Influence of Quercetin-Rich Food Intake on microRNA Expression in Lung Cancer Tissues

Tram K. Lam; Stephanie Shao; Yingdong Zhao; Francesco Marincola; Angela Pesatori; Pier Alberto Bertazzi; Neil E. Caporaso; Ena Wang; Maria Teresa Landi

doi:10.1158/1055-9965.EPI-12-0745

DOI: 10.1158/1055-9965.EPI-12-0745 Published December 2012

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Figure 1.
Mean expression levels for significant miR groups comparing the highest versus lowest tertile of quercetin-rich food intake in former smokers with adenocarcinoma.

Table 1.

Selected characteristics by sex-specific tertile (T1-T3) of quercetin-rich food^a intake in EAGLE, separately for histologic subtypes

	AD				SQ
Subject characteristics	T1 (n = 57)	T2 (n = 47)	T3 (n = 40)	P	T1 (n = 48)	T2 (n = 42)	T3 (n = 30)	P
Quercetin-rich food intake^b, median (IQR)	0.79 (0.42)	1.56 (0.46)	2.45 (0.53)		0.71 (0.37)	1.47 (0.44)	2.53 (0.67)
Age, mean (SD)	62.58 + 9.03	64.58 + 8.21	65.57 + 8.55	0.22^c	68.48 + 7.09	69.02 + 6.30	66.37+ 8.62	0.29^c
Male, n, %	38 (66.67)	25 (53.19)	20 (50.0)	0.20^d	46 (95.83)	42 (100)	30 (100)	0.22^d
BMI, mean (SD)	24.42 + 3.76	25.59 + 3.98	24.42 + 3.17	0.21^c	25.42 + 3.10	26.73 + 3.71	27.74 + 3.92	0.02^{^c}
Smoking status, %				0.01^{^d}				0.08^d
Never	4 (7.02)	16 (34.04)	7 (17.50)		0	1 (2.38)	0
Former	22 (38.60)	12 (25.53)	22 (55.0)		18 (37.50)	20 (47.62)	20 (66.67)
Current	31 (54.39)	19 (40.43)	11 (27.50)		30 (62.50)	21 (50.0)	10 (33.33)
Pack years, median (IQR) intake	40.0 (24.0)	34.5 (32.0)	33.0 (28.0)	0.63^e	54.0 (28.25)	46.25 (21.70)	42.50 (33.0)	0.22^e
Vegetables^b,^fmedian (IQR)	1.01 (0.43)	2.03 (0.79)	2.46 (1.93)	<0.01^{^e}	0.95 (0.51)	1.68 (0.94)	2.61 (1.83)	<0.01^{^e}
Fruits^b,^g, median (IQR)	0.96 (0.82)	1.78 (1.28)	2.80 (1.40)	<0.01^{^e}	1.03 (0.89)	1.55 (0.69)	3.00 (1.37)	<0.01^{^e}
Meats^b,^h, median (IQR)	0.70 (0.68)	1.07 (0.79)	0.94 (0.98)	0.01^{^e}	1.07 (0.94)	1.23 (1.27)	1.33 (0.62)	0.06^e
Lifetime alcohol^b, median (IQR)	23.13 (25.17)	14.79 (34.01)	7.41 (19.58)	0.01^{^e}	30.69 (24.69)	36.23 (20.30)	31.18 (31.97)	0.66^e

NOTE: Column percent totals may not sum to 100% because of rounding; bolded P values indicated statistical significance.

Abbreviations: T1–T3 = first tertile through third tertile; IQR, interquartile range; SD, standard deviation.

↵^aQuercetin-rich foods: summary measure of apples, grapes, onions, artichoke/fennel/celery, beans, apricots, plums, turnips, peppers, strawberries, tomatoes, and broccoli.
↵^bFrequency (food groups, servingsper day; alcohol, grams per day).
↵^cANOVA test.
↵^dχ² test.
↵^eNonparametric Kruskal–Wallis test.
↵^fTotal vegetables intake: summary measure of tomatoes, peppers, carrots, salad, peas, beans/chickpeas, mushrooms, broccoli, turnips, savoy, black cabbage, onions, cooked spinach/Swiss.
↵^gTotal fruits intake: summary measure of apples, pears, bananas, kiwis, oranges/grapefruits, mandarins/clementines, grapes, peaches/clingstones, apricots, plums, strawberries, melons, and fruit cocktails.
↵^hTotal meat intake: summary measure of cooked ham (prosciutto cotto), smoked ham (prosciutto crudo), cured ham (speck), salami, baloney (mortadella), wurstel, salted sliced beef, coppa, pancetta, and other types of processed meats.

