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. 2015 Dec 19;6:398–404. doi: 10.1016/j.dib.2015.12.026

Data describing the effects of dietary bioactive agents on colonic stem cell microRNA and mRNA expression

Manasvi S Shah a,b,h,i,1, Eunjoo Kim a,c,1, Laurie A Davidson a, Jason M Knight a,d,g, Roger S Zoh a,e, Jennifer S Goldsby a,g, Evelyn S Callaway a, Beyian Zhou f,g, Ivan Ivanov a,f,g, Robert S Chapkin a,b,g,
PMCID: PMC4707287  PMID: 26862588

Abstract

With the identification of Lgr5 as a definitive marker for intestinal stem cells, we used the highly novel, recently described, Lgr5-EGFP-IRES-cre ERT2knock in mouse model. Mice were injected with azoxymethane (AOM, a colon carcinogen) or saline (control) and fed a chemo-protective diet containing n-3 fatty acids and fermentable fiber (n-3 PUFA+pectin) or a control diet (n-6 PUFA + cellulose). Single cells were isolated from colonic mucosa crypts and three discrete populations of cells were collected via fluorescence activated cell sorting (FACS): Lgr5high (stem cells), Lgr5low (daughter cells) and Lgr5negative (differentiated cells). microRNA profiling and RNA sequencing were performed from the same sample and analyzed. These data refer to ‘Comparative effects of diet and carcinogen on microRNA expression in the stem cell niche of the mouse colonic crypt’ (Shah et al., 2016) [5].


Specifications table

Subject area Biology
More specific subject area Intestinal stem cells
Type of data Tables
How data was acquired The raw data was generated using an Illumina sequencer and was statistically analyzed and displayed in tabular format.
Data format Analyzed
Experimental factors The samples were collected from mice fed with either corn oil+ cellulose or fish oil+pectin. Both groups of mice were then either injected with Azoxymethane (carcinogen) or saline (control). Total = 20 mice in all.
Experimental features Colonic crypts were isolated from the stem cell reporter mice and were sorted based on GFP into high, low and negative cell populations using a BD FACS Aria II cytometer/sorter (BD Biosciences). Total RNA was isolated from the sorted cells using a miRVana miRNA isolation kit. After checking the quality of the RNA, libraries were subjected to RNA sequencing and microRNA profiling.
Data source location College Station, Texas, USA (30.6014oN, 96.3144oW)
Data accessibility All the datasets mentioned in this manuscript will be uploaded to the Gene Expression Omnibus (GEO) (accession no.- SRP061188) in NCBI.

Value of the data

  • These data will serve as a basis to compare Lgr5high stem cells that have been perturbed by inflammation, radiation or knockout of tumor suppressors/oncogenes.

  • These data describe expression values for miRNAs in Lgr5high, Lgr5low and Lgr5neg cells. For example, if a researcher wants to know if a certain miRNA is expressed in the colonic crypt, this database will provide that information. The data set also details the location of the miRNA throughout the entire crypt.

  • RNA sequencing data from Lgr5high, Lgr5low and Lgr5neg cells also provides the basis for determining the expression of different mRNAs throughout the crypt. This database will serve as a comparative platform for future studies to determine correlations between current and future datasets.

1. Data

The data presented in this article represent: (a) Ct values of miRNAs expressed in mouse colonic epithelial cells (Table 1), (b) differentially expressed miRNAs in Lgr5high versus Lgr5negative cells (i.e., stem cells vs. differentiated cells) (Table 2), (c) the effect of diet on miRNA expression in Lgr5high sorted cells (Table 3), (d) the effect of carcinogen on miRNA expression in Lgr5 high sorted cells (Table 4) and (e) the effect of diet and carcinogen combination on miRNA expression in GFPnegative sorted cells (Table 5).

Table 1.

