J mice were isolated as described in Peck et al., (2017) [63]. Isolated crypts were plated in Reduced Growth Factor Matrigel (Corning, Corning, NY, catalog #: 356,231) on Day 0. Advanced DMEM/F12 (Gibco, Gaithersburg, MD, catalog #: 12,634–028) was used for culture and supplemented with GlutaMAX (Gibco, Gaithersburg, MD, catalog #:35,050–061), Pen/Strep (Gibco, Gaithersburg, MD, catalog #:15,140), HEPES (Gibco, Gaithersburg, MD, catalog #:15,630–080), N2 supplement (Gibco, Gaithersburg, MD, catalog #:17,502–048), 50 ng/mL EGF (R&D Systems, Minneapolis, MN, catalog #: 2028-EG), 100 ug/mL Noggin (PeproTech, Rocky Hill, NJ, catalog #: 250–38), 250 ng/uL murine R-spondin (R&D Systems, catalog #: 3474-RS-050), and 10 mM Y27632 (Enzo Life Sciences, Farmingdale, NY, catalog #:ALX270-333-M025) miR-24-3p LNA inhibitor treatment: Cells were transfected with hsa-miR-24-3p miRCURY LNA miRNA Power Inhibitor (Qiagen, Germantown, MD, catalog #: YI04101706-DDA) or Power Negative Control A (Qiagen, Germantown, MD, catalog #: YI00199006-DDA) to a final concentration of 500 nM on Day 0 using gymnosis. Media was changed and cells were treated with 250 nM miR-24 LNA inhibitor or scramble. Cells were harvested and fixed in 4% (v/v) paraformaldehyde on Day 5. Tgf-B treatment: Recombinant Human TGF-B1 (PeproTech catalog #: 100–21) was added to enteroid media on Day 0 for final concentration of 0, 0.5, or 1 ng/mL. Enteroids were harvested on Day 3.
Cell line transfection
All cell lines were purchased from the American Type Culture Collection (ATCC) and plated in DMEM + 10% FBS media. HCT116 cells were plated at a density of 3,400 cells/well in a 96-well plate. Caco-2 cells were plated at a density of 20,000 cells/well in a 96-well plate. HT-29 cells were plated at a density of 3,400 to 6,800 cells/well in a 96-well plate. SW48 cells were plated at a density of 10,000 cells/well in a 96-well plate. Cells incubated for 24 h in a 37 °C incubator. Cells were transfected with hsa-miR-24-3p miRCURY LNA miRNA Power Inhibitor (Qiagen, Germantown, MD, catalog #: YI04101706-DDA) or scramble control to a final concentration of 100 nM using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, catalog #: L3000-008) according to manufacturer’s instructions. Either Power Negative Control A (Qiagen, Germantown, MD, catalog #: YI00199006-DDA) or Negative Control miRCURY LNA miRNA Mimic (Qiagen, Germantown, MD, catalog #: YM00479902-AGA) was used for scramble control. After 24-h, media was replaced with complete media. Ferrostatin-1 treatment: At the time of LNA transfection, cells were also treated with 0, 0.5, 2, 5, or 10 µM Ferrostatin-1 (Sigma-Aldrich, St. Louis, MO, catalog #: SML0583-5MG). After 24-h, media was replaced with complete media. Cells were harvested 48-h post-transfection.
Cell count assay
Forty-eight-H following transfection, cells in 96-well plate were washed with PBS and treated with 50 µL trypsin. Cells incubated for 5 min in 37 °C incubator. Cells were resuspended using 150 µL complete media and transferred to clean 1.5 mL Eppendorf tubes. Cell concentration was calculated by adding 10 µL of cell suspension to chip for Biorad TC20 Automated Cell Counter (Bio-Rad Laboratories, Richmond, CA).
CellTiter-Glo assays
Forty-eight-H following transfection, cells in 96-well plate were incubated at room temperature for 30 min. 100 µL of room temperature CellTiter-Glo reagent (Promega, Madison, WI) was added to each well and placed on cell rocker for 2 min to lyse the cells. Afterwards, plate was incubated at room temperature for 10 min. Luminescent signal was quantified using a Synergy 2 Microplate Reader (Biotek, Winooski, VT; area scan; Integration = 0:00:50; Sensitivity = 135).
EdU Assay
Forty-eight-H following transfection, cells in 96-well plate were incubated with 10 µM EdU at 37 °C in complete media for 1 h. Cells were then fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized using 0.5% Triton X-100 in PBS for 20 min. The Invitrogen Click-iT Plus EdU AlexaFluor 488 Imaging Kit (Invitrogen, Waltham, MA, C10637) was used to detect EdU according to manufacturer’s instructions. Nuclei were stained using DAPI (ThermoFisher Scientific, Waltham, MA, catalog #: D1306) and imaged using ZOE Fluorescent Cell Image (Bio-Rad Laboratories, Richmond, CA). Images were analyzed using FIJI. For EdU positive cells, threshold value was set to 10. For analyzing particles, counted those particles with size = 250-Infinity and circularity = 0.4–1.
TUNEL Assay
Forty-eight-H following transfection, cells in 96-well plate were washed twice with PBS and fixed using 4% paraformaldehyde for 15 min at room temperature. Permeabilization was performed by using 0.5% Triton X-100 in PBS for 20 min. Cells were washed twice with deionized water. Positive control wells were treated with 1X DNase I, Amplification Grade (ThermoFisher Scientific, Waltham, MA, catalog #: 18,068–015) solution according to manufacturer’s instructions. Labeling and detection of apoptotic cells was completed using the Invitrogen Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection 488 kit (Invitrogen, Waltham, MA, catalog #: C10617) according to manufacturer’s instructions. Nuclei were stained using DAPI (ThermoFisher Scientific, Waltham, MA, catalog #: D1306) and imaged using ZOE Fluorescent Cell Image (Bio-Rad Laboratories, Richmond, CA). Images were analyzed using FIJI. For TUNEL positive cells, threshold value was set to 14. For analyzing particles, counted those particles with size = 250-Infinity and circularity = 0.4–1.