Table 2.

MiRs that significantly (at P < 0.05) differentiate highest (T3) versus lowest (T1) consumers of quercetin-rich food intake, separately by histology

	T1 mean ± SD	T3 mean ± SD	Fold change^a	P^b
AD (n = 97)
hsa-miR-502	0.085 ± 0.353	0.202 ± 0.350	1.124	0.017
hsa-mir-564	0.565 ± 0.258	0.449 ± 0.273	0.890	0.030
hsa-miR-124a	0.232 ± 0.447	0.072 ± 0.423	0.852	0.044
hsa-miR-125a	0.625 ± 0.723	1.034 ± 0.715	1.505	0.045
SQ (n = 78)
hsa-miR-510	0.283 ± 0.361	0.147 ± 0.295	0.872	0.003
hsa-mir-605	2.118 ± 0.815	1.279 ± 0.765	0.432	0.004
hsa-miR-155	−5.113 ± 1.071	−4.777 ± 0.784	1.399	0.012
hsa-miR-373	−0.005 ± 0.420	−0.091 ± 0.330	0.917	0.014
hsa-miR-453	0.597 ± 0.324	0.491 ± 0.211	0.899	0.017
hsa-miR-502	0.318 ± 0.309	0.095 ± 0.223	0.801	0.017
hsa-miR-18b	−2.621 ± 0.735	−2.227 ± 0.724	1.483	0.020
hsa-miR-183	1.160 ± 0.495	0.779 ± 0.470	0.683	0.022
hsa-mir-573	0.267 ± 0.355	0.126 ± 0.406	0.869	0.024
hsa-miR-524^a	0.074 ± 0.259	−0.082 ± 0.341	0.855	0.036
hsa-mir-612	−0.171 ± 0.851	−0.104 ± 0.719	1.069	0.042
hsa-miR-363^a	−0.076 ± 0.778	0.124 ± 0.703	1.222	0.046

↵^aFold change is the ratio (T3/T1) of geometric means (>1.0 indicates upregulation and < 1.0 downregulation).
↵^bCoefficient P value from ANOVA model adjusted for age, sex, BMI, smoking status, non–quercetin-rich fruits and vegetables, red/processed meat, alcohol, and cigarette pack years.

Table 3.

Influence of quercetin-rich food intake (T3 vs. T1) on individual miR, stratified by histology and smoking status

