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. 2018 Jan;7(1):36-46.
doi: 10.1530/EC-17-0315. Epub 2017 Nov 13.

Estrogen and high-fat diet induced alterations in C57BL/6 mice endometrial transcriptome profile

Yali Cheng  1 , Qiaoying Lv  1 , Bingying Xie  1 , Bingyi Yang  1 , Weiwei Shan  1 , Chengcheng Ning  1 , Bing Li  1 , Liying Xie  1 , Chao Gu  1 , Xuezhen Luo  1 , Xiaojun Chen  2 , Qin Zhu  3
Affiliations

Estrogen and high-fat diet induced alterations in C57BL/6 mice endometrial transcriptome profile

Yali Cheng et al. Endocr Connect. 2018 Jan.

Abstract

Unopposed estrogen stimulation and insulin resistance are known to play important roles in endometrial cancer (EC), but the interaction between these two factors and how they contribute to endometrial lesions are not completely elucidated. To investigate the endometrial transcriptome profile and the associated molecular pathway alterations, we established an ovariectomized C57BL/6 mouse model treated with subcutaneous implantation of 17-β estradiol (E2) pellet and/or high-fat diet (HFD) for 12 weeks to mimic sustained estrogen stimulation and insulin resistance. Histomorphologically, we found that both E2 and E2 + HFD groups showed markedly enlarged uterus and increased number of endometrial glands. The endometrium samples were collected for microarray assay. GO and KEGG analysis showed that genes regulated by E2 and/or HFD are mainly responsible for immune response, inflammatory response and metabolic pathways. Further IPA analysis demonstrated that the acute phase response signaling, NF-κB signaling, leukocyte extravasation signaling, PPAR signaling and LXR/RXR activation pathways are mainly involved in the pathways above. In addition, the genes modulated reciprocally by E2 and/or HFD were also analyzed, and their crosstalk mainly focuses on enhancing one another's activity. The combination analysis of microarray data and TCGA database provided potential diagnostic or therapeutic targets for EC. Further validation was performed in mice endometrium and human EC cell lines. In conclusion, this study unraveled the endometrial transcriptome profile alterations affected by E2 and/or HFD that may disturb endometrial homeostasis and contribute to the development of endometrial hyperplasia.

Keywords: 17-β estradiol; C57BL/6 mice; endometrial cancer; high-fat diet; microarray.

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Figures

Figure 1
Figure 1
Uterine size and histological performance. (A) Body weight change at the beginning (9 weeks) and end (21 weeks) of treatment (n = 8/group). (B) Representative morphological images of uteri from mice with estrogen and/or high-fat diet treatment. Ctrl, Control; E2, Estrogen; HFD, High-fat Diet. Ruler units, 1 mm. (C) Hematoxylin-eosin (H&E) staining of endometrium from mice with estrogen and/or high-fat diet treatment. The boxed areas in the top panel (100×) are magnified in the bottom panel (200×). Scale bars, 100 μm. (D) Gland number and size evaluation. The left panel represents the change of gland number per field, and the right panel represents the percentage of gland (diameter > 50 µm) in total glands per field. *P < 0.05, **P < 0.01, ***P < 0.001, n.s, no significant.
Figure 2
Figure 2
Changes of mRNA profiles in mice treated with estrogen and/or high-fat diet. (A) Cluster analysis of dysregulated gene expression in the endometrium of mice. Samples in the heat map are labeled: GCS (100454, 100455, 98700) for Ctrl, GCS (100456–100458, 98701) for E2, GCS (100459, 100460, 98702) for HFD and GCS (98703–98705) for E2 + HFD. Red color represents highly expressed genes and green color represents lowly expressed genes. (B) The number of altered genes regulated by E2, HFD and E2 + HFD. The number of genes upregulated or downregulated (with >1.5- or <0.67- fold change compared to Ctrl group) are represented as red and green numbers, respectively. (C) GO analysis of E2, HFD and E2 + HFD regulated genes in mouse endometrial samples. The top ten functions are presented and overlapped functions are boxed. (D) KEGG analysis of E2, HFD and E2 + HFD regulated genes in mouse endometrial samples. The top ten pathways are presented and overlapped pathways are boxed.
Figure 3
Figure 3
Estrogen and high-fat diet synergically promote endometrial lesions. (A) The 173 commonly upregulated and downregulated genes of mouse endometrial microarray in E2, HFD and E2 + HFD groups compared to Ctrl group. (B) Heat map illustrating the expression patterns of the 173 commonly dysregulated genes in E2 and/or HFD. The red and green colors indicate high and low expression level of genes, respectively. (C) E2 and HFD control gene modulation reciprocally. The left pie chart is the effect of HFD on common differentially expressed genes in mouse endometrium. The right pie chart is the effect of E2 on common differential genes in mice endometrium. We regarded the fold change >1.5 as ‘enhanced’ and <0.67 as ‘repressed’.
Figure 4
Figure 4
Aberrantly expressed genes analysis based on TCGA data. (A) Genetic alterations. Red represents amplification, blue represents deep deletion and pink represents mRNA upregulation. Genetic alterations were found in 225 of 333 UCEC patients (68%). The aberrant expression threshold was defined as Z-score ± 2.0 from the TCGA RNA-Seq V2 data. (B) Comparisons of genes (PIGR, KCTD1, FMO2, ARG2, PMAIP1, CCND2, ALPL, SLC27A2) expression between UCEC cancer tissues and non-cancerous tissues involved in TCGA based on GEPIA. Y axis indicates the log2 (TPM + 1) for genes expression. Red bar shows the tumor tissues and green bar indicates the non-cancerous tissues. These figures were derived from Gene Expression Profiling Interactive Analysis (GEPIA). *P < 0.01. TPM, Transcripts per Kilobase Million.
Figure 5
Figure 5
Microarray validation in mouse endometrial samples and EC cell lines. (A) Microarray validation by qRT-PCR in mice endometrium. The expression levels of genes Tnfaip3, Neurl3, Ihh, Plk1, Ccnf, Peli3, Mdm2 and Skp2 from microarray analysis in comparison with qRT-PCR gene expression levels are presented. These genes were normalized to GAPDH and then calculated as fold changes of relative mRNA expression. (B and C) IHC staining of PGR, CCND1, PCNA, STMN1, LGR5, TNFAIP3, VIM and KRT4 (B) are consistent with the microarray data (C) in E2 and/or HFD group vs Ctrl group. Yellow, Ctrl group; Green, E2 group; Red, HFD group; Blue, E2 + HFD group. Scale bars, 100 µm. (D) Microarray validation by qRT-PCR in human endometrial cancer cell lines. The expression levels of genes TNFAIP3, NEURL3, IHH, PLK1, CCNF, PELI3, MDM2 and SKP2 from microarray analysis compared to qRT-PCR gene expression levels are presented. These genes were normalized to GAPDH and calculated as fold changes in relative mRNA expression.
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