Ct values of 103 miRNAs expressed in mouse colonic epithelial cells.

miRNA Ct values miRNA Ct values miRNA Ct values
mmu-miR-31 11.44 mmu-miR-671-3p 26.81 mmu-let-7g- 28.94
rno-miR-190b 14.92 mmu-miR-215 26.81 mmu-miR-103 28.95
mmu-miR-872 17.72 mmu-miR-151-3p 26.9 mmu-miR-320 29
mmu-miR-124 20.88 mmu-let-7b 26.96 mmu-miR-218 29.02
mmu-miR-128a 20.99 mmu-miR-203 26.97 mmu-miR-30d 29.2
mmu-miR-429 21.89 mmu-miR-484 27.02 mmu-miR-125b-5p 29.2
mmu-miR-148b 22.68 mmu-miR-29a 27.11 mmu-miR-205 29.63
mmu-miR-324-5p 22.88 mmu-miR-29b 27.24 mmu-miR-18a 29.63
mmu-miR-322 23.04 mmu-miR-340-5p 27.41 mmu-miR-195 29.68
mmu-miR-142-3p 23.43 mmu-miR-139-5p 27.43 mmu-miR-28 29.7
mmu-miR-192 23.51 mmu-miR-15b 27.43 mmu-miR-574-3p 29.83
mmu-miR-200a 23.75 mmu-miR-93 27.49 mmu-miR-101a 29.83
mmu-miR-423-5p 23.88 mmu-let-7e 27.55 mmu-miR-29c 30.01
mmu-miR-375 23.94 mmu-miR-20b 27.71 rno-miR-345-3p 30.02
mmu-miR-10b 24.03 mmu-miR-27b 28.01 mmu-miR-301b 30.12
mmu-miR-19b 24.16 mmu-miR-30a 28.04 mmu-miR-146b 30.29
mmu-miR-30c 24.31 mmu-miR-27a 28.04 mmu-miR-744 30.34
mmu-miR-92a 24.36 mmu-miR-148a 28.06 mmu-miR-331-3p 30.37
mmu-miR-191 24.43 mmu-miR-222 28.13 mmu-miR-186 30.38
mmu-miR-30b 24.75 mmu-miR-141 28.18 mmu-miR-196b 30.39
mmu-miR-24 24.78 mmu-miR-188-5p 28.19 mmu-miR-340-3p 30.44
mmu-miR-126-3p 24.79 mmu-miR-106b 28.22 mmu-miR-301a 30.45
mmu-miR-17 24.91 mmu-miR-19a 28.23 mmu-miR-130b 30.49
mmu-miR-194 24.92 mmu-let-7d 28.23 mmu-miR-193b 30.68
mmu-miR-200b 24.99 mmu-miR-25 28.24 mmu-miR-155 30.77
mmu-miR-34b-3p 25.05 rno-miR-196c 28.46 mmu-miR-152 30.8
mmu-miR-20a 25.16 mmu-miR-130a 28.46 mmu-miR-23b 30.98
mmu-miR-106a 25.16 mmu-miR-100 28.5 mmu-miR-183 31.39
mmu-miR-200c 25.38 mmu-miR-30e 28.57 mmu-miR-125a-5p 31.84
mmu-let-7c 25.74 mmu-miR-182 28.57 mmu-miR-181a 31.94
mmu-miR-16 25.98 mmu-miR-328 28.59 mmu-miR-181c 34.76
mmu-miR-10a 26.11 mmu-let-7i 28.68
mmu-miR-145 26.32 mmu-miR-26b 28.84
mmu-miR-26a 26.33 mmu-miR-140 28.86
mmu-miR-21 26.48 mmu-miR-146a 28.87
mmu-miR-99a 26.67 mmu-miR-224 28.92

Expression of miRNAs was quantified by reverse transcription using miRNA-specific primers followed by real-time PCR TaqMan low-density array analysis. Ct values represent means of 60 samples, mmu, mouse; rno, rat; Ct, cross threshold.

Table 2.

Differentially expressed miRNAs in Lgr5high versus Lgr5negative cells.

Up-regulated in Lgr5high
P-value Down-regulated in Lgr5high
P-value
miRNA Expression ratio Lgr5high/ Lgr5negative miRNA Expression ratio Lgr5high/ Lgr5negative
mmu-miR-342-3p 2.64 0.012 mmu-miR-652 0.32 0.000
mmu-miR-671-3p 2.29 0.008 mmu-miR-145 0.40 0.009
rno-miR-345-3p 2.14 0.022 mmu-miR-27a 0.42 0.000
rno-miR-190b 1.95 0.018 mmu-miR-215 0.42 0.000
mmu-miR-155 1.90 0.033 mmu-miR-532-5p 0.50 0.040
mmu-miR-191 1.81 0.001 mmu-miR-7b 0.60 0.027
mmu-miR-20b 1.68 0.011 mmu-miR-21 0.62 0.024
mmu-miR-17 1.68 0.000 mmu-miR-30d 0.62 0.017
mmu-miR-125a-5p 1.58 0.036 rno-miR-224 0.62 0.018
mmu-miR-186 1.58 0.006 mmu-miR-30a 0.66 0.000
mmu-miR-218 1.44 0.003 mmu-miR-200b 0.69 0.013
mmu-miR-10a 1.30 0.047 mmu-miR-203 0.76 0.003
mmu-miR-92a 1.29 0.018
mmu-miR-200a 1.27 0.031

