Tissue specimens

Flash-frozen cancer tissue specimens, obtained from black and white patients undergoing surgery for endometrial cancer at Duke University Medical Center, were deidentified and provided to our investigative team following exemption approvals. Tumor tissue was harvested at the time of surgery and flash frozen in liquid nitrogen within 60 minutes of removal. Fifty endometrial cancer specimens selected for the miR discovery analysis consisted of 41 white and 9 black women ( Table 1 ). All endometrial cancers were of endometrioid histology and staged according to International Federation of Gynecology and Obstetrics. A subset of 9 stage- and grade-matched cancer pairs from whites and blacks were used for a stratified discovery analysis. Seventeen normal endometrial tissue samples (9 whites and 8 blacks) were obtained from age-matched women undergoing hysterectomy for benign indications.

An independent set of 47 endometrial cancers (23 white and 24 black) matched by stage and grade was used to validate differential expression of a miRNA miR-337-3p identified in the discovery analysis ( Table 2 ). This expanded sample set also included advanced-stage endometrioid endometrial cancers. Racial status was self-described for all cases used for discovery and validation.

Tissue preparation

Laser microdissection (LMD) was used to preferentially isolate cancer cells or normal endometrial glandular epithelial cells to >95% homogeneity. miRNA was isolated from cells procured by LMD with TRIzol (Life Technologies, Carlsbad, CA) and mRNA was isolated by an additional level of purification with the Rneasy kit (Qiagen, Valencia, CA). The integrity of each of the RNA samples was confirmed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA).

miRNA analysis

Approximately 25 ng of total RNA was labeled using the Megaplex RT primer pool (Life Technologies), preamplified with miRNA-specific primers as specified by the manufacturer. Preamplified miRNA was then diluted and used in the TaqMan low-density arrays (TLDA) (Life Technologies) and thermocycled 40 times in a 7900HT thermocycler (Applied Biosystems, Foster City CA). Raw Ct values were extracted and calculated using SDS software (Life Technologies) and normalized to a reference Ct value that was derived from multiple positive endogenous controls on the TLDA.

External validation of miRNA expression using quantitative polymerase chain reaction

The expression of miR-337-3p was validated using an independent set of endometrial cancers specimen from black (n = 24) and white (n = 23) patients. The expression of miR-337-3p and RNU48 (endogenous control) were evaluated by qRT polymerase chain reaction (PCR) on the 7500 fast real-time PCR system (Applied Biosystems) in triplicate. Reverse transcription was performed with the TaqMan miRNA reverse transcription kit (Applied Biosystems) using 10 ng of total RNA.

Transcript expression analysis

Approximately 50 ng of total RNA was extracted from each sample and processed using the GeneChip 2-cycle cDNA synthesis kit (Affymetrix Inc, Santa Barbara, CA). Approximately 10 μg of amplified cRNA was labeled, hybridized to HG-U133 plus 2.0 GeneChips (Affymetrix Inc), washed, and scanned according to the manufacturer’s specifications (Affymetrix Inc).

Bioinformatics

Fifty endometrial cancers and 17 normal endometrial epithelial tissues were used to quantitatively assess the expression of 738 miRNAs arrayed on the TLDA following the manufacturers protocol (Applied Biosystems). The Ct (threshold cycle) values were normalized to an endogenous reference value using multiple endogenous controls. In all, 22 highly expressed positive controls were used to compute the endogenous reference Ct value (as the negative logarithm to the base 2 of their mean expression level). Normalized Ct values were obtained by subtracting endogenous reference Ct value. Unsupervised analysis of miRNA expression was performed using multidimensional scaling (MDS). The differential expressions were tested by parametric testing using analysis of variance (ANOVA) and nonparametric testing using Wilcoxon tests. All the above statistical analyses were performed using R software ( http://www.r-project.org/ ). All miRNAs on the array were tested and the miRNAs significant at the specified α threshold (0.005 for cancers) were selected, and we included only those having mean expressions above the detection limit (normalized ΔCt >–22). Total mRNA expressions were analyzed from a subset of the same endometrial cancers used for miRNA assays to enable comparison of mRNA and miRNA expression profiles. Expression Console software (Affymetrix Inc) was used to estimate mRNA transcript signal levels from scanned images by the 1-step Tukey biweight algorithm according to MAS5 algorithm. The signals on each array were normalized to a trimmed mean value of 500 excluding the lowest 2% and highest 2% of the signals. A probe set representing a unique gene bank sequence (Affymetrix Inc) is referred to as a transcript hereafter for convenience.