LNA24 transfection with leChRO-seq and RNA-seq cross comparison
HCT116 cells were plated in DMEM + 10% FBS media at a density of 102,000 cells/well in a 6-well plate. Cells incubated for 24 h in a 37 °C incubator and transfected with hsa-miR-24-3p miRCURY LNA miRNA Power Inhibitor (Qiagen, Germantown, MD, catalog #: YI04101706-DDA) or Power Negative Control A (Qiagen, Germantown, MD, catalog #: YI00199006-DDA) to a final concentration of 100 nM using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, catalog #: L3000-008). After 24-h, media was replaced with complete media. After 48-h post-transfection, cells were resuspended using 0.25% Trypsin (ThermoFisher Scientific, Waltham, MA, catalog #: 25,200–114). Wells from the same treatment condition were pooled together into a single tube during each experimental replicate. 20,000 cells were isolated for total RNA isolation using the Total RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) according to manufacturer’s instructions. RNA-seq and quantitative qPCR were performed as described previously.
The remaining cells (450,000 + cells) were flash frozen using 100% EtOH and dry ice until utilized for Length Extension Chromatin Run-On Sequencing (leChRO-seq) as previously described [28, 87]. Chromatin Isolation: Chromatin was isolated by treating cell pellet with 750µL 1X NUN buffer (20 mM HEPES, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M Urea, 1% NP-40, 1 mM DTT, and 50 units/mL RNase Cocktail Enzyme (ThermoFisher Scientific, Waltham, MA), and 1X 50X Protease Inhibitor Cocktail (Roche, Branchburg, NJ)). Samples were vortexed vigorously for 1 min to physically lyse the samples. An additional 750µL of 1X NUN buffer was added and samples were vortexed again for 1 min. Cell lysates were incubated in an Eppendorf Thermomixer (Eppendorf, Hamburg, Germany) at 12 °C and shaken at 2000 rpm for 30 min. Chromatin was pelleted by centrifuging samples at 12,500 × g for 30 min at 4 °C. Supernatant was removed and chromatin was washed 3 times with 1 mL 50 mM Tris–HCl (pH = 7.5) containing 40 units/mL SUPERase In RNase Inhibitor. After removing supernatant from final wash, 50 µL storage buffer was added to chromatin and samples were transferred to 1.5 mL Bioruptor Microtubes with Caps for Bioruptor (Diagenode, Denville, NJ). Samples were then loaded into Pico Biorupter (Diagenode, Denville, NJ) and sonicated on high for 10 cycles (1 cycle = 30 s on, 30 s off). Sonication was repeated until chromatin was solubilized (max 3 cycles). Samples were stored at -80 °C until further processing.
ChRO-seq library preparation and sequencing: 50 µL of 2X Biotin-11 Reaction mix (10 mM Tris–HCl pH = 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 400 µM ATP, 0.8 µM CTP, 400 µM GTP, 400 µM UTP, 40 µM Biotin-11-CTP (Perkin Elmer, Waltham, MA, NEL542001EA), 100 ng yeast tRNA (VWR, Radnor, PA, 80,054–306), 0.8 units/µL SUPERase In RNase Inhibitor, 1% sarkosyl) was added to 50 µL solubilized chromatin. Samples were placed in Eppendorf Thermomixer at 37 °C for 5 min and shaken at 750 rpm. Run-on was halted by adding 300 µL Trizol LS (Life Technologies, 10,296–010) and allowing the samples to incubate at room temperature for 3 min. RNA was purified using streptavidin beads (New England Biolabs, Ipswich, MA, S1421S) and ethanol precipitated with the co-precipitate GlycoBlue (Ambion, AM9515). Ligation of the 3’ adapter was done using the T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA, M0204L). Ligation of 5’ adaptor required (i) Removal of the 5’ cap using RNA 5’ pyrophosphohydrolase (RppH, New England Biolabs, Ipswich, MA, M0356S) (ii) Phosphorylation of the 5’ end using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, M0201L) (iii) 5’ adaptor ligation using T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA, M0204L). Generation of cDNA was done by using Superscript III Reverse Transcriptase (Life Technologies, 18,080–044). Amplification was completed by using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, M0491L). Single-end sequencing (5’ end, 75 bp) was performed at the Cornell Biotechnology Research Center using the NextSeq500 (Illumina, San Diego, CA) platform. Data analysis: To prepare bigwig files for further analyses, leChRO-seq libraries were aligned to the hg38 genome using the proseq2.0 pipeline (https://github.com/Danko-Lab/proseq2.0) in single-end mode (Chu et al., 2018) [28]. Annotation of leChRO-seq reads excluded reads within 500 bp downstream of the transcription start site (TSS) to account for RNA polymerase pausing at the gene promoters. Genes < 1000 bp were then excluded to account for the bias resulting from short gene bodies. ChRO-seq reads were normalized and differential expression analysis was performed using DESeq2.
Statistics
All statistical tests used are detailed in the figure legends. Either two-tailed Welch t-test (calculated using R) or two-tailed Student’s t-test (calculated using excel) was applied to datasets that were normalized (DESeq2, log2, rlog). Significance for data sets that did not statistically differ from a normal distribution (Shapiro–Wilk test p-value > 0.05) was calculated using a t-test. A two-sided Wilcoxon test was applied to non-parametric data sets unless where indicated. P-values < 0.05 are considered statistically significant. NS. = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.