AD
Former smokers (n = 44)					Current smokers (n = 42)
miR name	T1 mean ± SD	T3 mean ± SD	Fold change^a	P^b	miR name	T1 mean ± SD	T3 mean ± SD	Fold change^a	P^b
hsa-mir-641	−0.048 ± 0.548	−0.237 ± 0.879	0.828	0.003	hsa-mir-580	0.492 ± 0.340	0.226 ± 0.286	0.767	0.003
hsa-miR-29b	−1.110 ± 1.390	−1.021 ± 1.158	1.092	0.003	hsa-miR-215	−0.665 ± 0.403	−0.932 ± 0.456	0.766	0.004
hsa-miR-146a	−4.714 ± 0.805	−4.830 ± 1.334	0.890	0.006	hsa-miR-194	−0.726 ± 0.662	−1.159 ± 0.883	0.648	0.011
hsa-miR-500a	0.646 ± 0.360	0.471 ± 0.539	0.839	0.008	hsa-mir-598	−0.119 ± 0.498	−0.674 ± 0.538	0.574	0.016
hsa-let-7e	−1.121 ± 0.808	−0.882 ± 0.804	1.270	0.018	hsa-miR-518a-2^a	0.077 ± 0.343	0.004 ± 0.228	0.929	0.020
hsa-miR-134	0.404 ± 0.304	0.354 ± 0.322	0.952	0.020	hsa-miR-503	0.147 ± 0.682	−0.244 ± 0.451	0.677	0.037
hsa-miR-26b	−1.624 ± 1.514	−0.930 ± 1.157	2.003	0.021	hsa-miR-146b	−4.682 ± 1.234	−4.278 ± 1.388	1.497	0.043
hsa-miR-302c^a	0.107 ± 0.333	0.244 ± 0.305	1.147	0.023	hsa-miR-381	0.044 ± 0.432	−0.127 ± 0.269	0.843	0.047
hsa-miR-98	−1.804 ± 1.199	−1.798 ± 1.527	1.006	0.024
hsa-let-7c	−1.634 ± 1.559	−1.265 ± 1.326	1.446	0.024
hsa-miR-27a	−1.351 ± 1.218	−1.097 ± 1.002	1.290	0.025
hsa-let-7a	−2.044 ± 1.400	−1.663 ± 1.288	1.464	0.026
hsa-let-7g	−2.283 ± 1.344	−2.396 ± 1.469	0.893	0.026
hsa-let-7i	−2.153 ± 1.492	−1.810 ± 1.300	1.409	0.028
hsa-let-7f	−2.512 ± 1.587	−2.191 ± 1.433	1.377	0.030
hsa-miR-195	−2.106 ± 1.343	−2.141 ± 1.111	0.966	0.031
hsa-miR-16	−2.852 ± 1.482	−2.495 ± 1.081	1.429	0.032
hsa-miR-146b	−4.292 ± 1.012	−4.254 ± 1.336	1.039	0.034
hsa-miR-26a	−0.943 ± 1.723	−0.364 ± 1.552	1.783	0.034
hsa-miR-19b	−3.679 ± 1.048	−3.947 ± 1.108	0.764	0.036
hsa-mir-564	0.556 ± 0.263	0.495 ± 0.220	0.941	0.037
hsa-miR-20a	−4.321 ± 1.456	−4.262 ± 1.075	1.061	0.041
hsa-miR-106a	−3.747 ± 1.533	−3.702 ± 0.988	1.047	0.044
hsa-miR-34a	−0.896 ± 0.743	−0.779 ± 0.530	1.124	0.046
hsa-miR-92a	−3.729 ± 1.292	−3.614 ± 1.051	1.121	0.048
SQ
Former smokers (n = 38)					Current smokers (n = 40)
miR name	T1 mean ± SD	T3 mean ± SD	Fold change^a	P^b	miR name	T1 mean ± SD	T3 mean ± SD	Fold change^a	P^b
hsa-miR-492	−1.383 ± 0.540	−1.262 ± 0.394	1.129	0.012	hsa-miR-502	0.354 ± 0.222	0.108 ± 0.220	0.782	0.010
hsa-miR-510	0.408 ± 0.374	0.166 ± 0.307	0.785	0.021	hsa-mir-605	2.198 ± 0.861	1.105 ± 0.701	0.335	0.013
hsa-miR-491	−0.274 ± 0.798	−0.121 ± 0.638	1.165	0.023	hsa-miR-506	0.245 ± 0.222	−0.195 ± 0.615	0.644	0.017
hsa-mir-612	−0.264 ± 1.072	−0.109 ± 0.789	1.168	0.025	hsa-miR-183	1.282 ± 0.518	0.357 ± 0.390	0.397	0.028
hsa-miR-500a	0.387 ± 0.278	0.291 ± 0.385	0.908	0.028	hsa-miR-524^a	0.094 ± 0.294	−0.170 ± 0.151	0.768	0.029
hsa-mir-663	−0.169 ± 0.551	−0.117 ± 0.495	1.054	0.034
hsa-miR-503	−0.069 ± 0.710	−0.149 ± 0.417	0.923	0.034
hsa-miR-453	0.584 ± 0.324	0.482 ± 0.226	0.903	0.035
hsa-mir-654	−1.239 ± 0.422	−1.111 ± 0.366	1.137	0.041
hsa-mir-658	0.211 ± 0.793	0.123 ± 0.372	0.916	0.047

NOTE: miRs are ordered by P value within strata.

↵^aFold change is the ratio (T3/T1) of geometric means (>1.0 indicates upregulation and < 1.0 downregulation).
↵^bCoefficient P value from ANOVA model adjusted for age, sex, BMI, non–quercetin-rich fruits and vegetables, red/processed meat, alcohol, and cigarette packyears.