Expression of miRNAs were quantified as described in Table 1. GFPhigh stem cells (n=20, pooled samples); Lgr5negative cells (n=20, pooled samples). Only miRNAs with P<0.05 are shown. mmu, mouse; rno, rat.

Table 3.

Effect of carcinogen on miRNA expression in Lgr5high sorted cells.

A.
miRNA Expression ratio AOM-Lgr5high /Saline-Lgr5high P-value
mmu-miR-532-3p 2.61 0.020
rno-miR-196c 1.66 0.008
mmu-miR-331-3p 1.45 0.032
mmu-miR-92a 1.41 0.042
mmu-miR-100 0.19 0.050
mmu-miR-124 0.25 0.021
B.
miRNA Expression ratio AOM-Lgr5neg /Saline-Lgr5neg P-value

mmu-let-7e 1.97 0.008
mmu-miR-18a 1.84 0.050
mmu-miR-20b 1.59 0.029
mmu-miR-101a 1.52 0.045
mmu-let-7i 1.45 0.039
mmu-miR-375 1.26 0.046
mmu-miR-224 0.65 0.030
mmu-miR-193b 0.65 0.045
mmu-miR-10a 0.62 0.008

miRNA expression was quantified as described in Table 1. AOM, azoxymethane (n=20); saline (n=20); Lgr5high, stem cells; Lgr5neg, non-stem cells. Only miRNAs with P<0.05 are shown.

Table 4.

Effect of diet on miRNA expression in Lgr5high sorted cells.

A. Carcinogen
miRNA Expression ratio CCA-Lgr5high /FPA-Lgr5high P-value
miR-21 2.2 0.030
miR-26b 2.0 0.010
miR-200a 1.8 0.000
miR-10a 1.7 0.040
miR-26a 1.7 0.020
miR-29c 1.6 0.010
miR-30c 1.5 0.040
miR-203 1.5 0.040
miR-30a 1.4 0.020
miR-19b 1.3 0.040
miR-181a 0.6 0.030
miR-34b-3p 0.1 0.000
B. Saline
miRNA Expression ratio CCS-Lgr5high/FPS-Lgr5high P-value

mmu-miR-188-5p 5.3 0.027
mmu-miR-218 0.6 0.016
mmu-miR-125a-5p 0.5 0.047
mmu-miR-574-3p 0.5 0.028
mmu-miR-200c 0.5 0.047
mmu-miR-222 0.4 0.016
mmu-miR-429 0.3 0.034
mmu-miR-106a 0.1 0.047

Expression of miRNAs was quantified as described in Table 1. CCA, Corn oil+cellulose+azoxymethane (AOM) (n=5); FPA, Fish oil+pectin+AOM (n=5); CCS, Corn oil+cellulose+saline (n=5); FPS, Fish oil+pectin+saline (n=5); Lgr5high, stem cells. Only miRNAs with P ≤0.05 are shown.

Table 5.

Effect of diet and carcinogen combination on miRNA expression in GFPnegative sorted cells.

miRNA Expression ratio CCA- Lgr5neg/FPA- Lgr5neg P-value
miR-29b 3.8 0.008
let-7e 2.1 0.004
Let-7c 1.3 0.048
miR-19b 0.6 0.029
miR-484 0.5 0.019
miR-19a 0.4 0.034
miRNA Expression ratio CCA- Lgr5neg/FPA- Lgr5neg P-value
miR-29b 3.8 0.008
let-7e 2.1 0.004
Let-7c 1.3 0.048
miR-19b 0.6 0.029
miR-484 0.5 0.019
miR-19a 0.4 0.034

Expression of miRNAs was quantified as described in Table 1. CCA, Corn oil+cellulose+azoxymethane (AOM) (n=5); FPA, Fish oil+pectin+AOM (n=5); Lgr5neg, differentiated cells. Only miRNAs with P≤0.05 are shown.

