miR-200 family expression is downregulated upon neoplastic progression of Barrett’s esophagus

Table 1 Relative miRNA expression in Barrett’s esophagus, gastric and duodenal mucosal tissues

MiRNA	Duodenal (n = 10)	Barrett’s esophagus (n = 17)	Gastric (n = 15)	P value (Kruskal-Wallis test)
miR-141	0.076 (0.039,0.167)	0.026 (0.023, 0.036)	0.051 (0.042, 0.092)	0.000212
miR-200a	0.148 (0.067, 0.340)	0.148 (0.126, 0.177)	0.247 (0.154, 0.509)	0.03142
miR-200b	0.796 (0.606, 1.276)	0.833 (0.750, 0.993)	1.233 (1.089, 1.963)	0.00112
miR-200c	2.700 (1.890, 3.511)	1.049 (0.929, 1.170)	2.335 (1.792, 2.773)	< 0.000112
miR-429	0.095 (0.042, 0.070)	0.070 (0.061, 0.087)	0.078 (0.072, 0.153)	0.259

Relative expression for each epithelial tissue type. Relative expression values are median (95% CI). The group P value was the result of a Kruskal-Wallis test across the three tissue groups. Significant differences were identified by post hoc testing by the Holm-Bonferroni method for:

¹Duodenal versus Barrett’s esophagus mucosa - miR-141 (P = 0.0008) and miR-200c (P < 0.0001);

²Gastric versus Barrett’s esophagus mucosa - miR-141 (P = 0.0004) miR-200a (P = 0.0078), miR-200b (P = 0.0001), and miR-200c (P < 0.0001).

Table 2 Top molecular and cellular functions of miR-200 gene targets

miRNA	Molecular and cellular functions	Molecules involved	P value
	Cell cycle	41	1.90E-07-2.82E-02
	Gene expression	53	4.94E-07-2.82E-02
miR-141	Cellular movement	39	2.45E-05-2.82E-02
	Cellular assembly and organization	61	2.88E-04-2.82E-02
	Cellular growth and proliferation	68	2.88E-04-2.28E-02
	Gene expression	84	1.37E-09-2.32E-02
	Cellular growth and proliferation	108	5.65E-05-2.32E-02
miR-200c	Cell cycle	53	9.17E-05-2.32E-02
	Cell death	95	9.62E-05-2.32E-02
	Cellular assembly and organization	55	1.19E-04-2.32E-02

The top five functions associated with the predicted targets of miR-141 and miR-200c, as determined by Ingenuity Pathway Analysis, are shown in this table. This analysis used a right-tailed Fisher’s exact test to calculate the probability (P value above) that each cellular function ascribed to the predicted target gene list was due to chance alone. For each miRNA, the molecular and cellular functions are ranked according to their P value.

Table 3 miRNA expression in Barrett’s esophagus and esophageal adenocarcinoma

miRNA	BE (n = 17)	Adeno carcinoma (n = 20)	BEC (n = 9)	P value (Kruskal-Wallis test)
miR-141	0.026 (0.023, 0.036)	0.012 (0.011, 0.025)	0.015 (0.01, 0.04)	0.033981
miR-200a	0.148 (0.126, 0.177)	0.057 (0.013, 0.042)	0.08 (0.017, 0.201)	0.000791
miR-200b	0.833 (0.75, 0.993)	0.387 (0.316, 0.572)	0.399 (0.258, 1.13)	0.000681
miR-200c	1.05 (0.929, 1.170)	0.551 (0.461, 0.939)	0.662 (0.438, 0.965)	0.0032312
miR-429	0.07 (0.062, 0.087)	0.039 (0.029, 0.06)	0.042 (0.036, 0.09)	0.013551

Relative expression for each tissue type. Relative expression values are median (95% CI). BE = Barrett’s esophagus epithelium from individuals without cancer, BEC = Barrett’s epithelium taken proximal to adenocarcinoma and confirmed by histology to be free of invasive cancer. The group P value was the result of a Kruskal-Wallis test across the three tissue groups. Significant differences were identified by post hoc testing by Holm-Bonferroni method for:

¹Adenocarcinoma versus Barrett’s esophagus mucosa from individuals without cancer - miR-141 (P = 0.0126), miR-200a (P = 0.0001), miR-200b (P < 0.0001), and miR-200c (P = 0.0014) and miR-429 (P = 0.0031);

²Barrett’s esophagus mucosa from individuals with versus without cancer - miR-200c (P = 0.0191).

Table 4 ZEB1 and ZEB2 expression in Barrettt’s epithelium and esophageal adenocarcinoma

	Fold↑	miR-141	miR-200a	miR-200b	miR-200c	miR-429	miR-215
ZEB1	2.9	R = -0.2	R = -0.4	R = -0.5	R = -0.5	R = -0.3	R = -0.3
	P < 0.0001	P = 0.341	P = 0.046	P = 0.009	P = 0.006	P = 0.159	P = 0.152
ZEB2	1.5	R = -0.3	R = -0.3	R = -0.5	R = -0.5	R = -0.4	R = -0.4
	P = 0.029	P = 0.101	P = 0.085	P = 0.008	P = 0.015	P = 0.067	P = 0.044

The fold increase (↑) in median ZEB1 and ZEB2 expression in esophageal adenocarcinoma versus Barrett’s esophagus mucosa from individuals without cancer, and the P value derived using the Mann-Whitney test are given. Spearman correlations (R = Spearman’s rho) between ZEB1/2 expression and each miR-200 member, with associated P values are also shown.