Table 4.

Influence of quercetin-rich food intake (T3 vs. T1) on family of functional^a miR, stratified by histology and smoking status

		AD		SQs
		Former	Current	Former	Current
Family Function	miRNA members	P^a	P^a	P^a	P^a
Let-7 family	hsa-miR-let-7a	P < 0.001	P = 0.426	P = 0.366	P = 0.988
Tumor suppressor	hsa-miR-let-7b
	hsa-miR-let-7c
	hsa-miR-let-7d
	hsa-miR-let-7e
	hsa-miR-let-7f
	hsa-miR-let-7g
	hsa-miR-let-7i
	hsa-miR-98
	hsa-miR-202
miR-146 family	hsa-miR-146a	P = 0.002	P = 0.092	P = 0.753	P = 0.222
Tumor growth and invasion	hsa-miR-146b
miR-26 family	hsa-miR-26a	P = 0.010	P = 0.623	P = 0.588	P = 0.664
Apoptosis	hsa-miR-26b
miR-17 family	hsa-miR-20a	P = 0.031	P = 0.943	P = 0.766	P = 0.283
Tumor progression	hsa-miR-20b
	hsa-miR-106a
	hsa-miR-106b
	hsa-miR-17-5p
	hsa-miR-93
miR-29 family	hsa-miR-29a	P = 0.064	P = 0.373	P = 0.886	P = 0.137
DNA methylation	hsa-miR-29b
	hsa-miR-29c
miR-18 family	hsa-miR-18a	P = 0.705	P = 0.220	P = 0.156	P = 0.392
Tumor progression	hsa-miR-18b
miR-34 family	hsa-miR-34a	P = 0.142	P = 0.649	P = 0.275	P = 0.568
Tumor suppressor	hsa-miR-34c
miR-19 family	hsa-miR-19a	P = 0.072	P = 0.991	P = 0.608	P = 0.103
Tumor progression	hsa-miR-19b
miR-15/16 family	hsa-miR-503	P = 0.286	P = 0.073	P = 0.307	P = 0.763
Apoptosis	hsa-miR-15a
	hsa-miR-16
	hsa-miR-195
	hsa-miR-424

NOTE: Only results of miR families that had at least 1 miR that were significant at P < 0.05 from individual-based miR analyses (Table 2); bolded P values indicated results that remained significant after Bonferroni correction for multiple comparisons. Models adjusted for age, sex, BMI, non–quercetin-rich fruits and vegetables, red/processed meat, alcohol, and cigarette pack years.

*P value based on FCS as described in the Materials and Methods section.

↵^aRefer to Supplementary Table S5 for more detailed functions.

Additional Files

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Supplementary Data

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Supplementary Figure 1 - PDF file - 5418K, Hierarchical clustering heat map of miR expression levels for 25 miRs that displayed significant differential expression among former smokers with adenocarcinoma in the individual miR-based analysis. Average linkage was used to cluster miRs and samples with a distance metric of 1 minus centered correlation using BRB-ArrayTools version 4.2. Tertiles define subjects with low (Tertile 1) and high (Tertile 3) consumption of quercetin-rich food.
Supplementary Figure 2 - PDF file - 3580K, Hierarchical clustering heat map of miR expression levels for 10 miRs that displayed significant differential expression among former smokers with squamous cell carcinoma in the individual miR-based analysis. Average linkage was used to cluster miRs and samples with a distance metric of 1 minus centered correlation using BRB-ArrayTools version 4.2. Tertiles define subjects with low (Tertile 1) and high (Tertile 3) consumption of quercetin-rich food.
Supplementary Table 1 - PDF file - 51K, Selected characteristics of included and excluded EAGLE cases
Supplementary Table 2 - PDF file - 80K, List of miRs (N=198) retained from chip array analysis
Supplementary Table 3 - PDF file - 47K, Food groups and individual food items
Supplementary Table 4 - PDF file - 54K, All identified miR groups based on "seed" sequence (2-7nt)
Supplementary Table 5 - PDF file - 69K, Influence of quercetin-rich food intake (T3-vs-T1) on miR expression, by histology and smoking status