These data refer to our recently published paper ‘Comparative effects of diet and carcinogen on microRNA expression in the stem cell niche of the mouse colonic crypt’ (Shah et al., 2016) [5].

2. Experimental design, materials and methods

2.1. Experimental diets

Lgr5-EGFP-IRES-creERT2 mice were assigned to one of the two diet groups (fish oil / pectin or corn oil / cellulose), which differed only in the type of fat and fiber. Diets contained (g/100 g diet): dextrose, 51.00; casein, 22.40; D,L-methionine, 0.34; American Institute of Nutrition (AIN)-76 salt mix, 3.91; AIN-76 vitamin mix, 1.12; choline chloride, 0.13; pectin or cellulose, 6.00. The total fat content of each diet was 15% by weight with the n-6 PUFA diet containing 15.0 g corn oil/100 g diet (Dyets, Bethlehem, PA) and the n-3 PUFA diet containing 11.5 g fish oil/100 g diet (Omega Protein, Houston, TX) plus 3.5 g corn oil/100 g diet to prevent essential fatty acid deficiency. All diet ingredients except oils were obtained from Bio-serv (Frenchtown, NJ). To prevent the formation of oxidized lipids, diets were stored at −20 °C and provided fresh to animals every day.

2.2. Fluorescence activated cell sorting of colonic stem cells

Colonic crypts from individual mice were isolated as previously described Sato et al. [1] with minor modification. The intact colons were everted on a disposable mouse gauge needle (Instech Laboratories) and incubated with 20 mM EDTA in PBS at 37 °C for 30 min. Following transfer to chilled Ca/Mg free HBSS, colons were vigorously vortexed to release crypts. The crypts were then incubated with 50 ul of DNase (stock concentration – 20 units/ml) in 10 ml of trypsin solution and single cells were then passed through a 40 micron cell strainer. Cells were counted and resuspended to a final cell density of 2×106 cells/mL. FACS (Fluorescence activated cell sorting) was then carried out to isolate the Lgr5high expressing stem cells, Lgr5low expressing daughter cells and Lgr5negative cells isolated from the colon using a BD FACS Aria II cytometer /sorter (BD Biosciences). Cells from wild type mice were used to set the gates for sorting.

2.3. RNA analyses

Total RNA from Lgr5high, Lgr5low and Lgr5negative sorted cells was isolated. For this purpose, cells from individual mice from all 4 groups (total of 60 samples) were separately processed using the mirVana miRNA Isolation Kit according to manufacturer’s instructions (Ambion, Austin, TX). Expression of 368 mature miRNAs was determined using TaqMan Rodent MicroRNA A Array 2.0 (Life Technologies, Grand Island, NY) as we have previously described [2], [3]. For mRNA profiling, samples were randomized prior to RNASeq library preparation. Sequencing libraries from RNA (10 ng) were generated using the TruSeq RNA Sample Preparation kit (Illumina, San diego, CA). ERCC (Life Technologies, Grand Island, NY) was added at the appropriate level as per manufacturer instructions. The libraries were pooled and sequenced using an Illumina HiSeq 2500 at SeqWright Genomic Services (Houston, TX). Sequencing data was provided demultiplexed and aligned using STAR with default parameters [4] and referenced against Mus musculus (UCSC version mm10).

2.4. Statistical analyses

For miRNA and mRNA profiling, two sided t-tests with Welch correction for unequal variance were performed on select miRNA across the specific treatment comparisons of interest. Mann–Whitney U nonparametric tests were also performed as a control against non-normal data and similar P-values were obtained. Standard error bars were plotted in order to document the variation in the population mean. P values <0.05 were considered to be statistically significant, and genes were selected for analysis using prior knowledge without considering P-values. Therefore, no multiple testing correction procedure was used. Standardized differences for the miRNAs and mRNAs were computed and a two-sample t-test was utilized to compare them. Small p-values indicated strong evidence of the hypothesized trend.

Acknowledgments

We gratefully acknowledge grant support from the National Institutes of Health grants (CA168312, CA129444, P30ES023512 and F32DK107108) and the American Institute for Cancer Research 208460.

Footnotes

Appendix A

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.dib.2015.12.026.

Appendix A. Supplementary material

Supplementary material

mmc1.pdf (1.2MB, pdf)

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material

mmc1.pdf (1.2MB, pdf)

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