miRNA expression in black and white endometrial cancers

To determine the extent to which miRNAs might be differentially expressed between endometrial cancers from 9 black and 41 white patients, data were analyzed by MDS, the results of which indicated no clear distinction of miRNA expression according to race ( Figure 1 , A). This is in agreement with previously reported similar global mRNA expression profiles between whites and blacks in these cancers. The mRNA expression of endometrial cancers are known to depend on stage and grade, which are more dominant influences over race at the level of the transcriptome. We anticipated that substage and grade might also influence miRNA expression profiles. Therefore, ANOVA was performed using a linear model that included race and stage as variables to identify differentially expressed miRNAs between the endometrial cancers from white and black patients. Parametric testing by ANOVA considering race and stage as factors indicated only 5 miRNAs differentially expressed at P < .005. To decipher the influence of outliers on parametric testing, these results were compared to nonparametric testing. Wilcoxon rank sum test indicated 3 differentially expressed miRNAs at P < .005, but none of them were common to the 5 found by ANOVA. All the 8 differentially expressed miRNAs are listed in Table 3 .

Unsupervised and supervised analyses

Multidimensional scaling of microRNAs (miRNAs) of A , 50 cases, and B , 9 black and 9 white cases. C , Heat map of expressions of 18 miRNAs differentially regulated between black and white at P < .005 using balanced sample sizes.

Maxwell. microRNA expression in endometrial cancers. Am J Obstet Gynecol 2015 .

Table 3

Summary of microRNAs in blacks and whites found significant by parametric or nonparametric tests

miRNA	9 B and 41 W cancers				9 B and 9 W cancers				8 B and 9 W normal tissues
	B/W ratio	ANOVA P value a	Wilcoxon P value b	Significant c	B/W ratio	ANOVA P value a	Wilcoxon P value d	Significant c	B/W ratio	ANOVA P value	Wilcoxon P value b	Significant c
hsa-miR-767-3p	4.576	.0003	.017	Yes	6.223	.0412	.0040	Yes	0.378	.245	.423
hsa-miR-337-3p	3.187	.0299	.001	Yes	7.711	.0004	.00004	Yes	1.601	.603	.139
hsa-miR-544	2.616	.1092	.004	Yes	5.605	.0118	.00004	Yes	1.170	.903	.541
hsa-miR-600	2.508	.0046	.042	Yes	3.411	.0685	.0040	Yes	0.842	.553	.743
hsa-miR-377*	2.368	.0211	.0005	Yes	3.508	.0018	.0008	Yes	3.577	.244	.277
hsa-let-7d*	2.285	.0041	.016	Yes	3.179	.0200	.0028	Yes	3.651	.254	.167
hsa-miR-24	1.686	.0033	.016	Yes	2.260	.0001	.0106	Yes	1.141	.383	.277
hsa-miR-494	3.597	.0037	.012	Yes	3.844	.1255	.0244		1.295	.436	.139
hsa-miR-935	5.842	.0103	.008		8.240	.0847	.0028	Yes	1.499	.627	.606
hsa-miR-565	2.470	.1304	.005		3.611	.0003	.0003	Yes	1.422	.703	.277
hsa-miR-149	2.241	.0418	.020		3.074	.0070	.0028	Yes	1.057	.726	.541
hsa-miR-644	2.184	.1326	.048		5.699	.0003	.0012	Yes	1.352	.622	.673
hsa-miR-654-5p	2.145	.1097	.012		3.282	.0620	.0040	Yes	2.800	.442	.370
hsa-miR-645	2.056	.0500	.034		4.054	.0012	.0003	Yes	0.876	.868	.606
hsa-miR-924	1.816	.0124	.008		2.623	.0025	.0012	Yes	0.909	.796	.888
hsa-miR-200a*	1.703	.1209	.051		1.867	.0085	.0028	Yes	1.450	.178	.139
hsa-miR-632	1.681	.0434	.017		2.252	.0093	.0040	Yes	0.505	.274	.815
hsa-miR-23b*	1.655	.1788	.091		3.025	.0094	.0040	Yes	1.734	.594	.423
hsa-miR-668	1.646	.0442	.032		2.353	.0103	.0040	Yes	0.474	.418	.277
hsa-miR-545*	0.401	.1086	.655		0.747	.4969	.9314		14.70	.017	.074	Yes
hsa-miR-27a*	1.051	.8630	.747		1.015	.9702	1.0000		2.346	.022	.027	Yes
hsa-miR-335	1.098	.8012	.901		1.226	.4814	.6665		1.686	.021	.036	Yes
hsa-miR-324-3p	1.452	.0633	.081		1.686	.0285	.0625		1.423	.023	.036	Yes
hsa-miR-520c-3p	1.192	.6815	.261		1.770	.1692	.1615		0.425	.028	.046	Yes

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

1. Public reporting of assisted reproductive technology outcomes: past, present, and future
2. Impact of prolapse meshes on the metabolism of vaginal extracellular matrix in rhesus macaque
3. Inflammatory predictors of neurologic disability after preterm premature rupture of membranes
4. Computed tomography-guided preoperative localization of abdominal wall endometrioma

Stay updated, free articles. Join our Telegram channel

Join